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Traditional Protocols and Optimization Methods Lead to Absent Expression in a Mycoplasma Cell-Free Gene Expression Platform

Traditional Protocols and Optimization Methods Lead to Absent Expression in a Mycoplasma... Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome- minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Keywords: mycoplasma, cell-free expression system, ribonuclease, in vitro transcription, in vitro translation 2 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Introduction One of the greatest challenges in modern science is to bring inanimate matter to life. Achieving such a transition would shed light on the very principles by which complex sets of chemical reactions create living systems. This key milestone will create new directions for research into synthetic systems that could lead to completely novel, yet unexplored, complex systems that have the ability to evolve. There is general consensus that the minimal living system needs to be a cell. A key stumbling block in the quest to create a minimal living cell from the bottom-up is the sheer complexity of even the simplest living systems, and our lack of knowledge of the general design principles that would allow us to construct a roadmap towards the bottom-up construction of a minimal cell. Most efforts to the bottom-up construction of a synthetic cell are based on Escherichia coli cell-free expression (CFE) systems, either using cell lysate or the PURE (protein synthesis using recombinant elements) system (1, 2). While E. coli is a valuable model for many purposes, the organism is vastly complex. In contrast, more simplified cellular systems are now available through the application of top-down synthetic biology strategies to reduce genome content (3–5). One approach is to re-design and re-build the genomic software of a cell ex vivo and reintroduce it into a compatible recipient host through genome transplantation (6), thereby redirecting the cellular machinery to create a new cell with re-programmed attributes. The J. Craig Venter Institute (JCVI) pioneered this strategy and applied it to completely synthesize, then minimize, a genome based on a template sequence of a natural organism of the genus Mycoplasma (hereafter mycoplasmas): Mycoplasma mycoides subspecies capri (7). The initial cell construct, JCVI-syn1.0 (hereafter Syn1.0) has a genome of 1,079 kbp, similar to 3 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 that of its natural precursor. Extensive genome reduction of Syn1.0 resulted in the “minimal bacterial cell” JCVI-syn3.0 (hereafter Syn3.0), with a genome of 531 kbp comprising 473 genes (8). This is a vastly simpler organism than E. coli K-12, whose >4,600-kbp genome contains >4,200 genes (9). While JCVI-syn3.0 propagates in the laboratory, its slow growth rate and pleomorphic phenotype renders it unsuitable for some applications. To overcome this, a near-minimal cell JCVI-syn3A (hereafter Syn3A) restoring 19 genes that are not present in Syn3.0 has been constructed as a more robust laboratory model and is in use to study various aspects of cell biology (5, 10, 11). The additional genes include a subset required for proper cell division, others of completely unknown function and a rRNA operon that restores its redundant presence in the original Syn1.0 genome (10). Our strategy to construct a bacterial cell from non-living parts leverages the lessons learned from the genome transplantation process used to boot up isolated M. mycoides and minimized M. mycoides genomes. In genome transplantation, a donor genome that is constructed as a yeast centromeric plasmid is installed in a recipient cell in a way that it commandeers that cell to produce bacteria programmed only by the donor genome (6, 12). To date the only acceptable genome transplantation recipient cell is Mycoplasma capricolum subspecies capricolum (hereafter Mcap). The only donor genomes that work are all close phylogenetic relatives of Mcap (13). This is presumably necessary because the software of the donor genome has to be capable of properly interacting with the enzymatic machinery in the recipient cell cytoplasm, such as the ribosomes or polymerases. By using donor genomes isolated from yeast instead of M. mycoides bacteria, there is no possibility of a resulting transplant being a contaminating cell that somehow made it through the chromosome isolation process. By transplanting genomes from one species of mycoplasma 4 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 into a recipient cell from a different species, sequence analysis of any genome transplant can confirm whether the transplant cell genome is the result of recombination between the donor genome and recipient cell genome. Our plan to construct a synthetic cell from non- living parts is to fill micron sized lipid vesicles with cytoplasm derived from bacteria and then install a genome in that CFE containing vesicle in a way resulting in the enlivening of the assembled parts. Given the similarity of this plan to the genome transplantation technique, our intent is to use the same reagents, controls and safeguards that are part of the genome transplantation protocol. We plan to use a Syn3A genome obtained from a yeast as the genetic software of our synthetic cell and a CFE derived from Mcap. Here we report our attempts to develop a CFE system using these cells, starting with protocols developed for successful E. coli lysates. We will discuss how unexpected nuclease activity in the lysate initially prohibited transcription, even to the extent that Mcap lysate poisoned E. coli CFE. We overcame these problems, but despite our best efforts and testing every known procedure for lysate preparation, there appear to be as yet unknown aspects of mycoplasma biology, that prevent us from obtaining a functional lysate. Considering the intense interest in the bottom-up construction of synthetic cells in general, our work should serve as a general reminder of the many complexities that will be encountered in these endeavors. We are aware that our results do not bring a minimal genome-based synthetic cell closer. However, we identify several factors that limited our success that may inform such efforts in other organisms. We also discuss alternative approaches for packaging the cell machinery of minimal cells to create an operational bottom-up synthetic cell. 5 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1. Materials and methods 1.1 Reagents Chemicals and reagents were purchased from Sigma Aldrich (reagent-grade), except those specified below. 1.2 Microorganisms and cell culture For preparation of mycoplasma cell-free extracts we cultivated the mycoplasma strains Mycoplasma capricolum subsp. capricolum (Mcap) and JCVI-syn3A (Syn3A) (6, 8). Among mycoplasma species, Mcap presents a faster and more robust growth than Syn3A. This difference in growth rates is explained by the significant genome reduction in Syn3A (543 kb) compared to its parent genome (1,079 Mb, JCVI-syn1.0). As obligate parasites, mycoplasmas require a rich medium (including serum) for in vitro growth. Mycoplasma cells were cultured in adapted SP4 glucose broth including 17 vol% KnockOut™ serum (Gibco) as a replacement for fetal bovine serum (SP4-KO medium) (14, 15). For lysates prepared by digitonin treatment, cells were grown in modified Hayflick medium supplemented with 20 vol% heat- inactivated horse serum (ThermoFisher) (HS medium) (16). Cells were incubated at 37°C without agitation or aeration. A fully-grown culture of 500-mL volume required about 6 days from the inoculation of a 1-mL culture (in 14-mL tube), subsequent transfer into a 100-mL culture (in 250-mL flasks), and the final transfer into a 500-mL culture (in 2-L flask). Cell growth was determined by color change (phenol red) as proliferating cells accumulate acidic metabolic byproducts. E. coli lysates were prepared from the BL21 (DE3) Star™ pLysS strain containing plasmid to express rare tRNAs (pRARE) (Invitrogen) following a protocol previously described (17). 6 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.3 Lysate preparation E. coli CFE is currently the CFE system with the best protein production levels (18). The successful preparation of E. coli crude lysates for CFE system, known as the S30 protocol, started many decades ago (19–22) and it has been gradually improved (23, 24). Each E. coli lysate batch has a slightly different composition and consequently a distinct efficiency regarding the protein production. In this work, we used the same batch of E. coli extract within a single experiment but different batches between experiments (due to extract availability). Since there is no reported protocol for the preparation of mycoplasma lysates for CFE system, we adapted a recent S30 protocol (17) for the preparation of mycoplasma lysates. Mcap or Syn3A were harvested at stationary growth phase (culture medium pH around 5) by centrifugation at 5,000 g, 4°C for 15 minutes. Pellets were washed twice with cold S30A buffer (50 mM Tris pH 7.7, 14 mM Mg-glutamate, 60 mM K-glutamate), keeping the flasks as much as possible on ice (ideally also in the cold room). The original S30 protocol consists of (i) cell lysis (by French press or sonicator, Figure 1) followed by (ii) run-off incubation to release ribosomes from polysomes, and (iii) dialysis for buffer exchange (S30B buffer: 5 mM Tris pH 8.2, 14 mM Mg-glutamate, 60 mM K-glutamate). High-speed centrifugation steps are employed to clarify the lysate after the lysis, the run-off incubation, and the dialysis steps (at same speed). Since the original protocol generates clean supernatants at 30,000 g centrifugal force, this method is known as S30. Because of morphological differences between E. coli and mycoplasmas (e.g. size, cell membrane composition) we tested different methods for cell lysis: (i) sonication at 150- 1,000 J/mL (Q500, 40% amplitude, 3-mm tip, Fisherbrand); (ii) French press at 140-360 mPa 7 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 (FPG12800, Homogenizing Systems Ltd); (iii) liquid nitrogen grinding with mortar and pestle, 20 min. non-stop with frequent addition of liquid nitrogen; or (iv) osmotic shock by incubation in S30B buffer, supplemented with 250 mM NaCl (30 min., 23˚C); or (v) by treatment with detergents, see Methods 1.6. The lysate is typically centrifuged at 20,000 g for 20 minutes at 4°C to remove cellular debris (first clarification step). Next, the supernatant is incubated at 37°C from 15 to 80 minutes (run-off incubation), followed by a second clarification step (20,000 g, 4°C, 20 min). The supernatants were then dialyzed in 10 kDa MWCO cassettes (Slide-a-lyzer™ Thermofisher) against S30B buffer (0.5x final concentration) (17). Samples were clarified once more (20,000, 4°C, 20 min) is performed to remove cellular debris, and finally flash frozen in liquid nitrogen and stored at -80°C. 1.4 Mycoplasma cell surface trypsinization Mycoplasma cell cultures were centrifuged at 5,000 g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed with 75 mM HEPES pH 8.0 buffer. The washed pellet was again centrifuged (5,000 g, 10 min, 4°C) and the supernatant was discarded. The washed pellet was then resuspended in 75 mM HEPES pH 8.0 buffer supplemented with 100 µg trypsin (Merck Life Science) per 100 mg of cell pellet and incubated at 37°C for 30 min under mild agitation (100 rpm). Afterwards, trypsinized cells were centrifuged at 5,000 g for 10 min at 4°C and the supernatant was discarded. The final pellet was washed once with purified soybean trypsin inhibitor solution (Gibco, 1x defined trypsin inhibitor, #R007100), centrifuged at 5,000 g for 10 minutes at 4°C, and finally washed with 75 mM HEPES pH 8.0 buffer. Alternatively, trypsin was inhibited using SP4-KO medium. 8 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.5 Separation of cells and media from mycoplasma cultures by sucrose cushion Mycoplasma cell suspensions or cell culture media (400 µL, Mcap or Syn3A) were underlaid with a sucrose cushion (600 µL, 0.5M sucrose in 20 mM HEPES pH 8, filter-sterilized) and centrifuged at 3,500 g for 10 min. The supernatant (culture medium or cell suspension buffer) was carefully removed by vacuum aspiration using a Pasteur pipette. The cell pellet was carefully resuspended and used for subsequent experiments. 1.6 Lysate preparation by Triton™ X-100 or digitonin treatment Mcap or Syn3A were cultured in HS medium (2-L flasks) as described in section 1.2) and harvested at pH 5.5-6 by centrifugation at 5,000 g, 4°C for 15 minutes. Next, cell pellets were washed twice with fresh HS medium, and collected by centrifugation (7,000 g, 4°C, 15 minutes). Cells were lysed upon resuspension with a lysis buffer (1 mL /100 mg of cell pellet) containing 1 vol% of Triton™ X-100 (hereafter TX100) and incubation on ice for 15 minutes (with vortexing every 5 minutes). The lysis buffer also included 75 mM HEPES pH 8.0, 1x HALT™ protease inhibitor cocktail without EDTA (Thermofisher), 20 mM CaCl , 1 mM polyvinyl sulfonic acid Mw~2-5 kDa (PSVA) (25). The crude lysate was clarified by high-speed centrifugation (32,000 g, 4°C, 20 minutes). When recovering the supernatant, we avoided to collect the pellet or any floating debris. Afterward, the clarified lysate was concentrated 10- fold with a centrifugal filter unit (Merck Millipore Amicon™ 15 mL, 10 kDa MWCO), followed by dialysis (Spectra™ 3.5 kDa MWCO dialysis membrane) against 0.5x S30B buffer at 4°C for 2 hours. Run-off incubation was not performed. Finally, samples were flash frozen and stored at -80°C. 9 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 For the preparation of mycoplasma lysates using digitonin, we used a similar protocol described for TX100 except for the surfactant in the lysis buffer (1 mg/mL of digitonin instead of 1 vol% of TX100). To improve the solubility of digitonin in aqueous solution, we prepared a stock of 2 wt% digitonin in deionized water and heated it to 95°C for 5 minutes. The solution was allowed to cool down to room temperature before mixing with other components of the lysis buffer. 1.7 Total RNA analysis RNA was isolated from mycoplasma cells or lysate by the hot phenol extraction method (26). Isolated RNA fractions were resuspended in nuclease-free water and their concentration measured by spectrophotometry (Nanodrop™, Thermofisher). RNA was diluted to a concentration of 1 µg/µL and 5 µL of each sample was analyzed by performing denaturing PAGE (8% acrylamide/bis-acrylamide, 8 M urea, 0.5x Tris/Borate/EDTA buffer, 250 V for 45 TM min). Gels were stained with SYBR -Gold (Invitrogen). Alternatively, total RNA fractions were analyzed using chip-based capillary electrophoresis in agarose gel (RNA 6000 Nano kit, Agilent 2100 Bioanalyzer). For that, samples were diluted to a 500 ng/µL final concentration, of which 1 µL of each sample was analyzed in the chip. The Bioanalyzer total RNA plots show arbitrary fluorescence units (y-axis) versus migration time (x-axis). For better data interpretation, the x-axis was converted to RNA size using an RNA ladder as reference (25- TM 6,000 nt). RNA was converted to cDNA library (iScript , Bio-Rad) for the qPCR experiment TM ® (iQ SYBR Green Supermix, Bio-Rad). qPCR was performed with primers for 23S rRNA (see Table S4 for sequences). 10 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.8 Cell-free expression (CFE) Reactions mixtures were designed from the E. coli CFE system previously described (17). Bacterial lysates can show batch-to-batch variation in cell-free expression efficiency. Therefore, for each CFE experiment we used a single lysate batch to generate consistent results. The reaction mix comprised cell lysates (33 vol%), feeding buffer (18.5 vol%), and a suspension of DNA template (plasmid or linear DNA, 6-10 nM, 5-10 vol%) adjusting the final volume with deionized water or S30B buffer. GamS nuclease inhibitor (3 µM final concentration) was added when linear DNA was used as template (27). The feeding buffer (Table S1) contains the amino acid mixture (1.5 mM of all 20 amino acids initially dissolved in 5M KOH and finally adjusted to pH 6.52 with acetic acid) and energy solution (50 mM HEPES pH 8, 1.5 mM ATP/GTP, 0.9 mM CTP/UTP, 0.2 mg/mL E. coli or S. cerevisiae tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.07 mM folinic acid, 1 mM spermidine, 30 mM 3-PGA, 1 mM DL-dithiothreitol, DTT). Mg-glutamate (5-30 mM), K-glutamate (60-120 mM), RNAse inhibitor (60U/150 µL reaction mix volume of recombinant RNAse inhibitor, Thermofisher #N8080119), recombinant T7 RNA polymerase (120U / 150 µL of reaction mix volume) were added to all reactions. For some reactions polyethylene glycol 8000 (2 vol%, PEG8000, MW ~8,000 kDa), calcium salts (calcium chloride, calcium acetate, calcium glutamate, 0-25 mM) were added. Two alternative RNase inhibitors, Superase-In and RNase Off, were also used at 60 U/150 µL. Samples were analyzed in microplate reader (M200 or M10 Tecan, SpectraMax Gemini, fluorescence detection) using black 384-well plates with flat and transparent bottom (Greiner Bio-One, #781900, 11 µL sample per well). Green fluorescent protein (eGFP) was analyzed at 488/525 nm (excitation/emission) and red fluorescent protein (mCherry) at 11 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 560/610 nm. The plate was kept at 30°C for 4-24 hours (5-min data acquisition interval) at 30°C. Graphs were prepared using Origin 8 software. 1.9 Mycoplasma DNA constructs Translation was monitored by the expression of red fluorescent protein (mCherry) or enhanced-green fluorescent protein (eGFP) (Figure S1). DNA template coding for mCherry under the control of Pspi promoter was previously developed for mycoplasma in vivo experiments and provided by Prof. Yo Suzuki (28). The eGFP gene was optimized from the original E. coli sequence for mycoplasma usage (Table S5) using online codon usage database and automated DNA sequence optimization tool (29, 30). Cloning was performed by Golden Gate assembly using pRSET5d vector and transformed into XL-1 E. coli chemically competent cells (Agilent Technologies). Primers are listed in the supplementary information (Table S4). For tracking transcription, we used malachite green (MG, Addgene, pJBL7004) and Spinach2 RNA aptamers combined with their respective dyes (Table S5) (31–35). Spinach2 RNA aptamer utilized (5Z)-5-[(3,5-Difluoro-4-hydroxyphenyl) methylene]-3,5-dihydro-2-methyl-3- (2,2,2-trifluoroethyl)-4H-imidazol-4-one dye (DFHBI-1T) and MG aptamer complexed with malachite green dye. For tracking RNA degradation, we used Broccoli RNA aptamer with 3,5- difluoro-4-hydroxybenzylidene imidazolinone dye (DFHBI) (33, 36). The reaction mix contained 1-8 nM RNA aptamer template and 60 µM DFHBI-1T. 1.10 mRNA degradation assay 12 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 The mRNA substrate (coding for eGFP, ~800 nucleotides long, 1.5 µg) was mixed with mycoplasma or E. coli lysate (protein concentration around 3 mg/mL) in a 5-µL final volume sample (volume adjusted with deionized nuclease-free water) kept in ice. Next, the samples were incubated at 30°C for 1-10 minutes to assay mRNA degradation. To quench RNAse activity after incubation, samples were diluted with TES buffer to a final volume of 100 µL (10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 vol% SDS). The RNA fraction was purified using the hot phenol extraction method (26). Dried RNA pellets were resuspended in 5 µL deionized water and mixed with 11 µL of sample buffer (0.85 mM EDTA, 12.5 mM 4- morpholinepropanesulfonic acid (MOPS), 3.1 sodium acetate, 25 vol% formamide, 63 vol% formaldehyde solution (37 wt% in water) and 0.25 µL ethidium bromide. RNA samples were analyzed using denaturing 1 % agarose gels (60V for 100 minutes in 1x MOPS as running buffer: 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0). The mRNA substrate was synthesized by in vitro transcription in a reaction containing 40 mM Tris-HCl pH 8.1, 25 mM MgCl , 5 mM DDT, 1 mM spermidine, 4 mM rNTPs, 5 mM guanosine-5’-monophosphate (GMP), 5 nM T7p-GFP linear dsDNA PCR product, and T7 RNA polymerase 600U / 500 µL reaction mix volume. The IVT reaction mix was incubated at 37°C for 4 hours, followed by RNA precipitation with 97% ethanol and centrifugation for 15 minutes at 14,000 g. The mRNA pellet was washed once with 80% ethanol and then spin- dried to remove solvent. The dried pellet was resuspended in nuclease-free water (~800 ng/µL). 1.11 Statistical analysis 13 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Experiments were done in triplicate (n=3) whenever feasible. Error bars in the figures are the standard deviation of multiple experiments. Results Development of a mycoplasma CFE platform. CFE platforms have been derived from various prokaryotes and eukaryotes (18). However, no protocols for the preparation of a mycoplasma lysate for a CFE platform have been reported in literature. For the development of a robust CFE platform based on Mcap and Syn3A, we started out with systematically testing several conditions used in a well-established protocol for obtaining the robust CFE platform of E. coli, known as the S30 protocol (1, 17). It comprises cell lysis, extract clarification, run-off incubation, and dialysis (Figure 1). The S30 protocol has been used by other researchers as a starting point to produce lysates from non-model prokaryotes, basically using the same essential steps. To date, no organism was able to yield similar protein production levels as the E. coli CFE platform. In terms of protein synthesis capacity, lysates derived from Gram-negative bacteria such as Vibrio natriegens and Pseudomonas putida have lower yields, by approximately 35% and 90% respectively, in batch reactions of green fluorescent protein (eGFP) (37–39). When lysates were produced from Gram-positive bacteria, the protein yields were even lower. For instance, Bacillus megaterium and Bacillus subtilis remained below 4% of E. coli CFE’s full protein synthesis capacity (18, 40, 41). Based on the 60-year history of the S30 protocol development and the phylogenetic differences between E. coli and mycoplasmas, we were aware of the complexity of creating the mycoplasma CFE platform from scratch. 14 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 We initially hypothesized that the mycoplasma lysate preparation would compare to the S30 lysate protocol with adjustments due to the unique cell membrane of mycoplasmas (i.e. absent cell wall and cholesterol-rich). We initially tested physical cell disruption methods (i.e. sonication, French press, liquid nitrogen grinding, osmotic shock), since these methods have been successful in producing active lysates for most prokaryote CFE platforms, including Gram-negative (E. coli, Vibrio natriegens) and Gram-positive bacteria (Bacilli). Because of the absence of a cell wall in mycoplasmas, we also tested surfactant-based cell lysis methods (i.e. by Triton™ X-100 or digitonin treatment). In addition to the lysis method, we also analyzed the importance of centrifugation speeds for lysate clarification, run-off incubation, and dialysis on the lysate’s capacity to support transcription and translation (Table 1). For CFE batch reactions, lysates were mixed with a feeding buffer and DNA template as described in methods. Starting from the optimal concentrations for the E. coli CFE platform, we systematically tested a range of concentrations for key components in mycoplasma CFE (e.g. Mg-glutamate, K-glutamate, DNA template, PEG8000) (Table 2). For testing transcription, we used DNA templates coding for the RNA aptamers malachite green and Spinach2 controlled by an exogenous T7 promoter. In presence of the respective dyes (malachite green dye complexed with malachite green aptamer and DFHBI-1T complexed with Spinach2 aptamer), we measured fluorescence of RNA aptamer-dye complexes. For testing protein production, we prepared DNA templates coding for enhanced-green fluorescent protein (eGFP) and red fluorescent protein (mCherry) controlled by an exogenous T7 promoter (Figure S1A) or endogenous promoter (Figure S1B). 15 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 To ensure maximum translation efficiency, eGFP and mCherry gene sequences were codon- optimized for mycoplasma usage (28–30). We tested different cell disruption methods to find the best conditions for mycoplasma cell disruption. The functionality of each lysate was assessed in terms of its capacity to support transcription and translation. Cell disruption efficiency depends on the lipid composition of cell membrane and the energy level employed for cell disruption (for mechanical disruption methods) (42, 43). Compared to other Gram-positive (e.g. Bacilli) and Gram-negative bacteria (e.g. E. coli, Vibrio), mycoplasma cells are relatively small (approximately 0.4 µm versus 2 µm E. coli) and their plasma membranes rely on cholesterol and cholesterol esters for mechanical stability rather than a cell wall (44, 45). Therefore, methods available to lyse E. coli or Bacilli may not be readily applicable to mycoplasma. First, we tested a range of energy levels for mycoplasma cell disruption by sonication (150, 300, 500, 1,000 J). Sonicated Mcap lysates did not show CFE regardless of the energy level employed for lysis or centrifugal force (3,000, 12,000 g) (Figure S2 A-O). Lysis by osmotic shock (Figure S2P) or liquid nitrogen grinding (Figure S2Q) also failed to support CFE for Mcap as well as Syn3A. Different energy regeneration molecules (PEP or 3-PGA) (Figure S2 A-L) or adding a molecular crowder (PEG8000) (Figure S2 A-O, Figure S3) failed to improve expression of DNA template coding for eGFP controlled by the T7 promoter in Mcap CFE. Similar results were observed using a DNA template coding for the mCherry sequence controlled by the Pspi promoter (Figure S3) (28). 16 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 To understand the basis for our failure in setting up a working mycoplasma CFE system we next investigated Mcap and Syn3A extracts for evidence of in vitro transcription only. The rationale for using Syn3A was that the minimized Syn3A genome contained only about half the number of genes present in Mcap, and we might be able to produce a minimal cell (i.e. Syn3A) extract being capable of CFE due to the absence of a non-essential nuclease or protease gene. Using linear double-stranded DNA templates encoding a T7 RNA polymerase promoter, a low level of transcription was detected for the malachite green RNA aptamer expressed in E. coli lysate, but Mcap and Syn3A lysates prepared by liquid nitrogen grinding did not give a clear signal (Figure S4), indicating no expression of malachite green aptamers in Syn3A or Mcap. These experiments suggested that poor transcription rates or rapid degradation of DNA-RNAs or a combination thereof prevented us from obtaining a functional CFE system using mycoplasma extracts. To investigate the possible impact of RNA degradation on mycoplasma CFE, we measured the fluorescence signal of pre-transcribed Broccoli aptamer complexed with DFHBI dye in various extracts as a function of time (36). DFHBI-Broccoli rapidly degraded over time with a more pronounced decrease in Mcap lysate obtained by sonication than in Syn3A lysate obtained by sonication (Figure S5B), irrespective of the amplitude used (Figure S5 A/B). In a kinetic assay, DFHBI-Broccoli degraded in Mcap lysate produced by sonication but not in Syn3A lysate produced by sonication (Figure S5C). Degradation of DFHBI-Broccoli was even faster in the Mcap lysate produced by liquid nitrogen grinding (Figure S5D). Nitrogen- ground Syn3A lysate degraded DFHBI-Broccoli over time but at a much slower rate. E. coli lysates controls made by sonication and liquid nitrogen grinding both degraded DFHBI- Broccoli. Different lysate preparation techniques might be responsible for the differences in 17 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 DFHBI-Broccoli degradation that we see from Syn3A lysates made by sonication vs. liquid nitrogen grinding. Liquid nitrogen grinding avoids overheating proteins during extraction, which could lead to denaturation and degradation. However, because the fluorescent signal from the aptamer-dye complex depends on the RNA folding, it cannot be ruled out that the low level of fluorescent signal is caused by improper folding in lysates as well. This could well explain the unexpected low signal of DFHBI-Broccoli in the E. coli lysate that was used as positive control. In summary, most of our mycoplasma extracts presented low levels of transcription and rapid degradation of reporter RNAs (except for Syn3A lysate, which degraded the DFHBI- Broccoli complex at a slower rate) were observed as well as for E. coli lysate. As a reference, the fluorescence signal of RNA-dye complex can be 2-10 times higher in E. coli CFE systems, depending on the type of RNA aptamer, dye, buffer composition, and plate reader settings (46). The inability to develop a mycoplasma CFE system starting from successful protocols for E. coli lysates made us wonder if some yet unknown aspects of mycoplasma biology might pose additional challenges to the development of a mycoplasma CFE system. Therefore, we next explored the mycoplasma metabolism to identify factors that might affect the production of a functional CFE system. Gene expression assay with E. coli lysate led to investigation of the critical impact of nuclease activity in mycoplasma CFE. Membrane-associated nucleases enable mycoplasmas to scavenge the extracellular environment for nucleotides. These membrane-associated nucleases might impair mycoplasma CFE by degrading essential nucleic acids involved in transcription and translation. Even though the enzymatic activity of membrane-associated 18 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 nucleases for some mycoplasma species are described in literature (47–52), little is known about their structure and inhibition mechanisms. Mcap and Syn3A share 20 common nucleases with annotated function (Table S2). The only common nuclease for Mcap and Syn3A associated with the cell membrane is ribonuclease Y, which is a degradosome protein C-terminally anchored to the inner membrane related to RNA turnover mechanisms (53). It is possible that both Mcap and Syn3A contain unannotated surface nucleases. Many of the genes of unknown function in both organisms encode membrane associated proteins. To test mycoplasma lysates for factors incompatible with CFE, we added mycoplasma lysate directly to an E. coli CFE (Figure 2A). Since we aimed to measure the effect of mycoplasma lysate in normal E. coli CFE reaction, we employed a plasmid coding for eGFP controlled by T7 promoter codon-optimized for E. coli. Indeed, with increasing mycoplasma lysate fraction, eGFP expression by E. coli CFE was gradually decreased. Importantly, eGFP expression in E. coli CFE mixed with Syn3A lysate could be restored by adding the surface nuclease inhibitor CaCl (Figure 2B, Table S3). Even though CaCl is described as a specific inhibitor of Mcap 2 2 nuclease activity (49) (what is true for Mcap is likely true for M. mycoides), we did not discard the possibility of other roles for CaCl in the CFE environment. In this instance, the effect of adding an extra component to the CFE reaction mix (i.e. mycoplasma lysate), likely did not have a significant effect on the GFP yields observed. As evidence, the GFP yields in a reaction containing 15 mM CaCl (50% Syn3A lysate in E. coli CFE reaction) were similar to the yields obtained in 100% E. coli CFE, which was around the maximum yield obtained for this reaction. 19 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 These results suggested a possible role of nucleases present in the mycoplasma lysates in 'poisoning' CFE and a possible remedy by adding CaCl . A murine RNAse inhibitor (NEB) was used throughout the experiments but did not increase eGFP signal. Two other commercially available RNase inhibitors, Superase-In™ (ThermoFisher) and RNase Off™ (Biovision), were also examined for their capacity to inhibit RNA nuclease activity in mycoplasma lysates but neither could produce measurable eGFP expression (Figure S13A-B). Rescue of eGFP expression in E. coli CFE poisoned with mycoplasma lysate was unsuccessful using any of the RNases (Figure S13C). Encouraged by the CaCl result, we tested expression in 100% Syn3A CFE reaction with CaCl . 2 2 However, no protein production was detected (Figure S6A). Calcium acetate also failed to support eGFP synthesis in either a 100% Syn3A or 100% Mcap CFE reaction (Figure S6B). We also observed that the poisoning effect could not be removed by higher centrifugation speeds. Syn3A lysates prepared by French press and clarified at different centrifugation speeds (20-80,000 g) (Figure S7) depleted the production of eGFP in E. coli/mycoplasma mixed CFE system, as observed in Figure 2A. However, because calcium-mediated nuclease inhibition restored eGFP expression in E. coli CFE mixed with Syn3A lysate, we reasoned that the nuclease content in mycoplasma lysates was perhaps too high for mycoplasma CFE. We next explored procedures for removing these poisoning components from mycoplasma lysates. Trypsinization of mycoplasma cells removed the poisoning effect and suggested surface nuclease activity. Because the extracellular mycoplasma surface could be a major source of 20 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 nucleases that incapacitated CFE of the extracts, we trypsinized mycoplasma cells before cell lysis to inactivate surface nucleases. Mcap lysate prepared from trypsinized cells (hereafter trypsin-Mcap) did not inactivate expression in the E. coli CFE platform (Figure 3A). Instead, the eGFP expression in E. coli CFE mixtures containing up to 50 vol% trypsin-Mcap lysate was similar to eGFP expression observed for control experiments using S30B buffer as diluent. The increase in expression by adding up to 50 vol% of diluent into E. coli CFE system indicates an improved expression of eGFP at lower concentrations of E. coli lysate, which might be due to specific optimal volume fraction of lysate in the CFE reaction mix. Although trypsin-Mcap lysate did not inactivate E. coli CFE, translation remained disabled for the mycoplasma CFE platform containing 100% trypsin-Mcap lysate, even in the presence of calcium glutamate (0-20 mM) (Figure S8). Only a small transcription level (Spinach2 aptamer) was detected (Figure 3B) compared to the signal obtained in E. coli CFE system, which was at least two-fold higher. RNA degraded during mycoplasma lysate preparation. At this point, we reasoned that development of a functional Mcap and/or Syn3A CFE was impeded by rapid degradation of RNAs by mycoplasma nucleases, leading to complete inactivation of the translational machinery early on during lysate preparation. Therefore, we decided to investigate the total RNA content at various stages of lysate preparation with the goal of understanding more about the lack of CFE. Analysis of the total RNA content by gel electrophoresis of several mycoplasma lysates revealed high degradation levels throughout the lysate preparation (Figure 4). Mcap lysate obtained by sonication and subsequent centrifugation at 20,000 g showed significant RNA degradation directly after cell lysis (Figure 4A, red line). Even the 16S and 23S ribosomal RNAs (rRNAs) (Figure 4, black line), which were expected to be 21 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 protected by ribosomal proteins, were greatly degraded after cell lysis. As the lysate preparation proceeded to run-off incubation and dialysis, RNA was completely degraded (Figure 4A, blue and green lines respectively). Trypsin-Mcap lysate also degraded rRNA (Figure 4B), which suggested that trypsinization was insufficient to deplete all surface ribonucleases from Mcap cells. We performed a similar analysis for Syn3A and trypsin-Syn3A lysates, but after lysis, RNA was found completely degraded as well. Surface nucleases degraded foreign RNA. To determine whether the mycoplasma cell membrane and associated proteins were the source of the RNAse activity, we assessed degradation of RNA by whole mycoplasma cells using total RNA from E. coli lysate as target. Whole Mcap or Syn3A cells were first separated from culture medium using centrifugation through a sucrose cushion (0.5M sucrose in 75 mM HEPES pH 8). During incubation at 37°C for 60 minutes, both purified Mcap and Syn3A cells degraded the longer rRNAs from E. coli into fragments smaller than 1,500 bases (Figure 5, red lines), indicating RNAse activity of the cell surface or exterior. A control experiment shows that rRNAs from E. coli lysate incubated at 37°C remain relatively stable within the same time scale (Figure S9A). Considering that both Mcap and Syn3A were incubated at similar conditions, Syn3A cells showed lower ribonuclease activity as 16S and 23S bands remained visible whereas the same bands were absent with Mcap cell suspensions (under same incubation conditions). This result suggested rRNA degradation caused by nucleases present at the cell surface of Mcap and Syn3A. The degradation of rRNA from E. coli lysate was enhanced when incubated with mycoplasma lysates (Figure S9 C-J). 22 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Cell trypsinization was insufficient to deplete ribonuclease activity; whole trypsin-Mcap and trypsin-Syn3A cells purified by sucrose cushion still degraded RNA (Figure 5, blue lines). As observed for purified RNA aptamers (Figure S5) and endogenous Mcap and trypsin-Mcap RNAs (Figure 4), mycoplasma cells rapidly degraded foreign E. coli RNA, whether cells were trypsinized or not. Also, the cell culture supernatant degraded total RNA from E. coli, confirming ribonuclease activity from released nucleases (Figure S9B, green line). Our controls, a 0.5 M sucrose solution and fresh mycoplasma growth medium, did not degrade E. coli RNA (Figure S9B, blue and red lines, respectively). Considering the unique biological features of mycoplasmas, the development of its CFE system will require new methods specially for cell lysis and lysate clarification. We next tested cell disruption methods that are unusual for E. coli or Bacilli CFE systems. Lysis by digitonin improved rRNA content in Mcap and Syn3A lysates. All cell disruption methods tested thus far were based on successful strategies to prepare E. coli or Bacilli CFE systems. Considering the unique biological features of mycoplasmas, we investigated alternative methods for cell lysis and lysate clarification. As mycoplasmas do not have a cell wall, rather only a cholesterol-rich membrane, we explored milder disruption methods using detergents to solubilize the mycoplasma membranes. Triton™ X-100 (hereafter TX100) is a non-ionic surfactant previously used for membrane solubilization of M. laidlawii and M. mobile (54, 55). When we used TX100 to solubilize Mcap membranes (TX100-Mcap lysate), a small amount of 16S rRNA was observed, whereas 23S rRNA was completely degraded (Figure S10, red line). Although the TX100-Mcap lysate had a slightly higher rRNA content 23 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 than lysates prepared by sonication or French press, the lysate still did not support translation. Next, we tested digitonin, a detergent used for the lysis of mycoplasma cells (56–58) and subcellular fractionation of eukaryotic cells (59). Whereas TX100 completely solubilizes membranes, digitonin permeabilizes the membrane by precipitating cholesterol present in the mycoplasma membrane. Remarkably, lysates prepared from digitonin-treated Mcap cells (digitonin-Mcap) showed a higher content of rRNA compared to any other mycoplasma lysate (Figure 6, blue line). Apparently, purification of the extracted lysate from the larger permeabilized membranes was more effective in reducing RNAse activity of the lysate. Syn3A cells treated with digitonin (digitonin-Syn3A) (Figure 6, red line) showed an even higher rRNA content than digitonin-Mcap, possibly due to the absence of some unidentified RNAses. However, despite the higher rRNA content, digitonin-Syn3A lysate still failed to support CFE, even in the presence of calcium chloride as a nuclease inhibitor (Figure S11). Altogether, our results clearly show extensive nuclease activity, possibly originating from the interior as well as the exterior of Mcap and Syn3A, which apparently is detrimental for developing a robust mycoplasma CFE system, although perhaps not the only impediment. Lysates prepared by digitonin treatment show reduced RNAse activity. To compare the RNAse activity among different mycoplasma lysates, we followed degradation of a mRNA substrate over time. A denaturing agarose gel (Figure 7) showed rapid RNA degradation in Mcap lysate, followed by trypsin-Syn3A lysate and Syn3A lysate obtained by sonication. These results correlate with our previous observations regarding 24 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 RNAse activity in mycoplasma lysates (Figure 5). The mRNA degradation by digitonin-Syn3A lysate could not be observed directly in this experiment because of overlap with abundant rRNAs. However, although the mRNA band overlapped with the rRNA bands, its presence indicated much lower RNAse activity in digitonin-Syn3A lysate. Further analysis of the RNA degradation by digitonin-Syn3A lysate compared with E. coli lysate confirmed reduced degradation of mRNA and rRNA in digitonin-Syn3A lysate (Figure S12A). Despite these improvements, digitonin-derived lysates remained non-functional for eGFP production (Figure S12B). Also, only a small level of transcription, similar to trypsin-Mcap lysate could be detected (Figure S12C, Figure 3B). Discussion Features of mycoplasmas in general, while ideal for constructing synthetic minimal cells to study cellular life processes, may contribute to the pitfalls in developing CFE systems based on how such systems were developed for E. coli or other eubacteria. Our plan to construct a living synthetic cell from nonliving parts is dependent on developing CFE systems derived from mycoplasma bacteria. Discoveries about the limitations of the genome transplantation technique, where a synthetic genome from one species of bacteria is installed in a bacterial cell of a different species to create a new cell with the genotype and phenotype of the synthetic genome, convinced us that to construct a synthetic cell by booting up a mycoplasma genome we would need to use a CFE system from a closely related mycoplasma species (60). While we expected difficulties in our program to produce a synthetic cell, we did not expect producing a mycoplasma CFE system to be problematic. 25 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 CFE have been created for a broad variety of Gram-positive and Gram-negative bacteria (61) and there are no literature reports describing bacterial species for which no CFE could be developed. In retrospect, we should have been more cognizant that it took many years to develop today’s efficient CFE systems based on E. coli, and that CFE using bacteria other than E. coli produce only a small fraction of the amounts of proteins that can be obtained using E. coli CFE (18). The methods developed to make effective CFE systems for E. coli and other bacteria, when applied to both Mcap and near minimal bacterium Syn3A, resulted in cytoplasmic extracts in which we were unable to produce useful mounts of in vitro transcribed RNA. Our data indicate that mycoplasma RNase activity is at least one of the causes of this failure, but they do not rule out the possibility that there could be other factors confounding development of a mycoplasma CFE. In an attempt to understand this, we looked at the principal differences between the biology of conventional bacteria, such as E. coli or B. subtilis, and the mycoplasmas. In our view, there are three salient differences:  First, mycoplasmas cannot synthesize DNA or RNA precursors. Thus, they must import bases or nucleosides or and nucleotides. All of which are included in mycoplasma growth media, but in nature where mycoplasmas are typically respiratory and urogenital parasites of mammals, reptiles, birds and fish, these molecules must be gleaned from all available sources to support mycoplasma growth. Nucleases that degrade nucleic acids to molecules more easily imported could be critical for mycoplasma survival in nature (although genes encoding such nucleases would likely have been non-essential for laboratory growth and thus not included in 26 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 the minimal cell genome). The existence of membrane associated ribonuclease activity for several mycoplasma species is documented (47–52, 62, 63).  Second, mycoplasma cells are much smaller than most other bacteria. The volume of a 1.5 µm long E. coli cell is ~40 times greater than the volume of a typical 400 nm diameter Mcap cell. These size differences lead to a variety physiological differences. Notably, since ribosome and protein concentrations systematically shift with cell size the ratio of these two macromolecules varies by a factor of three between mycoplasmas and E. coli (64). Similarly, smaller cells have much higher surface to volume ratios than larger cells affecting the flux of nutrients to the cell and the requirements for transporters (65). These drastic differences in ratios and macromolecular requirements may introduce novel tuning considerations for mycoplasmas in how to properly adjust their concentrations of various macromolecules. Similarly, the lifestyle and size of mycoplasma likely implies sensitivity and lack of robustness to noise for two reasons. First, as obligate parasites mycoplasmas often experiences the homeostatic environment of their host compared with the large and chemically diverse environmental fluctuations experienced by many other bacteria. In fact, genome reduction is often argued to be associated with more consistent environments (66, 67). Second, the cellular environment of mycoplasmas is defined by truly discrete abundances of many of the macromolecules and this may mean that typical cellular physiology is defined by precisely tuned concentrations compared with larger bacteria. This may mean that the physiological and regulatory dynamics of mycoplasmas may be evolved to precisely regulate certain abundances and it may be the case that CFE systems are not precise enough to capture this tuning. 27 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022  Third and perhaps most important, mycoplasmas have no cell walls. Cells are enveloped by phospholipid bilayer membranes containing cholesterol. The methods used to produce CFE for conventional bacteria, such as sonication, French press or digitonin treatment followed by centrifugation to separate cytoplasm from the cell envelope may not work for cells lacking cell walls. We will investigate whether the CFE system methods used to purify the cytoplasm from conventional bacteria that have cells walls are not effective at eliminating membrane bound nuclease activity in bacterial cells where the lipid bilayer membrane is not tethered to the cell wall. The 30-34,000 g, 10–15-minute centrifugation step on which S30 extracts are named that we used to separate cytoplasm from cell envelope may not be working. RNases associated with membrane fragments generated by digitonin treatment or French press may not be spun away from the cytoplasm during the centrifugation. Each of these mycoplasma attributes could have contributed to the presence of nuclease activity in our efforts to make mycoplasma CFE systems. As for the actual Syn3A gene product(s) responsible for the problematic ribonuclease activity, we know it was present on the surfaces of cells. This was because intact mycoplasma cells were capable of poisoning E. coli CFE systems. Cell surface associated RNase activity correlated with the mycoplasma need to scavenge and import external nucleotides. It was noteworthy that Syn3A, which contains a subset of the nuclease encoding genes present in Mcap, was capable of supporting better in vitro transcription than Mcap (Figure S5). Furthermore, lysates prepared by digitonin treatment of Syn3A cells presented the lowest RNAse activity among 28 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 all mycoplasma lysates prepared in this study. This suggested to us that at least one or more nuclease-encoding genes were deleted as a result of genome minimization. Armed with data showing the nuclease activity was membrane associated, we looked carefully at the only two annotated Mcap and Syn3A genes that encode membrane- associated ribonucleases (Table S2). RNAse Y, is part of the degradosome complex and highly abundant in the cell (53, 68). The other gene, provisionally annotated as ribonuclease HII, which is involved in the degradation of the ribonucleotide moiety on RNA-DNA hybrid molecules carrying out endonucleolytic cleavage to 5'-phospo-monoester (69). However, that gene, which is not an exact match to characterized ribonuclease HII enzymes, may have evolved in mycoplasmas for a different purpose. Still, in Syn3A, there are fewer than 10 copies of the putative ribonuclease HII per cell (5). Because both of these potential ribonucleases are essential, it is not practical to genetically engineer away their nuclease activity. Because the RNase activity was so potent, we hypothesize the likely issue is the degradosome, which is an enzymatic complex comprising multiple RNases that is responsible for recycling most of the cells RNA and even in a tiny Syn3A cell there are hundreds of degradosome complexes (53). Alternatively, the source of the ribonuclease activity causing our problems may be one of the more than 90 Syn3A genes of unknown function; however, none of those genes have domains suggesting ribonuclease activity. Even though nucleases seem to be a major cause of the mycoplasma CFE inefficacy, the existence of other issue cannot be completely ruled out. The attempts to produce a mycoplasma lysate using surfactants points out the importance of (i) maintaining the overall structure of the membrane during cell lysis and (ii) quickly removing cellular debris by centrifugation. Those principles will lead to a lower level of 29 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 membrane shearing or solubilization leads, which means a lower release of surface nucleases into the final lysate. Also, milder cell fragmentation with digitonin treatment could have allowed more efficient removal of cellular debris by centrifugation. The high energy employed for cell disruption in sonication and French press methods may be one of the reasons for high nuclease activity. Another way to deplete nucleases in the lysate would be by down-regulating or knocking out nuclease-related genes in mycoplasma cells. This is however complicated for mycoplasma, because several of the annotated nucleases are either essential or quasi-essential genes, which poses additional challenges to obtain such mutants. As an alternative to mycoplasma lysate-based CFE systems, mycoplasma DNA (codon- optimized for mycoplasma usage (70) could be used to produce the enzymes needed to construct a reconstituted cell-free protein expression system. Referred to as PURE (Protein synthesis Using Recombinant Elements) systems (2), this technology might be adapted to use mycoplasma enzymes. This approach would help circumvent the nucleases derived from the mycoplasma cell during lysate preparation. Since PURE technology was initially designed as an E. coli cell-free system, a PURE system for mycoplasma would likely require considerable fine tuning. Of course, it may be that despite that attractiveness of mycoplasmas as chasses for construction of synthetic cells, this problem of creating CFE systems with mycoplasmas is insurmountable. Efforts to construct a synthetic bacterial cell from non-living parts may need to use RNA and protein expression systems comprised of materials obtained from a more conventional bacterium. The observation in genome transplantation that the donor genome must come from a species very closely related to the recipient cell suggests that we 30 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 could not boot up a mycoplasma genome using a CFE from a conventional bacterium (60). So, while we search for either genetically altered mycoplasma strain absent the RNase activity or a preparation technique that will produce an effective mycoplasma CFE system, we will also consider using a genome and CFE from a conventional small genome bacterium, such as Lactococcus lactis, as a chassis for construction of a synthetic cell from non-living parts. Conclusion While our attempts to produce a functional mycoplasma lysate described here were unsuccessful, it is important to consider that more than 40 years of research was necessary to produce a robust and typically high-yielding E. coli CFE system (1, 71). With that in mind, our team or another may still be able to overcome the technical challenges we describe here in order to produce functional CFE system based on mycoplasmas. Material Availability All bacterial strains and non-commercially available materials described in this paper are available to qualified researchers after completion of a material transfer agreement. A template for that material transfer agreement “JCVI-CodexDNA MTA for minimal cell.template.docx” is included among the Supplementary Materials. Data Availability Supplementary Data is available at SYNBIO online. 31 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Online Supplement The supplementary information includes 13 figures and 5 tables providing more detail about experiments described in the text. This also includes oligonucleotide sequences and listings of other mycoplasma species that may have relevant ribonuclease. Funding AS, FHTN, AJJ, HAH, and WTSH were supported by The Netherlands Organization for Scientific Research through the “BaSyC - Building a Synthetic Cell” Gravitation grant (024.003.019) of the Dutch Ministry of Education, Culture, and Science. CRD, CPK, KSW, KPA, and JIG were supported by the United States National Science Foundation Division of Molecular and Cellular Biosciences grant 1840301. KPA was supported by John Templeton Foundation grant 61184. DMCB was supported by the Brazilian Agricultural Research Corporation (Embrapa, Brazil). Conflict of Interest Disclosure No potential conflict of interest was reported by the authors. References 1. Garenne,D., Thompson,S., Brisson,A., Khakimzhan,A. and Noireaux,V. (2021) The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth. Biol. (Oxford, England), 6, ysab017. 2. Shimizu,Y., Inoue,A., Tomari,Y., Suzuki,T., Yokogawa,T., Nishikawa,K. and Ueda,T. 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Biol., 22, 158–62. Figure Legends Figure 1. Summary of conditions tested for the preparation of mycoplasma lysate and its functional assessment. The lysate preparation starts with harvesting mycoplasma cells (upper left). For some preparations, cells were trypsinized before cell disruption. To obtain crude lysates, we initially tested different cell lysis methods: sonication, French press, osmotic shock, and liquid nitrogen grinding (lower left). In the first centrifugation step, most of cell debris are decanted and the supernatant (clarified lysate) proceeds to run-off nd rd incubation, 2 centrifugation, dialysis (in S30B buffer), and 3 centrifugation. The final 38 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 lysate is then flash frozen and stored at -80°C (lower right). To test lysate functionality, we performed CFE reactions and measured transcription and translation by tracking fluorescent probes. Some lysate preparations included cell trypsinization before lysis (purple). * The second and third centrifugation speeds are the same as the first centrifugation step. Figure 2. Effect of adding mycoplasma lysate and CaCl to E. coli CFE. (A) Expression of eGFP decreases by adding increasing amounts of Syn3A or Mcap lysate from 0% (100 vol% E. coli lysate) to 100% (100 vol% mycoplasma lysate). (B) Addition of CaCl to ~15 mM restored eGFP expression in a 1:1 E. coli: Syn3A CFE reaction. An E. coli codon-optimized plasmid under control of a T7-promotor was used for E. coli CFE reaction of eGFP. Figure 3. Effect of trypsinization of Mcap cells in the production of mRNA and eGFP protein in CFE reactions. (A) E. coli CFE of eGFP, mixed with trypsin-Mcap lysate or S30B buffer at 39 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 different ratios. We observed a decrease in eGFP expression above 75 vol% of either S30B buffer or trypsin-Mcap lysate. (B) Transcription of Spinach2 aptamer in trypsin-Mcap lysate. The fluorescence signal from Spinach2-DFHBI-1T complex was detected, which indicated mRNA production. However, translation was not observed in CFE reaction containing 100% trypsin-Mcap lysate. Figure 4. Integrity of native RNA in mycoplasma lysates prepared by sonication and centrifuged at 20,000 g. RNA profiles obtained from (A) Mcap and (B) trypsin-Mcap cells before lysis (black lines), after lysis (red lines), after run-off incubation (blue lines), and after dialysis (green lines). 40 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Figure 5. RNA degradation induced by surface ribonucleases of (A) Mcap and (B) Syn3A cells. The control sample (black) shows intact RNA isolated from E. coli lysate. Addition of mycoplasma cells (red), and trypsinized mycoplasma cells (blue), caused E. coli rRNA to degrade into fragments smaller than 1,500 nt. Compared to Mcap, Syn3A cells presented less active surface ribonucleases. 41 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Figure 6. Digitonin permeabilizes mycoplasma cell membrane, allowing cytosol to leak (left). RNA in lysates prepared from mycoplasma cells treated with digitonin (right). Black line: RNA isolated from Syn3A cells before lysis. Syn3A cells yielded lysates with higher rRNA content (red line) compared to Mcap lysate (blue line). Figure 7. Comparative RNAse activity among several mycoplasma lysates confirmed a higher RNAse activity in Mcap lysate compared to Syn3A. The mRNA substrate (1.5 µg) was incubated at 30°C with mycoplasma lysate (protein concentration around 3 mg/mL) for 1-10 minutes. We analyzed RNA samples were analyzed using denaturing 1 % agarose gel electrophoresis. The high rRNA content in the digitonin-Syn3A lysate suggested the presence of intact ribosomes. Mcap, Syn3A, and trypsin-Syn3A lysates were prepared by sonication and digitonin-Syn3A lysate by digitonin treatment. Table 1. Conditions tested for the cell lysis step for preparing mycoplasma lysates. 42 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Run-off Lysis method Lysis condition # of cycles Dialysis incubation 1, 2, 4, 5, 7 Sonication 20, 35, 40, 50% amplitude +/- +/- (30 sec on, 30/60 sec off) 2 washes, resuspend Osmotic shock 250 mM NaCl + + (30 min, 23°C) Liquid nitrogen Liquid nitrogen added grinding flash frozen pellet + - grinding every min (for 20 min) French press 1,000, 2,000 psi 1, 3 +/- +/- 1 vol% Triton™ X-100 Surfactant-based 1-3 cycles (15 min, ice) - + 1 mg/mL digitonin Table 2. CFE reaction mix compositions tested for mycoplasma CFE platform. The standard concentration ranges for E. coli CFE are shown in the right column. Component Mycoplasma CFE Optimal E. coli CFE* Mg-glutamate 0-25 mM 2.5-10 mM* K-glutamate 60-120 mM 60-80 mM DNA template 6-10 nM 5-8 nM* 1x S30B, 0.5x S30B, 1x S30B, 0.5x S30B, Lysate buffer 75 mM HEPES pH 8.0 deionized water Lysate fraction 33-50 vol% 33 vol%* Dilution solvent 1x S30B, 0.5x S30B, deionized water deionized water recombinant RNAse A/B/C inhibitors, RNAse inhibitor - RNAsecure®, polyvinyl sulfonic acid (PVSA) *The optimal concentrations of Mg-glut, DNA template, and lysate fraction varied from batch to batch. Therefore, we display concentration ranges in which we observed the best protein yields. Graphical abstracts 43 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Synthetic Biology Oxford University Press

Traditional Protocols and Optimization Methods Lead to Absent Expression in a Mycoplasma Cell-Free Gene Expression Platform

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Abstract

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome- minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Keywords: mycoplasma, cell-free expression system, ribonuclease, in vitro transcription, in vitro translation 2 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Introduction One of the greatest challenges in modern science is to bring inanimate matter to life. Achieving such a transition would shed light on the very principles by which complex sets of chemical reactions create living systems. This key milestone will create new directions for research into synthetic systems that could lead to completely novel, yet unexplored, complex systems that have the ability to evolve. There is general consensus that the minimal living system needs to be a cell. A key stumbling block in the quest to create a minimal living cell from the bottom-up is the sheer complexity of even the simplest living systems, and our lack of knowledge of the general design principles that would allow us to construct a roadmap towards the bottom-up construction of a minimal cell. Most efforts to the bottom-up construction of a synthetic cell are based on Escherichia coli cell-free expression (CFE) systems, either using cell lysate or the PURE (protein synthesis using recombinant elements) system (1, 2). While E. coli is a valuable model for many purposes, the organism is vastly complex. In contrast, more simplified cellular systems are now available through the application of top-down synthetic biology strategies to reduce genome content (3–5). One approach is to re-design and re-build the genomic software of a cell ex vivo and reintroduce it into a compatible recipient host through genome transplantation (6), thereby redirecting the cellular machinery to create a new cell with re-programmed attributes. The J. Craig Venter Institute (JCVI) pioneered this strategy and applied it to completely synthesize, then minimize, a genome based on a template sequence of a natural organism of the genus Mycoplasma (hereafter mycoplasmas): Mycoplasma mycoides subspecies capri (7). The initial cell construct, JCVI-syn1.0 (hereafter Syn1.0) has a genome of 1,079 kbp, similar to 3 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 that of its natural precursor. Extensive genome reduction of Syn1.0 resulted in the “minimal bacterial cell” JCVI-syn3.0 (hereafter Syn3.0), with a genome of 531 kbp comprising 473 genes (8). This is a vastly simpler organism than E. coli K-12, whose >4,600-kbp genome contains >4,200 genes (9). While JCVI-syn3.0 propagates in the laboratory, its slow growth rate and pleomorphic phenotype renders it unsuitable for some applications. To overcome this, a near-minimal cell JCVI-syn3A (hereafter Syn3A) restoring 19 genes that are not present in Syn3.0 has been constructed as a more robust laboratory model and is in use to study various aspects of cell biology (5, 10, 11). The additional genes include a subset required for proper cell division, others of completely unknown function and a rRNA operon that restores its redundant presence in the original Syn1.0 genome (10). Our strategy to construct a bacterial cell from non-living parts leverages the lessons learned from the genome transplantation process used to boot up isolated M. mycoides and minimized M. mycoides genomes. In genome transplantation, a donor genome that is constructed as a yeast centromeric plasmid is installed in a recipient cell in a way that it commandeers that cell to produce bacteria programmed only by the donor genome (6, 12). To date the only acceptable genome transplantation recipient cell is Mycoplasma capricolum subspecies capricolum (hereafter Mcap). The only donor genomes that work are all close phylogenetic relatives of Mcap (13). This is presumably necessary because the software of the donor genome has to be capable of properly interacting with the enzymatic machinery in the recipient cell cytoplasm, such as the ribosomes or polymerases. By using donor genomes isolated from yeast instead of M. mycoides bacteria, there is no possibility of a resulting transplant being a contaminating cell that somehow made it through the chromosome isolation process. By transplanting genomes from one species of mycoplasma 4 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 into a recipient cell from a different species, sequence analysis of any genome transplant can confirm whether the transplant cell genome is the result of recombination between the donor genome and recipient cell genome. Our plan to construct a synthetic cell from non- living parts is to fill micron sized lipid vesicles with cytoplasm derived from bacteria and then install a genome in that CFE containing vesicle in a way resulting in the enlivening of the assembled parts. Given the similarity of this plan to the genome transplantation technique, our intent is to use the same reagents, controls and safeguards that are part of the genome transplantation protocol. We plan to use a Syn3A genome obtained from a yeast as the genetic software of our synthetic cell and a CFE derived from Mcap. Here we report our attempts to develop a CFE system using these cells, starting with protocols developed for successful E. coli lysates. We will discuss how unexpected nuclease activity in the lysate initially prohibited transcription, even to the extent that Mcap lysate poisoned E. coli CFE. We overcame these problems, but despite our best efforts and testing every known procedure for lysate preparation, there appear to be as yet unknown aspects of mycoplasma biology, that prevent us from obtaining a functional lysate. Considering the intense interest in the bottom-up construction of synthetic cells in general, our work should serve as a general reminder of the many complexities that will be encountered in these endeavors. We are aware that our results do not bring a minimal genome-based synthetic cell closer. However, we identify several factors that limited our success that may inform such efforts in other organisms. We also discuss alternative approaches for packaging the cell machinery of minimal cells to create an operational bottom-up synthetic cell. 5 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1. Materials and methods 1.1 Reagents Chemicals and reagents were purchased from Sigma Aldrich (reagent-grade), except those specified below. 1.2 Microorganisms and cell culture For preparation of mycoplasma cell-free extracts we cultivated the mycoplasma strains Mycoplasma capricolum subsp. capricolum (Mcap) and JCVI-syn3A (Syn3A) (6, 8). Among mycoplasma species, Mcap presents a faster and more robust growth than Syn3A. This difference in growth rates is explained by the significant genome reduction in Syn3A (543 kb) compared to its parent genome (1,079 Mb, JCVI-syn1.0). As obligate parasites, mycoplasmas require a rich medium (including serum) for in vitro growth. Mycoplasma cells were cultured in adapted SP4 glucose broth including 17 vol% KnockOut™ serum (Gibco) as a replacement for fetal bovine serum (SP4-KO medium) (14, 15). For lysates prepared by digitonin treatment, cells were grown in modified Hayflick medium supplemented with 20 vol% heat- inactivated horse serum (ThermoFisher) (HS medium) (16). Cells were incubated at 37°C without agitation or aeration. A fully-grown culture of 500-mL volume required about 6 days from the inoculation of a 1-mL culture (in 14-mL tube), subsequent transfer into a 100-mL culture (in 250-mL flasks), and the final transfer into a 500-mL culture (in 2-L flask). Cell growth was determined by color change (phenol red) as proliferating cells accumulate acidic metabolic byproducts. E. coli lysates were prepared from the BL21 (DE3) Star™ pLysS strain containing plasmid to express rare tRNAs (pRARE) (Invitrogen) following a protocol previously described (17). 6 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.3 Lysate preparation E. coli CFE is currently the CFE system with the best protein production levels (18). The successful preparation of E. coli crude lysates for CFE system, known as the S30 protocol, started many decades ago (19–22) and it has been gradually improved (23, 24). Each E. coli lysate batch has a slightly different composition and consequently a distinct efficiency regarding the protein production. In this work, we used the same batch of E. coli extract within a single experiment but different batches between experiments (due to extract availability). Since there is no reported protocol for the preparation of mycoplasma lysates for CFE system, we adapted a recent S30 protocol (17) for the preparation of mycoplasma lysates. Mcap or Syn3A were harvested at stationary growth phase (culture medium pH around 5) by centrifugation at 5,000 g, 4°C for 15 minutes. Pellets were washed twice with cold S30A buffer (50 mM Tris pH 7.7, 14 mM Mg-glutamate, 60 mM K-glutamate), keeping the flasks as much as possible on ice (ideally also in the cold room). The original S30 protocol consists of (i) cell lysis (by French press or sonicator, Figure 1) followed by (ii) run-off incubation to release ribosomes from polysomes, and (iii) dialysis for buffer exchange (S30B buffer: 5 mM Tris pH 8.2, 14 mM Mg-glutamate, 60 mM K-glutamate). High-speed centrifugation steps are employed to clarify the lysate after the lysis, the run-off incubation, and the dialysis steps (at same speed). Since the original protocol generates clean supernatants at 30,000 g centrifugal force, this method is known as S30. Because of morphological differences between E. coli and mycoplasmas (e.g. size, cell membrane composition) we tested different methods for cell lysis: (i) sonication at 150- 1,000 J/mL (Q500, 40% amplitude, 3-mm tip, Fisherbrand); (ii) French press at 140-360 mPa 7 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 (FPG12800, Homogenizing Systems Ltd); (iii) liquid nitrogen grinding with mortar and pestle, 20 min. non-stop with frequent addition of liquid nitrogen; or (iv) osmotic shock by incubation in S30B buffer, supplemented with 250 mM NaCl (30 min., 23˚C); or (v) by treatment with detergents, see Methods 1.6. The lysate is typically centrifuged at 20,000 g for 20 minutes at 4°C to remove cellular debris (first clarification step). Next, the supernatant is incubated at 37°C from 15 to 80 minutes (run-off incubation), followed by a second clarification step (20,000 g, 4°C, 20 min). The supernatants were then dialyzed in 10 kDa MWCO cassettes (Slide-a-lyzer™ Thermofisher) against S30B buffer (0.5x final concentration) (17). Samples were clarified once more (20,000, 4°C, 20 min) is performed to remove cellular debris, and finally flash frozen in liquid nitrogen and stored at -80°C. 1.4 Mycoplasma cell surface trypsinization Mycoplasma cell cultures were centrifuged at 5,000 g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed with 75 mM HEPES pH 8.0 buffer. The washed pellet was again centrifuged (5,000 g, 10 min, 4°C) and the supernatant was discarded. The washed pellet was then resuspended in 75 mM HEPES pH 8.0 buffer supplemented with 100 µg trypsin (Merck Life Science) per 100 mg of cell pellet and incubated at 37°C for 30 min under mild agitation (100 rpm). Afterwards, trypsinized cells were centrifuged at 5,000 g for 10 min at 4°C and the supernatant was discarded. The final pellet was washed once with purified soybean trypsin inhibitor solution (Gibco, 1x defined trypsin inhibitor, #R007100), centrifuged at 5,000 g for 10 minutes at 4°C, and finally washed with 75 mM HEPES pH 8.0 buffer. Alternatively, trypsin was inhibited using SP4-KO medium. 8 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.5 Separation of cells and media from mycoplasma cultures by sucrose cushion Mycoplasma cell suspensions or cell culture media (400 µL, Mcap or Syn3A) were underlaid with a sucrose cushion (600 µL, 0.5M sucrose in 20 mM HEPES pH 8, filter-sterilized) and centrifuged at 3,500 g for 10 min. The supernatant (culture medium or cell suspension buffer) was carefully removed by vacuum aspiration using a Pasteur pipette. The cell pellet was carefully resuspended and used for subsequent experiments. 1.6 Lysate preparation by Triton™ X-100 or digitonin treatment Mcap or Syn3A were cultured in HS medium (2-L flasks) as described in section 1.2) and harvested at pH 5.5-6 by centrifugation at 5,000 g, 4°C for 15 minutes. Next, cell pellets were washed twice with fresh HS medium, and collected by centrifugation (7,000 g, 4°C, 15 minutes). Cells were lysed upon resuspension with a lysis buffer (1 mL /100 mg of cell pellet) containing 1 vol% of Triton™ X-100 (hereafter TX100) and incubation on ice for 15 minutes (with vortexing every 5 minutes). The lysis buffer also included 75 mM HEPES pH 8.0, 1x HALT™ protease inhibitor cocktail without EDTA (Thermofisher), 20 mM CaCl , 1 mM polyvinyl sulfonic acid Mw~2-5 kDa (PSVA) (25). The crude lysate was clarified by high-speed centrifugation (32,000 g, 4°C, 20 minutes). When recovering the supernatant, we avoided to collect the pellet or any floating debris. Afterward, the clarified lysate was concentrated 10- fold with a centrifugal filter unit (Merck Millipore Amicon™ 15 mL, 10 kDa MWCO), followed by dialysis (Spectra™ 3.5 kDa MWCO dialysis membrane) against 0.5x S30B buffer at 4°C for 2 hours. Run-off incubation was not performed. Finally, samples were flash frozen and stored at -80°C. 9 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 For the preparation of mycoplasma lysates using digitonin, we used a similar protocol described for TX100 except for the surfactant in the lysis buffer (1 mg/mL of digitonin instead of 1 vol% of TX100). To improve the solubility of digitonin in aqueous solution, we prepared a stock of 2 wt% digitonin in deionized water and heated it to 95°C for 5 minutes. The solution was allowed to cool down to room temperature before mixing with other components of the lysis buffer. 1.7 Total RNA analysis RNA was isolated from mycoplasma cells or lysate by the hot phenol extraction method (26). Isolated RNA fractions were resuspended in nuclease-free water and their concentration measured by spectrophotometry (Nanodrop™, Thermofisher). RNA was diluted to a concentration of 1 µg/µL and 5 µL of each sample was analyzed by performing denaturing PAGE (8% acrylamide/bis-acrylamide, 8 M urea, 0.5x Tris/Borate/EDTA buffer, 250 V for 45 TM min). Gels were stained with SYBR -Gold (Invitrogen). Alternatively, total RNA fractions were analyzed using chip-based capillary electrophoresis in agarose gel (RNA 6000 Nano kit, Agilent 2100 Bioanalyzer). For that, samples were diluted to a 500 ng/µL final concentration, of which 1 µL of each sample was analyzed in the chip. The Bioanalyzer total RNA plots show arbitrary fluorescence units (y-axis) versus migration time (x-axis). For better data interpretation, the x-axis was converted to RNA size using an RNA ladder as reference (25- TM 6,000 nt). RNA was converted to cDNA library (iScript , Bio-Rad) for the qPCR experiment TM ® (iQ SYBR Green Supermix, Bio-Rad). qPCR was performed with primers for 23S rRNA (see Table S4 for sequences). 10 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 1.8 Cell-free expression (CFE) Reactions mixtures were designed from the E. coli CFE system previously described (17). Bacterial lysates can show batch-to-batch variation in cell-free expression efficiency. Therefore, for each CFE experiment we used a single lysate batch to generate consistent results. The reaction mix comprised cell lysates (33 vol%), feeding buffer (18.5 vol%), and a suspension of DNA template (plasmid or linear DNA, 6-10 nM, 5-10 vol%) adjusting the final volume with deionized water or S30B buffer. GamS nuclease inhibitor (3 µM final concentration) was added when linear DNA was used as template (27). The feeding buffer (Table S1) contains the amino acid mixture (1.5 mM of all 20 amino acids initially dissolved in 5M KOH and finally adjusted to pH 6.52 with acetic acid) and energy solution (50 mM HEPES pH 8, 1.5 mM ATP/GTP, 0.9 mM CTP/UTP, 0.2 mg/mL E. coli or S. cerevisiae tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.07 mM folinic acid, 1 mM spermidine, 30 mM 3-PGA, 1 mM DL-dithiothreitol, DTT). Mg-glutamate (5-30 mM), K-glutamate (60-120 mM), RNAse inhibitor (60U/150 µL reaction mix volume of recombinant RNAse inhibitor, Thermofisher #N8080119), recombinant T7 RNA polymerase (120U / 150 µL of reaction mix volume) were added to all reactions. For some reactions polyethylene glycol 8000 (2 vol%, PEG8000, MW ~8,000 kDa), calcium salts (calcium chloride, calcium acetate, calcium glutamate, 0-25 mM) were added. Two alternative RNase inhibitors, Superase-In and RNase Off, were also used at 60 U/150 µL. Samples were analyzed in microplate reader (M200 or M10 Tecan, SpectraMax Gemini, fluorescence detection) using black 384-well plates with flat and transparent bottom (Greiner Bio-One, #781900, 11 µL sample per well). Green fluorescent protein (eGFP) was analyzed at 488/525 nm (excitation/emission) and red fluorescent protein (mCherry) at 11 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 560/610 nm. The plate was kept at 30°C for 4-24 hours (5-min data acquisition interval) at 30°C. Graphs were prepared using Origin 8 software. 1.9 Mycoplasma DNA constructs Translation was monitored by the expression of red fluorescent protein (mCherry) or enhanced-green fluorescent protein (eGFP) (Figure S1). DNA template coding for mCherry under the control of Pspi promoter was previously developed for mycoplasma in vivo experiments and provided by Prof. Yo Suzuki (28). The eGFP gene was optimized from the original E. coli sequence for mycoplasma usage (Table S5) using online codon usage database and automated DNA sequence optimization tool (29, 30). Cloning was performed by Golden Gate assembly using pRSET5d vector and transformed into XL-1 E. coli chemically competent cells (Agilent Technologies). Primers are listed in the supplementary information (Table S4). For tracking transcription, we used malachite green (MG, Addgene, pJBL7004) and Spinach2 RNA aptamers combined with their respective dyes (Table S5) (31–35). Spinach2 RNA aptamer utilized (5Z)-5-[(3,5-Difluoro-4-hydroxyphenyl) methylene]-3,5-dihydro-2-methyl-3- (2,2,2-trifluoroethyl)-4H-imidazol-4-one dye (DFHBI-1T) and MG aptamer complexed with malachite green dye. For tracking RNA degradation, we used Broccoli RNA aptamer with 3,5- difluoro-4-hydroxybenzylidene imidazolinone dye (DFHBI) (33, 36). The reaction mix contained 1-8 nM RNA aptamer template and 60 µM DFHBI-1T. 1.10 mRNA degradation assay 12 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 The mRNA substrate (coding for eGFP, ~800 nucleotides long, 1.5 µg) was mixed with mycoplasma or E. coli lysate (protein concentration around 3 mg/mL) in a 5-µL final volume sample (volume adjusted with deionized nuclease-free water) kept in ice. Next, the samples were incubated at 30°C for 1-10 minutes to assay mRNA degradation. To quench RNAse activity after incubation, samples were diluted with TES buffer to a final volume of 100 µL (10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 vol% SDS). The RNA fraction was purified using the hot phenol extraction method (26). Dried RNA pellets were resuspended in 5 µL deionized water and mixed with 11 µL of sample buffer (0.85 mM EDTA, 12.5 mM 4- morpholinepropanesulfonic acid (MOPS), 3.1 sodium acetate, 25 vol% formamide, 63 vol% formaldehyde solution (37 wt% in water) and 0.25 µL ethidium bromide. RNA samples were analyzed using denaturing 1 % agarose gels (60V for 100 minutes in 1x MOPS as running buffer: 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0). The mRNA substrate was synthesized by in vitro transcription in a reaction containing 40 mM Tris-HCl pH 8.1, 25 mM MgCl , 5 mM DDT, 1 mM spermidine, 4 mM rNTPs, 5 mM guanosine-5’-monophosphate (GMP), 5 nM T7p-GFP linear dsDNA PCR product, and T7 RNA polymerase 600U / 500 µL reaction mix volume. The IVT reaction mix was incubated at 37°C for 4 hours, followed by RNA precipitation with 97% ethanol and centrifugation for 15 minutes at 14,000 g. The mRNA pellet was washed once with 80% ethanol and then spin- dried to remove solvent. The dried pellet was resuspended in nuclease-free water (~800 ng/µL). 1.11 Statistical analysis 13 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Experiments were done in triplicate (n=3) whenever feasible. Error bars in the figures are the standard deviation of multiple experiments. Results Development of a mycoplasma CFE platform. CFE platforms have been derived from various prokaryotes and eukaryotes (18). However, no protocols for the preparation of a mycoplasma lysate for a CFE platform have been reported in literature. For the development of a robust CFE platform based on Mcap and Syn3A, we started out with systematically testing several conditions used in a well-established protocol for obtaining the robust CFE platform of E. coli, known as the S30 protocol (1, 17). It comprises cell lysis, extract clarification, run-off incubation, and dialysis (Figure 1). The S30 protocol has been used by other researchers as a starting point to produce lysates from non-model prokaryotes, basically using the same essential steps. To date, no organism was able to yield similar protein production levels as the E. coli CFE platform. In terms of protein synthesis capacity, lysates derived from Gram-negative bacteria such as Vibrio natriegens and Pseudomonas putida have lower yields, by approximately 35% and 90% respectively, in batch reactions of green fluorescent protein (eGFP) (37–39). When lysates were produced from Gram-positive bacteria, the protein yields were even lower. For instance, Bacillus megaterium and Bacillus subtilis remained below 4% of E. coli CFE’s full protein synthesis capacity (18, 40, 41). Based on the 60-year history of the S30 protocol development and the phylogenetic differences between E. coli and mycoplasmas, we were aware of the complexity of creating the mycoplasma CFE platform from scratch. 14 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 We initially hypothesized that the mycoplasma lysate preparation would compare to the S30 lysate protocol with adjustments due to the unique cell membrane of mycoplasmas (i.e. absent cell wall and cholesterol-rich). We initially tested physical cell disruption methods (i.e. sonication, French press, liquid nitrogen grinding, osmotic shock), since these methods have been successful in producing active lysates for most prokaryote CFE platforms, including Gram-negative (E. coli, Vibrio natriegens) and Gram-positive bacteria (Bacilli). Because of the absence of a cell wall in mycoplasmas, we also tested surfactant-based cell lysis methods (i.e. by Triton™ X-100 or digitonin treatment). In addition to the lysis method, we also analyzed the importance of centrifugation speeds for lysate clarification, run-off incubation, and dialysis on the lysate’s capacity to support transcription and translation (Table 1). For CFE batch reactions, lysates were mixed with a feeding buffer and DNA template as described in methods. Starting from the optimal concentrations for the E. coli CFE platform, we systematically tested a range of concentrations for key components in mycoplasma CFE (e.g. Mg-glutamate, K-glutamate, DNA template, PEG8000) (Table 2). For testing transcription, we used DNA templates coding for the RNA aptamers malachite green and Spinach2 controlled by an exogenous T7 promoter. In presence of the respective dyes (malachite green dye complexed with malachite green aptamer and DFHBI-1T complexed with Spinach2 aptamer), we measured fluorescence of RNA aptamer-dye complexes. For testing protein production, we prepared DNA templates coding for enhanced-green fluorescent protein (eGFP) and red fluorescent protein (mCherry) controlled by an exogenous T7 promoter (Figure S1A) or endogenous promoter (Figure S1B). 15 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 To ensure maximum translation efficiency, eGFP and mCherry gene sequences were codon- optimized for mycoplasma usage (28–30). We tested different cell disruption methods to find the best conditions for mycoplasma cell disruption. The functionality of each lysate was assessed in terms of its capacity to support transcription and translation. Cell disruption efficiency depends on the lipid composition of cell membrane and the energy level employed for cell disruption (for mechanical disruption methods) (42, 43). Compared to other Gram-positive (e.g. Bacilli) and Gram-negative bacteria (e.g. E. coli, Vibrio), mycoplasma cells are relatively small (approximately 0.4 µm versus 2 µm E. coli) and their plasma membranes rely on cholesterol and cholesterol esters for mechanical stability rather than a cell wall (44, 45). Therefore, methods available to lyse E. coli or Bacilli may not be readily applicable to mycoplasma. First, we tested a range of energy levels for mycoplasma cell disruption by sonication (150, 300, 500, 1,000 J). Sonicated Mcap lysates did not show CFE regardless of the energy level employed for lysis or centrifugal force (3,000, 12,000 g) (Figure S2 A-O). Lysis by osmotic shock (Figure S2P) or liquid nitrogen grinding (Figure S2Q) also failed to support CFE for Mcap as well as Syn3A. Different energy regeneration molecules (PEP or 3-PGA) (Figure S2 A-L) or adding a molecular crowder (PEG8000) (Figure S2 A-O, Figure S3) failed to improve expression of DNA template coding for eGFP controlled by the T7 promoter in Mcap CFE. Similar results were observed using a DNA template coding for the mCherry sequence controlled by the Pspi promoter (Figure S3) (28). 16 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 To understand the basis for our failure in setting up a working mycoplasma CFE system we next investigated Mcap and Syn3A extracts for evidence of in vitro transcription only. The rationale for using Syn3A was that the minimized Syn3A genome contained only about half the number of genes present in Mcap, and we might be able to produce a minimal cell (i.e. Syn3A) extract being capable of CFE due to the absence of a non-essential nuclease or protease gene. Using linear double-stranded DNA templates encoding a T7 RNA polymerase promoter, a low level of transcription was detected for the malachite green RNA aptamer expressed in E. coli lysate, but Mcap and Syn3A lysates prepared by liquid nitrogen grinding did not give a clear signal (Figure S4), indicating no expression of malachite green aptamers in Syn3A or Mcap. These experiments suggested that poor transcription rates or rapid degradation of DNA-RNAs or a combination thereof prevented us from obtaining a functional CFE system using mycoplasma extracts. To investigate the possible impact of RNA degradation on mycoplasma CFE, we measured the fluorescence signal of pre-transcribed Broccoli aptamer complexed with DFHBI dye in various extracts as a function of time (36). DFHBI-Broccoli rapidly degraded over time with a more pronounced decrease in Mcap lysate obtained by sonication than in Syn3A lysate obtained by sonication (Figure S5B), irrespective of the amplitude used (Figure S5 A/B). In a kinetic assay, DFHBI-Broccoli degraded in Mcap lysate produced by sonication but not in Syn3A lysate produced by sonication (Figure S5C). Degradation of DFHBI-Broccoli was even faster in the Mcap lysate produced by liquid nitrogen grinding (Figure S5D). Nitrogen- ground Syn3A lysate degraded DFHBI-Broccoli over time but at a much slower rate. E. coli lysates controls made by sonication and liquid nitrogen grinding both degraded DFHBI- Broccoli. Different lysate preparation techniques might be responsible for the differences in 17 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 DFHBI-Broccoli degradation that we see from Syn3A lysates made by sonication vs. liquid nitrogen grinding. Liquid nitrogen grinding avoids overheating proteins during extraction, which could lead to denaturation and degradation. However, because the fluorescent signal from the aptamer-dye complex depends on the RNA folding, it cannot be ruled out that the low level of fluorescent signal is caused by improper folding in lysates as well. This could well explain the unexpected low signal of DFHBI-Broccoli in the E. coli lysate that was used as positive control. In summary, most of our mycoplasma extracts presented low levels of transcription and rapid degradation of reporter RNAs (except for Syn3A lysate, which degraded the DFHBI- Broccoli complex at a slower rate) were observed as well as for E. coli lysate. As a reference, the fluorescence signal of RNA-dye complex can be 2-10 times higher in E. coli CFE systems, depending on the type of RNA aptamer, dye, buffer composition, and plate reader settings (46). The inability to develop a mycoplasma CFE system starting from successful protocols for E. coli lysates made us wonder if some yet unknown aspects of mycoplasma biology might pose additional challenges to the development of a mycoplasma CFE system. Therefore, we next explored the mycoplasma metabolism to identify factors that might affect the production of a functional CFE system. Gene expression assay with E. coli lysate led to investigation of the critical impact of nuclease activity in mycoplasma CFE. Membrane-associated nucleases enable mycoplasmas to scavenge the extracellular environment for nucleotides. These membrane-associated nucleases might impair mycoplasma CFE by degrading essential nucleic acids involved in transcription and translation. Even though the enzymatic activity of membrane-associated 18 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 nucleases for some mycoplasma species are described in literature (47–52), little is known about their structure and inhibition mechanisms. Mcap and Syn3A share 20 common nucleases with annotated function (Table S2). The only common nuclease for Mcap and Syn3A associated with the cell membrane is ribonuclease Y, which is a degradosome protein C-terminally anchored to the inner membrane related to RNA turnover mechanisms (53). It is possible that both Mcap and Syn3A contain unannotated surface nucleases. Many of the genes of unknown function in both organisms encode membrane associated proteins. To test mycoplasma lysates for factors incompatible with CFE, we added mycoplasma lysate directly to an E. coli CFE (Figure 2A). Since we aimed to measure the effect of mycoplasma lysate in normal E. coli CFE reaction, we employed a plasmid coding for eGFP controlled by T7 promoter codon-optimized for E. coli. Indeed, with increasing mycoplasma lysate fraction, eGFP expression by E. coli CFE was gradually decreased. Importantly, eGFP expression in E. coli CFE mixed with Syn3A lysate could be restored by adding the surface nuclease inhibitor CaCl (Figure 2B, Table S3). Even though CaCl is described as a specific inhibitor of Mcap 2 2 nuclease activity (49) (what is true for Mcap is likely true for M. mycoides), we did not discard the possibility of other roles for CaCl in the CFE environment. In this instance, the effect of adding an extra component to the CFE reaction mix (i.e. mycoplasma lysate), likely did not have a significant effect on the GFP yields observed. As evidence, the GFP yields in a reaction containing 15 mM CaCl (50% Syn3A lysate in E. coli CFE reaction) were similar to the yields obtained in 100% E. coli CFE, which was around the maximum yield obtained for this reaction. 19 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 These results suggested a possible role of nucleases present in the mycoplasma lysates in 'poisoning' CFE and a possible remedy by adding CaCl . A murine RNAse inhibitor (NEB) was used throughout the experiments but did not increase eGFP signal. Two other commercially available RNase inhibitors, Superase-In™ (ThermoFisher) and RNase Off™ (Biovision), were also examined for their capacity to inhibit RNA nuclease activity in mycoplasma lysates but neither could produce measurable eGFP expression (Figure S13A-B). Rescue of eGFP expression in E. coli CFE poisoned with mycoplasma lysate was unsuccessful using any of the RNases (Figure S13C). Encouraged by the CaCl result, we tested expression in 100% Syn3A CFE reaction with CaCl . 2 2 However, no protein production was detected (Figure S6A). Calcium acetate also failed to support eGFP synthesis in either a 100% Syn3A or 100% Mcap CFE reaction (Figure S6B). We also observed that the poisoning effect could not be removed by higher centrifugation speeds. Syn3A lysates prepared by French press and clarified at different centrifugation speeds (20-80,000 g) (Figure S7) depleted the production of eGFP in E. coli/mycoplasma mixed CFE system, as observed in Figure 2A. However, because calcium-mediated nuclease inhibition restored eGFP expression in E. coli CFE mixed with Syn3A lysate, we reasoned that the nuclease content in mycoplasma lysates was perhaps too high for mycoplasma CFE. We next explored procedures for removing these poisoning components from mycoplasma lysates. Trypsinization of mycoplasma cells removed the poisoning effect and suggested surface nuclease activity. Because the extracellular mycoplasma surface could be a major source of 20 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 nucleases that incapacitated CFE of the extracts, we trypsinized mycoplasma cells before cell lysis to inactivate surface nucleases. Mcap lysate prepared from trypsinized cells (hereafter trypsin-Mcap) did not inactivate expression in the E. coli CFE platform (Figure 3A). Instead, the eGFP expression in E. coli CFE mixtures containing up to 50 vol% trypsin-Mcap lysate was similar to eGFP expression observed for control experiments using S30B buffer as diluent. The increase in expression by adding up to 50 vol% of diluent into E. coli CFE system indicates an improved expression of eGFP at lower concentrations of E. coli lysate, which might be due to specific optimal volume fraction of lysate in the CFE reaction mix. Although trypsin-Mcap lysate did not inactivate E. coli CFE, translation remained disabled for the mycoplasma CFE platform containing 100% trypsin-Mcap lysate, even in the presence of calcium glutamate (0-20 mM) (Figure S8). Only a small transcription level (Spinach2 aptamer) was detected (Figure 3B) compared to the signal obtained in E. coli CFE system, which was at least two-fold higher. RNA degraded during mycoplasma lysate preparation. At this point, we reasoned that development of a functional Mcap and/or Syn3A CFE was impeded by rapid degradation of RNAs by mycoplasma nucleases, leading to complete inactivation of the translational machinery early on during lysate preparation. Therefore, we decided to investigate the total RNA content at various stages of lysate preparation with the goal of understanding more about the lack of CFE. Analysis of the total RNA content by gel electrophoresis of several mycoplasma lysates revealed high degradation levels throughout the lysate preparation (Figure 4). Mcap lysate obtained by sonication and subsequent centrifugation at 20,000 g showed significant RNA degradation directly after cell lysis (Figure 4A, red line). Even the 16S and 23S ribosomal RNAs (rRNAs) (Figure 4, black line), which were expected to be 21 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 protected by ribosomal proteins, were greatly degraded after cell lysis. As the lysate preparation proceeded to run-off incubation and dialysis, RNA was completely degraded (Figure 4A, blue and green lines respectively). Trypsin-Mcap lysate also degraded rRNA (Figure 4B), which suggested that trypsinization was insufficient to deplete all surface ribonucleases from Mcap cells. We performed a similar analysis for Syn3A and trypsin-Syn3A lysates, but after lysis, RNA was found completely degraded as well. Surface nucleases degraded foreign RNA. To determine whether the mycoplasma cell membrane and associated proteins were the source of the RNAse activity, we assessed degradation of RNA by whole mycoplasma cells using total RNA from E. coli lysate as target. Whole Mcap or Syn3A cells were first separated from culture medium using centrifugation through a sucrose cushion (0.5M sucrose in 75 mM HEPES pH 8). During incubation at 37°C for 60 minutes, both purified Mcap and Syn3A cells degraded the longer rRNAs from E. coli into fragments smaller than 1,500 bases (Figure 5, red lines), indicating RNAse activity of the cell surface or exterior. A control experiment shows that rRNAs from E. coli lysate incubated at 37°C remain relatively stable within the same time scale (Figure S9A). Considering that both Mcap and Syn3A were incubated at similar conditions, Syn3A cells showed lower ribonuclease activity as 16S and 23S bands remained visible whereas the same bands were absent with Mcap cell suspensions (under same incubation conditions). This result suggested rRNA degradation caused by nucleases present at the cell surface of Mcap and Syn3A. The degradation of rRNA from E. coli lysate was enhanced when incubated with mycoplasma lysates (Figure S9 C-J). 22 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Cell trypsinization was insufficient to deplete ribonuclease activity; whole trypsin-Mcap and trypsin-Syn3A cells purified by sucrose cushion still degraded RNA (Figure 5, blue lines). As observed for purified RNA aptamers (Figure S5) and endogenous Mcap and trypsin-Mcap RNAs (Figure 4), mycoplasma cells rapidly degraded foreign E. coli RNA, whether cells were trypsinized or not. Also, the cell culture supernatant degraded total RNA from E. coli, confirming ribonuclease activity from released nucleases (Figure S9B, green line). Our controls, a 0.5 M sucrose solution and fresh mycoplasma growth medium, did not degrade E. coli RNA (Figure S9B, blue and red lines, respectively). Considering the unique biological features of mycoplasmas, the development of its CFE system will require new methods specially for cell lysis and lysate clarification. We next tested cell disruption methods that are unusual for E. coli or Bacilli CFE systems. Lysis by digitonin improved rRNA content in Mcap and Syn3A lysates. All cell disruption methods tested thus far were based on successful strategies to prepare E. coli or Bacilli CFE systems. Considering the unique biological features of mycoplasmas, we investigated alternative methods for cell lysis and lysate clarification. As mycoplasmas do not have a cell wall, rather only a cholesterol-rich membrane, we explored milder disruption methods using detergents to solubilize the mycoplasma membranes. Triton™ X-100 (hereafter TX100) is a non-ionic surfactant previously used for membrane solubilization of M. laidlawii and M. mobile (54, 55). When we used TX100 to solubilize Mcap membranes (TX100-Mcap lysate), a small amount of 16S rRNA was observed, whereas 23S rRNA was completely degraded (Figure S10, red line). Although the TX100-Mcap lysate had a slightly higher rRNA content 23 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 than lysates prepared by sonication or French press, the lysate still did not support translation. Next, we tested digitonin, a detergent used for the lysis of mycoplasma cells (56–58) and subcellular fractionation of eukaryotic cells (59). Whereas TX100 completely solubilizes membranes, digitonin permeabilizes the membrane by precipitating cholesterol present in the mycoplasma membrane. Remarkably, lysates prepared from digitonin-treated Mcap cells (digitonin-Mcap) showed a higher content of rRNA compared to any other mycoplasma lysate (Figure 6, blue line). Apparently, purification of the extracted lysate from the larger permeabilized membranes was more effective in reducing RNAse activity of the lysate. Syn3A cells treated with digitonin (digitonin-Syn3A) (Figure 6, red line) showed an even higher rRNA content than digitonin-Mcap, possibly due to the absence of some unidentified RNAses. However, despite the higher rRNA content, digitonin-Syn3A lysate still failed to support CFE, even in the presence of calcium chloride as a nuclease inhibitor (Figure S11). Altogether, our results clearly show extensive nuclease activity, possibly originating from the interior as well as the exterior of Mcap and Syn3A, which apparently is detrimental for developing a robust mycoplasma CFE system, although perhaps not the only impediment. Lysates prepared by digitonin treatment show reduced RNAse activity. To compare the RNAse activity among different mycoplasma lysates, we followed degradation of a mRNA substrate over time. A denaturing agarose gel (Figure 7) showed rapid RNA degradation in Mcap lysate, followed by trypsin-Syn3A lysate and Syn3A lysate obtained by sonication. These results correlate with our previous observations regarding 24 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 RNAse activity in mycoplasma lysates (Figure 5). The mRNA degradation by digitonin-Syn3A lysate could not be observed directly in this experiment because of overlap with abundant rRNAs. However, although the mRNA band overlapped with the rRNA bands, its presence indicated much lower RNAse activity in digitonin-Syn3A lysate. Further analysis of the RNA degradation by digitonin-Syn3A lysate compared with E. coli lysate confirmed reduced degradation of mRNA and rRNA in digitonin-Syn3A lysate (Figure S12A). Despite these improvements, digitonin-derived lysates remained non-functional for eGFP production (Figure S12B). Also, only a small level of transcription, similar to trypsin-Mcap lysate could be detected (Figure S12C, Figure 3B). Discussion Features of mycoplasmas in general, while ideal for constructing synthetic minimal cells to study cellular life processes, may contribute to the pitfalls in developing CFE systems based on how such systems were developed for E. coli or other eubacteria. Our plan to construct a living synthetic cell from nonliving parts is dependent on developing CFE systems derived from mycoplasma bacteria. Discoveries about the limitations of the genome transplantation technique, where a synthetic genome from one species of bacteria is installed in a bacterial cell of a different species to create a new cell with the genotype and phenotype of the synthetic genome, convinced us that to construct a synthetic cell by booting up a mycoplasma genome we would need to use a CFE system from a closely related mycoplasma species (60). While we expected difficulties in our program to produce a synthetic cell, we did not expect producing a mycoplasma CFE system to be problematic. 25 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 CFE have been created for a broad variety of Gram-positive and Gram-negative bacteria (61) and there are no literature reports describing bacterial species for which no CFE could be developed. In retrospect, we should have been more cognizant that it took many years to develop today’s efficient CFE systems based on E. coli, and that CFE using bacteria other than E. coli produce only a small fraction of the amounts of proteins that can be obtained using E. coli CFE (18). The methods developed to make effective CFE systems for E. coli and other bacteria, when applied to both Mcap and near minimal bacterium Syn3A, resulted in cytoplasmic extracts in which we were unable to produce useful mounts of in vitro transcribed RNA. Our data indicate that mycoplasma RNase activity is at least one of the causes of this failure, but they do not rule out the possibility that there could be other factors confounding development of a mycoplasma CFE. In an attempt to understand this, we looked at the principal differences between the biology of conventional bacteria, such as E. coli or B. subtilis, and the mycoplasmas. In our view, there are three salient differences:  First, mycoplasmas cannot synthesize DNA or RNA precursors. Thus, they must import bases or nucleosides or and nucleotides. All of which are included in mycoplasma growth media, but in nature where mycoplasmas are typically respiratory and urogenital parasites of mammals, reptiles, birds and fish, these molecules must be gleaned from all available sources to support mycoplasma growth. Nucleases that degrade nucleic acids to molecules more easily imported could be critical for mycoplasma survival in nature (although genes encoding such nucleases would likely have been non-essential for laboratory growth and thus not included in 26 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 the minimal cell genome). The existence of membrane associated ribonuclease activity for several mycoplasma species is documented (47–52, 62, 63).  Second, mycoplasma cells are much smaller than most other bacteria. The volume of a 1.5 µm long E. coli cell is ~40 times greater than the volume of a typical 400 nm diameter Mcap cell. These size differences lead to a variety physiological differences. Notably, since ribosome and protein concentrations systematically shift with cell size the ratio of these two macromolecules varies by a factor of three between mycoplasmas and E. coli (64). Similarly, smaller cells have much higher surface to volume ratios than larger cells affecting the flux of nutrients to the cell and the requirements for transporters (65). These drastic differences in ratios and macromolecular requirements may introduce novel tuning considerations for mycoplasmas in how to properly adjust their concentrations of various macromolecules. Similarly, the lifestyle and size of mycoplasma likely implies sensitivity and lack of robustness to noise for two reasons. First, as obligate parasites mycoplasmas often experiences the homeostatic environment of their host compared with the large and chemically diverse environmental fluctuations experienced by many other bacteria. In fact, genome reduction is often argued to be associated with more consistent environments (66, 67). Second, the cellular environment of mycoplasmas is defined by truly discrete abundances of many of the macromolecules and this may mean that typical cellular physiology is defined by precisely tuned concentrations compared with larger bacteria. This may mean that the physiological and regulatory dynamics of mycoplasmas may be evolved to precisely regulate certain abundances and it may be the case that CFE systems are not precise enough to capture this tuning. 27 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022  Third and perhaps most important, mycoplasmas have no cell walls. Cells are enveloped by phospholipid bilayer membranes containing cholesterol. The methods used to produce CFE for conventional bacteria, such as sonication, French press or digitonin treatment followed by centrifugation to separate cytoplasm from the cell envelope may not work for cells lacking cell walls. We will investigate whether the CFE system methods used to purify the cytoplasm from conventional bacteria that have cells walls are not effective at eliminating membrane bound nuclease activity in bacterial cells where the lipid bilayer membrane is not tethered to the cell wall. The 30-34,000 g, 10–15-minute centrifugation step on which S30 extracts are named that we used to separate cytoplasm from cell envelope may not be working. RNases associated with membrane fragments generated by digitonin treatment or French press may not be spun away from the cytoplasm during the centrifugation. Each of these mycoplasma attributes could have contributed to the presence of nuclease activity in our efforts to make mycoplasma CFE systems. As for the actual Syn3A gene product(s) responsible for the problematic ribonuclease activity, we know it was present on the surfaces of cells. This was because intact mycoplasma cells were capable of poisoning E. coli CFE systems. Cell surface associated RNase activity correlated with the mycoplasma need to scavenge and import external nucleotides. It was noteworthy that Syn3A, which contains a subset of the nuclease encoding genes present in Mcap, was capable of supporting better in vitro transcription than Mcap (Figure S5). Furthermore, lysates prepared by digitonin treatment of Syn3A cells presented the lowest RNAse activity among 28 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 all mycoplasma lysates prepared in this study. This suggested to us that at least one or more nuclease-encoding genes were deleted as a result of genome minimization. Armed with data showing the nuclease activity was membrane associated, we looked carefully at the only two annotated Mcap and Syn3A genes that encode membrane- associated ribonucleases (Table S2). RNAse Y, is part of the degradosome complex and highly abundant in the cell (53, 68). The other gene, provisionally annotated as ribonuclease HII, which is involved in the degradation of the ribonucleotide moiety on RNA-DNA hybrid molecules carrying out endonucleolytic cleavage to 5'-phospo-monoester (69). However, that gene, which is not an exact match to characterized ribonuclease HII enzymes, may have evolved in mycoplasmas for a different purpose. Still, in Syn3A, there are fewer than 10 copies of the putative ribonuclease HII per cell (5). Because both of these potential ribonucleases are essential, it is not practical to genetically engineer away their nuclease activity. Because the RNase activity was so potent, we hypothesize the likely issue is the degradosome, which is an enzymatic complex comprising multiple RNases that is responsible for recycling most of the cells RNA and even in a tiny Syn3A cell there are hundreds of degradosome complexes (53). Alternatively, the source of the ribonuclease activity causing our problems may be one of the more than 90 Syn3A genes of unknown function; however, none of those genes have domains suggesting ribonuclease activity. Even though nucleases seem to be a major cause of the mycoplasma CFE inefficacy, the existence of other issue cannot be completely ruled out. The attempts to produce a mycoplasma lysate using surfactants points out the importance of (i) maintaining the overall structure of the membrane during cell lysis and (ii) quickly removing cellular debris by centrifugation. Those principles will lead to a lower level of 29 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 membrane shearing or solubilization leads, which means a lower release of surface nucleases into the final lysate. Also, milder cell fragmentation with digitonin treatment could have allowed more efficient removal of cellular debris by centrifugation. The high energy employed for cell disruption in sonication and French press methods may be one of the reasons for high nuclease activity. Another way to deplete nucleases in the lysate would be by down-regulating or knocking out nuclease-related genes in mycoplasma cells. This is however complicated for mycoplasma, because several of the annotated nucleases are either essential or quasi-essential genes, which poses additional challenges to obtain such mutants. As an alternative to mycoplasma lysate-based CFE systems, mycoplasma DNA (codon- optimized for mycoplasma usage (70) could be used to produce the enzymes needed to construct a reconstituted cell-free protein expression system. Referred to as PURE (Protein synthesis Using Recombinant Elements) systems (2), this technology might be adapted to use mycoplasma enzymes. This approach would help circumvent the nucleases derived from the mycoplasma cell during lysate preparation. Since PURE technology was initially designed as an E. coli cell-free system, a PURE system for mycoplasma would likely require considerable fine tuning. Of course, it may be that despite that attractiveness of mycoplasmas as chasses for construction of synthetic cells, this problem of creating CFE systems with mycoplasmas is insurmountable. Efforts to construct a synthetic bacterial cell from non-living parts may need to use RNA and protein expression systems comprised of materials obtained from a more conventional bacterium. The observation in genome transplantation that the donor genome must come from a species very closely related to the recipient cell suggests that we 30 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 could not boot up a mycoplasma genome using a CFE from a conventional bacterium (60). So, while we search for either genetically altered mycoplasma strain absent the RNase activity or a preparation technique that will produce an effective mycoplasma CFE system, we will also consider using a genome and CFE from a conventional small genome bacterium, such as Lactococcus lactis, as a chassis for construction of a synthetic cell from non-living parts. Conclusion While our attempts to produce a functional mycoplasma lysate described here were unsuccessful, it is important to consider that more than 40 years of research was necessary to produce a robust and typically high-yielding E. coli CFE system (1, 71). With that in mind, our team or another may still be able to overcome the technical challenges we describe here in order to produce functional CFE system based on mycoplasmas. Material Availability All bacterial strains and non-commercially available materials described in this paper are available to qualified researchers after completion of a material transfer agreement. A template for that material transfer agreement “JCVI-CodexDNA MTA for minimal cell.template.docx” is included among the Supplementary Materials. Data Availability Supplementary Data is available at SYNBIO online. 31 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Online Supplement The supplementary information includes 13 figures and 5 tables providing more detail about experiments described in the text. This also includes oligonucleotide sequences and listings of other mycoplasma species that may have relevant ribonuclease. Funding AS, FHTN, AJJ, HAH, and WTSH were supported by The Netherlands Organization for Scientific Research through the “BaSyC - Building a Synthetic Cell” Gravitation grant (024.003.019) of the Dutch Ministry of Education, Culture, and Science. CRD, CPK, KSW, KPA, and JIG were supported by the United States National Science Foundation Division of Molecular and Cellular Biosciences grant 1840301. KPA was supported by John Templeton Foundation grant 61184. DMCB was supported by the Brazilian Agricultural Research Corporation (Embrapa, Brazil). Conflict of Interest Disclosure No potential conflict of interest was reported by the authors. References 1. Garenne,D., Thompson,S., Brisson,A., Khakimzhan,A. and Noireaux,V. (2021) The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth. Biol. (Oxford, England), 6, ysab017. 2. Shimizu,Y., Inoue,A., Tomari,Y., Suzuki,T., Yokogawa,T., Nishikawa,K. and Ueda,T. 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Biol., 22, 158–62. Figure Legends Figure 1. Summary of conditions tested for the preparation of mycoplasma lysate and its functional assessment. The lysate preparation starts with harvesting mycoplasma cells (upper left). For some preparations, cells were trypsinized before cell disruption. To obtain crude lysates, we initially tested different cell lysis methods: sonication, French press, osmotic shock, and liquid nitrogen grinding (lower left). In the first centrifugation step, most of cell debris are decanted and the supernatant (clarified lysate) proceeds to run-off nd rd incubation, 2 centrifugation, dialysis (in S30B buffer), and 3 centrifugation. The final 38 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 lysate is then flash frozen and stored at -80°C (lower right). To test lysate functionality, we performed CFE reactions and measured transcription and translation by tracking fluorescent probes. Some lysate preparations included cell trypsinization before lysis (purple). * The second and third centrifugation speeds are the same as the first centrifugation step. Figure 2. Effect of adding mycoplasma lysate and CaCl to E. coli CFE. (A) Expression of eGFP decreases by adding increasing amounts of Syn3A or Mcap lysate from 0% (100 vol% E. coli lysate) to 100% (100 vol% mycoplasma lysate). (B) Addition of CaCl to ~15 mM restored eGFP expression in a 1:1 E. coli: Syn3A CFE reaction. An E. coli codon-optimized plasmid under control of a T7-promotor was used for E. coli CFE reaction of eGFP. Figure 3. Effect of trypsinization of Mcap cells in the production of mRNA and eGFP protein in CFE reactions. (A) E. coli CFE of eGFP, mixed with trypsin-Mcap lysate or S30B buffer at 39 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 different ratios. We observed a decrease in eGFP expression above 75 vol% of either S30B buffer or trypsin-Mcap lysate. (B) Transcription of Spinach2 aptamer in trypsin-Mcap lysate. The fluorescence signal from Spinach2-DFHBI-1T complex was detected, which indicated mRNA production. However, translation was not observed in CFE reaction containing 100% trypsin-Mcap lysate. Figure 4. Integrity of native RNA in mycoplasma lysates prepared by sonication and centrifuged at 20,000 g. RNA profiles obtained from (A) Mcap and (B) trypsin-Mcap cells before lysis (black lines), after lysis (red lines), after run-off incubation (blue lines), and after dialysis (green lines). 40 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Figure 5. RNA degradation induced by surface ribonucleases of (A) Mcap and (B) Syn3A cells. The control sample (black) shows intact RNA isolated from E. coli lysate. Addition of mycoplasma cells (red), and trypsinized mycoplasma cells (blue), caused E. coli rRNA to degrade into fragments smaller than 1,500 nt. Compared to Mcap, Syn3A cells presented less active surface ribonucleases. 41 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Figure 6. Digitonin permeabilizes mycoplasma cell membrane, allowing cytosol to leak (left). RNA in lysates prepared from mycoplasma cells treated with digitonin (right). Black line: RNA isolated from Syn3A cells before lysis. Syn3A cells yielded lysates with higher rRNA content (red line) compared to Mcap lysate (blue line). Figure 7. Comparative RNAse activity among several mycoplasma lysates confirmed a higher RNAse activity in Mcap lysate compared to Syn3A. The mRNA substrate (1.5 µg) was incubated at 30°C with mycoplasma lysate (protein concentration around 3 mg/mL) for 1-10 minutes. We analyzed RNA samples were analyzed using denaturing 1 % agarose gel electrophoresis. The high rRNA content in the digitonin-Syn3A lysate suggested the presence of intact ribosomes. Mcap, Syn3A, and trypsin-Syn3A lysates were prepared by sonication and digitonin-Syn3A lysate by digitonin treatment. Table 1. Conditions tested for the cell lysis step for preparing mycoplasma lysates. 42 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022 Run-off Lysis method Lysis condition # of cycles Dialysis incubation 1, 2, 4, 5, 7 Sonication 20, 35, 40, 50% amplitude +/- +/- (30 sec on, 30/60 sec off) 2 washes, resuspend Osmotic shock 250 mM NaCl + + (30 min, 23°C) Liquid nitrogen Liquid nitrogen added grinding flash frozen pellet + - grinding every min (for 20 min) French press 1,000, 2,000 psi 1, 3 +/- +/- 1 vol% Triton™ X-100 Surfactant-based 1-3 cycles (15 min, ice) - + 1 mg/mL digitonin Table 2. CFE reaction mix compositions tested for mycoplasma CFE platform. The standard concentration ranges for E. coli CFE are shown in the right column. Component Mycoplasma CFE Optimal E. coli CFE* Mg-glutamate 0-25 mM 2.5-10 mM* K-glutamate 60-120 mM 60-80 mM DNA template 6-10 nM 5-8 nM* 1x S30B, 0.5x S30B, 1x S30B, 0.5x S30B, Lysate buffer 75 mM HEPES pH 8.0 deionized water Lysate fraction 33-50 vol% 33 vol%* Dilution solvent 1x S30B, 0.5x S30B, deionized water deionized water recombinant RNAse A/B/C inhibitors, RNAse inhibitor - RNAsecure®, polyvinyl sulfonic acid (PVSA) *The optimal concentrations of Mg-glut, DNA template, and lysate fraction varied from batch to batch. Therefore, we display concentration ranges in which we observed the best protein yields. Graphical abstracts 43 Downloaded from https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysac008/6590323 by DeepDyve user on 24 May 2022

Journal

Synthetic BiologyOxford University Press

Published: May 21, 2022

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