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BioBrick-based ‘Quick Gene Assembly’ in vitro

BioBrick-based ‘Quick Gene Assembly’ in vitro Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: ‘Quick Gene Assembly (QGA)’. The time required for finishing a se- quential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for ‘gene construction based on engineering principles’ at the present time, and is also a method suitable for automation in the near future. Key words: BioBrick; engineering principle; gene assembly; gene designing; magnetic beads. fragments with others. To overcome these limitations, new effi- 1. Introduction cient and reliable biochemical gene assembly methods are still Recent advances in genomics have allowed a great deal of ge- required. nome and cDNAs information in public databases to be re- Recent common DNA assembly methods can be categorized trieved, catalogued, and accessed by all whom require this data. into two major groups based on the molecular mechanisms em- Such public databases are widely used among molecular and ployed (1). The first group of common methods relies on the use synthetic biologists as fundamental open sources to obtain ge- of a type II restriction enzyme digestion of DNA fragments fol- netic information. In order to obtain genetic samples until now, lowed by ligation. This group includes the three-antibiotic (3A) researchers requested DNA clones after sending a material assembly method (2), the BASIC method (3), the Golden Gate as- transfer agreement, before being sent biological materials from sembly method (4,5) employing type IIS restriction enzyme, stock centers. However, recent improvements in de novo DNA BsaI, the Golden Braid system (6), the Modular cloning system synthesis have contributed to the removal of these steps, and (7), the VEGAS method (8) that were developed by improving the dramatically reduced the time required to obtain DNA con- Golden Gate assembly method, the MASTER ligation method (9) structs. Nevertheless, current systems for de novo DNA synthe- employing type IIM restriction enzyme, MspJI (10–13). The 3A as- sis still have technological limitations. For example, they are sembly method is one of the typical and rational protocols for not widely applicable for (i) making tandemly repeated DNA se- gene construction that supports the biological standardized ge- quence units, (ii) for constructing unbiased DNA libraries con- netic parts that are flanked between a common upstream and taining DNA fragments with sequence multiplicity in a downstream nucleotide sequences in an appropriate plasmid specific region of target genes, or (iii) for replacement of DNA DNA vector (BioBrick)-based gene designs. BioBricks, biological Submitted: 27 December 2016; Received (in revised form): 13 April 2017; Accepted: 13 April 2017 V C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1 2| Synthetic Biology, 2017, Vol. 2, No. 1 standardized genetic parts, were previously proposed by Knight a novel BioBrick-based DNA assembly method, ‘Quick Gene (14). All BioBrick parts are flanked between a common upstream Assembly (QGA)’, for overcoming the limitations in the tech- nucleotide sequence (called a ‘prefix’) and a common down- niques described above. stream nucleotide sequence (called a ‘suffix’) in an appropriate plasmid DNA vector (15). Knight also proposed and realized a 2. Materials and methods unified concept of BioBrick, in which each genetic fragment can be fused upstream or downstream by a simple ligation reaction 2.1 DNA primers, reagents, magnetic beads, and other after restriction digestion of BioBricks, using only a limited tools number of restriction enzymes. This assembly method relies on a combination of positive and negative selection to achieve a Nucleotide sequences of DNA oligomers prepared for this study high frequency of correctly assembled clones. The greatest ad- were as follows, QGA adaptor antisense strand: 5’-Phosphate- vantage of this method is the avoidance of DNA fragment purifi- CTAGAAGCGGCCGCGAATTC-(dA) -3’, QGA adaptor sense cation using column and agarose gel in all steps. However, in strand: 5’-TTGAATTCGCGGCCGCTT-3’, 100-bp upstream primer: order to perform the 3A assembly method, the destination vec- 5’-AACCTATAAAAATAGGCGTATCAC-3’, and 200-bp down- tor, into which two BioBrick parts will be assembled, must have stream primer: 5’-CCCCTGATTCTGTGGATAACCGTATTACCG-3’. a different antibiotic resistance marker from the plasmids Restriction endonucleases, XbaI (20 U/ll), SpeI-HF (20 U/ll), SpeI encoding the two input parts. Therefore, it requires time and ef- (10 U/ll), EcoRI (20 U/ll), and solution for digestion (CutSmart) fort to construct a large set of BioBricks fused to plasmid DNA were purchased from New England Biolabs Japan Inc (Tokyo, vectors with distinct drug resistant markers. Moreover, the Japan). DNA polymerase, KOD-Plus-Neo, and the solution for number of BioBricks assembled in one cycle of the experiment PCR were purchased from TOYOBO Co Ltd (Osaka, Japan). The li- is limited to two parts. Another typical protocol for gene con- gation kit, Mighty Mix, was purchased from TaKaRa BIO INC struction in the first group is the Golden Gate assembly method, (Shiga, Japan). Triton X-100 was purchased from Sigma-Aldrich which employs the type IIS restriction enzyme, BsaI. This en- Japan (Tokyo, Japan). Trehalose dehydrate was purchased from zyme can cut outside of its recognition site and generates a Wako Pure chemical Industries Ltd (Osaka, Japan). The mag- unique 5’ overhang structure composed of four complementary netic beads, SiMAG-Oligo-dT, were purchased from Chemicell nucleotides. By using this type of enzyme, digested fragments (Berlin, Germany). The neodymium magnet, ND-8R, was pur- can be ligated to generate expected products lacking recognition chased from Magna Co Ltd (Tokyo, Japan). The kit for purifica- sites of the restriction enzyme between each genetic part. This tion of DNA fragments, FastGene Gel/PCR Extraction Kit, was assembly method can be achieved by adding all DNA fragments purchased from NIPPON Genetics Co Ltd (Tokyo, Japan). and reagents required into one tube. Successful assembly of up to 12 DNA fragments into a destination vector in one reaction 2.2 Preparation of DNA fragments containing BioBricks was demonstrated (16). However, this method requires manipu- lations for design and synthesis of various DNA primers con- One microliter of template DNA (1 ng/ll) containing BioBrick, taining a BsaI recognition site and different cutting motif at 1 llof 10 lM 100-bp upstream primer (10 pmol), 1 llof 10 lM 200- both ends of each DNA fragment. Cutting sequences at the ends bp downstream primer (10 pmol), 11.3 ll of Distilled Water (DW), of DNA fragments must be sufficiently different to avoid unex- 2 ll of 10xPCR solution, 2 ll of 2 mM deoxyribonucleotide tri- pected insertion, deletion, duplication or reversal of genetic phosphate (dNTPs), 1.2 ll of 25 mM MgSO , and 0.5 ll of 1 U/50 ll parts (5). This method employs a one-pot/one-step protocol. DNA polymerase (KOD-Plus-Neo) were mixed for amplification However, it requires much more time and effort to check the fi- of DNA fragments by PCR. Amplified DNA fragments were puri- delity of assembly and to select the correct assembly clone. fied by using a FastGene PCR Extraction Kit. Amplified DNA frag- The second group of common assembly methods is based on ments were digested by XbaI, separated by agarose gel (1%) sequence homology at the ends of each DNA fragment and in- electrophoresis, and purified by using a FastGene Gel Extraction cludes techniques such as the in vitro Gibson assembly method Kit. Concentration of each DNA fragment solution was adjusted (17,18), the overlapping extension polymerase chain reaction to 0.1 pmol/ll by adding 10 mM Tris-HCl (pH ¼ 8.0) containing (OE-PCR) method (19), the circular polymerase extension clon- 1 mM EDTA (TE). ing method (20,21), the sequence and ligation-independent cloning method (22), the seamless ligation cloning extract 2.3 Preparation of adapter DNA solution method (23), the Urasil-Specific Excision Reagent (USER) method (24) and the in vivo DNA assembler-yeast method (25), and so on Five microliters of 100 lM QGA adaptor antisense strand (26–30). In the Gibson assembly method, T5 exonuclease chews (500 pmol), 5 ll of 100 lM QGA adaptor sense strand (500 pmol), back 5’-ends to generate single-strand complementary cohesive and 40 ll of TE containing 0.4 M NaCl were mixed and heated at ends. After specific annealing among DNA fragments, Phusion 80 C for 3 min. The mixture was left for 10 min until tempera- DNA polymerase fills in the gaps and Taq DNA ligase seals the ture went down to 25 C and diluted 10 times by adding TE con- nicks covalently (17). This assembly method is suitable for mak- taining 0.4 M NaCl to make ‘Adapter Solution’. ing large constructs from long DNA fragments. It was reported that full-length genome (583 kbp) derived from Mycoplasma geni- 2.4 Ligation of adapter to first BioBrick talium was assembled from separate fragments by this method (18). However, this protocol is not suitable for the assembly of Two microliters of 0.1 pmol/ll 1st BioBrick solution (0.2 pmol), short DNA fragments of less than 100 bp in length or tandemly 0.2 ll of 1 pmol/ll ‘Adapter Solution’ (0.2 pmol), 2.8 ll of deion- repeated DNA fragments, as T5 exonuclease removes dozens of ized water, and 5 ll of Mighty Mix, ligation mixture containing nucleotides from 5’-ends. polyethylene glycol for elevation of ligation efficiency (31,32), As described above, each common assembly method is were mixed and incubated at 16 C for 15 min followed by mix- widely used and has significant advantages, but each technique ing by pipetting up and down and additional incubation at 16 C also has its limitations. Here, we will show the development of for 15 min. K. Yamazaki et al. | 3 2.5 Preparation of magnetic beads 2.11 Amplification of assembled DNA fragment by PCR Two microliters of magnetic beads (SiMAG-Oligo-dT) were sus- Twenty microliters of TE was added to the beads and the beads pended in TE (50 ll) containing 0.4 M NaCl by agitating the tube were suspended by agitation. A total of 0.2 ll of the suspension, by touching it to the head of a sonic toothbrush (OMRON 1 ll (10 pmol) of 10 lM QGA adaptor sense strand, 1 ll (10 pmol) HEALTHCARE Co., Ltd, Kyoto Japan), the beads were collected by of 10 lM 200-bp downstream primer, 11.3 ll of deionized water, attracting them to one side of a 0.2 ml polypropylene tube using 2 ll of 10xPCR solution, 2 ll of 2 mM dNTPs, 1.2 llof25mM a neodymium magnet (4 mm in diameter), and the supernatant MgSO , and 0.5 ll of KOD-Plus-Neo (1 U/50 ll) were mixed. At was removed. first, the assembled DNA fragments were dissociated from the beads by incubating them at 94 C for 2 min, then the assembled the DNA fragments were amplified through step-down PCR (5 2.6 Fixation of adapter-conjugated first BioBrick to cycles of incubation at 98 C for10 s followed by incubation at magnetic beads 74 C for 1 min, 5 cycles of incubation at 98 C for 10 s followed by incubation at 72 C for 1 min, 5 cycles of incubation at 98 C Ten microliters of adapter-conjugated first BioBrick solution, for 10 s followed by incubation at 70 C for 1 min, 10 cycles of in- 10 ll of TE containing 0.8 M NaCl, and the beads previously cubation at 98 C for 10 s followed by incubation at 68 C for rinsed by the above protocol were mixed by agitation. The sus- 1 min). The amplified DNA fragments were purified by using a pension was incubated at 60 C for 5 min and left for 35 min until FastGene PCR Extraction Kit. The DNA fragments were digested by the temperature decreased to 25 C at a rate of 1 C/min. The EcoRI and SpeI, digested DNA fragments were separated on 1% aga- beads were resuspended by agitation and incubated for a fur- rose gel, then purified by using a FastGene Gel Extraction Kit. A ther 25 min at 25 C. The beads were collected using the magnet, total of 0.1 pmol of EcoRI/SpeI digested linear assembled DNA frag- and the supernatant containing excess-free DNA fragments was ments were ligated with 0.035 pmol of EcoRI/SpeIdigestedlinear removed. plasmid DNA. The ligated DNA constructs were transformed into Escherichia coli DH5a (HIT competent cells, RBC Bioscience), and 2.7 Beads rinsing cells were spread on an Luria-Bertani Broth (LB)-plate containing the appropriate antibiotic for screening transformed clones. Twenty microliters of TE containing 0.1% Triton X-100 was added to the beads and the beads were suspended by agitation. The beads were collected using a magnet to remove superna- 2.12 Notes tant by pipetting. Then, the beads were rinsed by repeating the (1) Usage of primer set (100-bp upstream primer and 200-bp process of suspending them in 20 ll of TE by agitation, and col- downstream primer) for gene amplification is recommended to lection by magnet to remove the supernatant. increase the length of the DNA fragment, since recovery of short DNA fragments using affinity columns (for preparation of DNA fragment) is inefficient when fusion of a short BioBrick DNA 2.8 Removal of 200-bp downstream DNA fragment fragments are required. This process is also helpful to make ‘Digestion Premix’ containing 1 llof 10 RE solution, 3 llof2M identification of completely digested DNA fragments easier. trehalose, 5.5 ll of DW, and 0.5 ll of restriction endonuclease, (2) All DNA fragments used in this experiment should be SpeI-HF, was added to the beads, and suspended by pipetting up highly purified. Usage of glycogen for DNA purification should and down. The suspension was incubated at 37 C for 15 min, be avoided (33). followed by mixing by pipetting up and down and further incu- (3) All steps for beads rinsing should be performed by agita- bation at 37 C for 15 min. Then, the beads were collected by tion of the tube by touching to the head of sonic toothbrush to magnet to remove the supernatant. The beads were rinsed avoid loss of beads by adsorption onto the inner surface of plas- twice, as described in the ‘Beads-rinsing’ section above. ticware during pipetting up and down. (4) All steps for mixing enzyme solutions should be per- formed by pipetting up and down to avoid reduction of enzyme 2.9 Fusion of second BioBrick to first BioBrick activity. ‘Ligation Premix’ containing 2 ll of second BioBrick solution (5) The beads should always be fixed on one side of the inner (0.2 pmol), 3 ll of deionized water, and 5 ll of Mighty Mix was surface of the polypropylene tube by a magnet when a superna- added to the beads and mixed by pipetting up and down. The tant is removed by pipetting. mixture was incubated at 16 C for 15 min, followed by mixing (6) We used a very small neodymium magnet (4 mm in diam- by pipetting up and down and further incubation at 16 C for eter) to avoid magnetization of magnetic beads. 15 min. The beads were rinsed twice, as described in ‘Beads- rinsing’. The 200-bp downstream DNA fragment was removed by SpeI restriction enzyme, as described in the ‘Removal of 200- 3. Results bp downstream DNA fragment’, section, followed by ‘Beads- 3.1 Schematic representation of QGA rinsing’ twice. First, (i) all DNA fragments containing BioBricks were prepared by PCR amplification using a common primer set consisting of 2.10 Fusion of more downstream BioBricks 100-bp upstream and 200-bp downstream DNA primers. (ii) Downstream BioBricks were fused by repeating the protocol de- Second, all DNA fragments were digested by restriction enzyme scribed in the ‘Fusion of second BioBrick to first BioBrick’ sec- (XbaI) to unmask the upstream end of each fragment and make tion. The final round of BioBrick fusion does not contain a step them competent for ligation to their upstream fragment, as for the removal of the 200-bp downstream DNA fragment, since shown in Figure 1a. (iii) The upstream end of the first BioBrick the downstream fragment is required for amplification of as- should be immobilized by fixing it to the origin of gene assem- sembled gene by PCR as a primer annealing site. bly, and its downstream end should be kept masked 4| Synthetic Biology, 2017, Vol. 2, No. 1 dT (Chemicell), we succeeded in achieving a dramatic reduction of the time required for the sequential precipitation and rinsing steps. Since oligo-dT conjugated to the beads can hybridize to oligo-dA, we made oligo-dA the central part of the adapter DNA fragment required to fix the first BioBrick to the beads. Utilizing the magnetic properties of the beads allows for much faster iso- lation and purification of the assembled DNA fragments with- out the need for multiple sequential centrifugation steps. Replacing the precipitation and rinsing steps by magnetic bead isolation using a magnet largely contributed to the simplifica- tion of the whole protocol. 3.2 Possible number of cycles for accurate assembly A short DNA fragment containing a promoter-BioBrick, P tetR (BBa_R0040, 54 bp) was used as a building block to test the possi- ble number of cycles for accurate assembly. As seen in Figure 2a, the sizes of PCR products derived from the amplification of tem- plates generated after each cycle of assembly was expected to in- crease stepwise with an increase in the cycle number of assembly. The PCR product size after each cycle should be 200-bp longer than the DNA fragments composed of only fused BioBricks after each DNA fragment is fused to a common 200-bp down- stream fragment. All BioBricks were connected by a 6-bp inter- vening scar site sequence (ACTAGA). This scar site occurs when the ends resulting from a 5’-overhang restriction digestion by SpeI and XbaI are ligated. The reduction of efficiency of the complete assembly was evaluated by measuring the rate of light intensity associated with the largest PCR products on a gel image using software, ‘Gel Analyzer 2010a (http://www.gelanalyzer.com/down load.html)’. Light intensity associated with the largest product in each lane (from lane 1 to lane 4 in Figure 2b)was thehighest among products. The efficiency of the complete assembly reac- tion was calculated by comparing the light intensity of the largest DNA product with other products. The efficiency decreased de- pending on the number of assembly cycles, where observations of 61.8% (after 2 cycles), 44.3% (after 3 cycles), and 31.1% (after 4 Figure 1. Schematic representation of the QGA protocol. (a) Preparation of a cycles) were made. The efficiency of the complete assembly reac- DNA fragment containing a BioBrick part. P: prefix DNA fragment, BB: BioBrick, tion after 5 cycles was calculated as 22.8%. These observations S: suffix DNA fragment, 100 bp UP-F: 100 base pair upstream forward primer, 200 bp DN-R: 200 base pair downstream reverse primer, circled P: phosphate res- suggest that the majority of the products are full length and re- idue, X: XbaI restriction site. (b) Structural change of extending the DNA mole- sult from up to four cycles of assembly. The potential of this as- cule in each step of QGA. MAGB: magnetic beads, E: EcoRI restriction site, S: SpeI sembly method was tested by repeating more than five BioBrick restriction site, S/X: mixed restriction site (scar), TTT...TTT: oligo-dT conjugated part assembly cycles. The largest products, corresponding to to magnetic beads, AAA...AAA: oligo-dA in DNA adaptor. eight BioBrick parts are still visible, even after eight cycles of as- sembly (data not shown). The largest product was isolated from (dephosphorylated) to avoid unexpected fusion with itself when agarose for further ligation with a plasmid vector when only per- fectly assembled constructs were required. sequentially assembling multiple BioBrick parts. (iv) The down- stream 5’-terminus should be removed by restriction enzyme digestion using SpeI to unmask its downstream end for ligation 3.3 Conditions affect higher yield of correctly assembled to the second BioBrick. (v) The second BioBrick is ligated down- product stream of the first BioBrick. The number of BioBricks fused to the downstream end can be increased by repeating this diges- To evaluate each factor affecting the yield of the largest product tion/ligation cycle, as is illustrated in Figure 1b. among all PCR products, only one factor was changed from the The protocol described above requires frequent replacement optimum condition described in Materials and Methods section. of reaction solutions for the treatment of both ends of all The assembled products generated under optimum condition BioBricks. The most common method for replacement was eth- are shown in lane 1 in Figure 3 as a positive control. In lane 2, anol precipitation of the DNA fragments. In this case, at least 10 the high-fidelity restriction enzyme SpeI(SpeI-HF) in digestion precipitation by ethanol addition steps, 10 rinsing by 70% etha- premix was replaced by a standard SpeI enzyme. The yield of nol steps, 20 centrifugation steps, and 10 drying-up steps are re- the largest product was reduced to 78.6% when the yield ob- quired, just to complete the assembly of 5 BioBricks. These tained in the optimum condition was set as 100%. The yield was protocols require a long period of time, and DNA sample yield further reduced to 72.9% when optimum conditions (30 min at tends to decrease dramatically after sequentially repeated etha- 16 C) were changed to incubation for 10 min at 25 C, as shown nol precipitation and rinsing steps. By taking advantage of the in lane 3. The yield was reduced to 66.3% when a rinsing by TE usage of ‘magnetic beads’ conjugated to oligo-dT, SiMAG-Oligo- containing 0.1% Triton X-100 was replaced by rinsing with TE, K. Yamazaki et al. | 5 Figure 4. Negative effects of enzyme solution mixing from pipetting up and down compared to the agitation method. Distribution of amplified DNA frag- ments by PCR when assembled DNA fragments were used as a template after each cycle. Cycle numbers of QGA are indicated above each lane. Copy numbers of PtetR fragments are indicated at the right side of each band. 3.4 Optimization of mixing steps Mixing enzyme solutions by pipetting up and down is generally considered to be one of the best methods to avoid enzyme de- naturation. However, a considerable reduction in the number of Figure 2. Size analysis of PCR products after an increase in the number of QGA magnetic beads and a decrease in the yield of the DNA fixed to cycles. (a) Predicted assembled structures of products after increase of cycles of them were observed through repetition of this manipulation. To QGA. Cycle numbers of QGA and predicted sizes of assembled DNA fragments address this problem, we tested agitating the test tube to avoid after each cycle are indicated at the left of each construct. PtetR: promotor BioBrick of tetracycline resistance gene. (b) Distribution of amplified DNA frag- the loss of beads by adsorption onto the inner surface of the ments by PCR when assembled DNA fragments after each cycle were used as a plastic pipette tips, and the shear forces associated with re- template. QGA cycle numbers are indicated above each lane. Copy numbers of peated pipetting up and down. Complete replacement of the PtetR fragments are indicated at the right side of each band. pipetting up and down manipulation by the agitation method considerably reduced the yield of the largest assembled product after assembly, as shown in Figure 4. The efficiency of complete assembly after cycles 1, 2, 3, 4, and 5 were reduced, step by step, from 100% to 57.9%, 38.9%, 21.3%, and 16.2%, respectively. These observations suggest that repeated agitation contributes to avoiding the loss of magnetic beads; however, the manipulation may cause a reduction of enzyme activity. Therefore, the vibra- tion method should be employed for beads rinsing, and the pipetting up and down manipulation should be used for mixing enzyme solutions, such as the ‘Digestion Premix’ and ‘Ligation Premix’ solutions. Figure 3. Negative effects on the QGA process resulting from the change of each condition away from optimum conditions. Lane 1: assembled products under optimum conditions. Lane 2: replacement of the restriction enzyme from SpeI- 3.5 Effects of increase of BioBrick length in QGA HF (NEB) to SpeI (NEB). Lane 3: changing of ligation condition from ‘at 16 C for 30 min’ to ‘at 25 C for 10 min’. Lane 4: removal of 0.1% Triton X-100 from the The results shown in Figures 2–4 indicate that assembly of a rinsing solution. Lane 5: removal of 0.6 M trehalose from the digestion premix. longer DNA fragment from five or more short Biobricks by QGA Lane 6: combination of all changes tested from lanes 2–5. Copy numbers of PtetR was achieved. To test if this protocol is applicable for the gene fragment are indicated at the right side of each band. assembly of longer BioBricks, a DNA fragment (300 bp) contain- ing five tandem repeats of the short BioBrick part BBa_R0040 as shown in lane 4. Moreover, production of immature product, was used as a building block for the assembly. The efficiency of the smallest product, increased 1.35 times. These observations complete assembly from longer BioBricks after five cycles was suggest that the presence of a low concentration of detergent in calculated to be 10.4%, shown in Figure 5, lane 5. Although the the rinsing solution is essential for the optimum removal of so- efficiency of the complete assembly was rather low, the amount lution components used in the previous step. The yield of the of DNA fragment isolated from an agarose gel was enough to be largest product was reduced to 41.4% when 0.6 M trehalose was used for an enzyme digestion followed by ligation and transfor- removed from ‘Digestion Premix’, as shown in lane 5. mation of bacterial cells. These observations suggest that genes Furthermore, production of immature product, the smallest up to 1500 bp in length can be assembled from 5 BioBricks bio- product, increased 2.4 times. Trehalose is a glucose disaccharide chemically in 24 h. synthesized in Saccharomyces cerevisiae and E. coli, and is known to retain the potential to elevate an enzyme reaction in vitro 3.6 Assembly of a functional gene through ligation of a (34,35). Combining all these changes dramatically reduced the promoter, protein coding sequence, and terminator yield to 29.2%, as shown in lane 6. These observations shown in Figure 5 suggest that the addition of 0.1% Triton X-100 to TE for To test the potential of the assembly system to construction rinsing and the addition of 0.6 M trehalose to the digestion pre- functional genes from distinct BioBricks, Green fluorescent pro- mix are essential for successful BioBrick assembly. However, tein (GFP), and Red fluorescent protein (RFP) coding sequences use of the high-fidelity restriction enzyme SpeI-HF and little were used as reporter genes. Schematic representations of DNA change of ligation conditions were not essential for higher yield constructs on the process to finish complete gene assembly are of the largest product. shown in Figure 6a together with their lengths. The efficiency of 6| Synthetic Biology, 2017, Vol. 2, No. 1 encoding transcription units for expression of GFP or RFP were digested by restriction enzymes, EcoRI and SpeI, ligated with plasmid vector, pSB1A3 which has previously been digested by EcoRI and SpeI, and the ligated samples were subjected for trans- formation of bacteria strain DH5a. Images of colonies appeared on agarose LB plates containing ampicillin (100 mg/mL), which are shown in Figure 6c. GFP and RFP expression was monitored under white light. Ratios of Figure 5. Effects of the increase of BioBrick length in QGA. Distribution of ampli- fied DNA fragments by PCR (length of each PCR product containing 5, 10, 15, 20, green colonies to all colonies and red colonies to all colonies and 25 tandem repeats of PtetR is 514 bp, 814 bp, 1114 bp, 1414 bp, and 1714 bp, were calculated by counting the total number of colonies. respectively) when assembled DNA fragments were used as a template after Colonies of 92% or 90% successfully produced GFP or RFP, re- each cycle. Cycle numbers of QGA correspond to lane numbers. Copy numbers spectively. These results suggest that the majority of the largest of PtetR fragments are indicated at the right side of each band. products after the assembly and gene amplification have a po- tential for successful expression. 4. Discussion We have often encountered a requirement to construct hun- dreds of recombinant genes and genetic circuits to investigate the function of various biological systems at the molecular level, or to generate novel biological systems in the field of syn- thetic biology. This process always requires continuous effort, especially given the increasing demand for new constructs in research. Therefore, the reduction of the time required for each design and construction cycle for gene assembly, is a critical is- sue facing the field as a whole. First, the existence of a common engineering principle based gene-design-concept is important to reduce the time required for the design cycle of genes, since the sharing of ideas and fre- quent discussions among researchers and students is crucial to achieve the best designs. The most well-known gene-design- concept based on engineering principles is the ‘standardized ge- netic part (BioBrick)-based gene design protocol’ proposed by Knight (14). To take full advantage of the gene-design-concept, making good use of a highly reliable BioBrick collection is essen- tial. Continuous effort to evaluate the quality of each BioBrick part, accumulation of measurement and characterization infor- mation of each BioBrick part, and the sharing of these data are critical to improve the quality and quantity of the collection. Furthermore, BioBrick parts should be widely used under differ- ent conditions and in different organisms to evaluate the per- formance of each part. The performance of each BioBrick part should be measured under a variety of conditions, and the in- formation should be shared widely and publically. With this accumulated body of knowledge, BioBricks can be used appro- priately under various conditions for researchers to achieve the Figure 6. Assembly of functional genes from distinct BioBricks. (a) Schematic best designs possible. representation of the QGA process from a single DNA construct to the finished Second, the development of an efficient gene assembly complete gene assembly is shown together with their lengths. P: prefix DNA fragment, PtetR: TetR promotor, RBS: ribosome binding site, GFP: DNA fragment method is also important for the construction of hundreds of re- encoding green fluorescent protein (BBa_I13500), RFP: DNA fragment encoding combinant genes, and this development could lead to a reduc- red fluorescent protein (BBa_K093005), dT: double terminator (BBa_B0015), S: suf- tion in the time required for the ‘construction cycle’ of genes. fix DNA fragment. (b) Sizes of PCR products amplified from assembled genes. Furthermore, the method should be based on engineering prin- White arrowheads indicate the expected DNA fragments. The numbers above ciples so that the technique can be applied to automated ge- each lane are corresponding to the numbers shown in panel A. (c) Images of col- netic part assembly. In the introduction section, we described onies on agarose LB plates containing 100 lg/mL ampicillin. Expression of GFP and RFP was monitored under white light. Scale bars are 2 mm. several other gene assembly protocols as well as their advan- tages and limitations. In this study, we succeeded in making a novel BioBrick-based DNA assembly method, and named it the assembly of functional genes from three BioBricks was ‘Quick Gene Assembly (QGA)’. This method enables us to as- monitored by observing a change in the sizes of PCR products semble at least five BioBricks in vitro in 12 h. All steps (including amplified from assembled genes, as shown in Figure 6b. White amplification of DNA fragments by PCR, and assembly of the arrow heads indicate intermediary products and the assembled plasmid DNA vector) can be finished in 24 h. DNA fragments, as we expected. The sizes of the assembled The advantages of QGA are listed as follows: (i) Use of only DNA fragments were successfully increased. The largest PCR one universal DNA primer set is enough to prepare any kind of products (in lanes 3 and 6 in Figure 6b) containing regions BioBrick or other building block for the assembly of genetic K. Yamazaki et al. | 7 14. Knight,T. (2006) Idempotent Vector Design for Standard Assembly parts. (ii) Genetic parts can be fused correctly in as short a time as 12 h. (iii) We can successfully assemble repetitive DNA se- of Biobricks. MIT Synthetic Biology Working Group. quences or short DNA fragments (less than 100 bp). (iv) Filling 15. Registry of standard biological parts. http://partsregistry.org gaps among building blocks and plasmid DNA vector by DNA (27th December 2016, date last accessed). polymerase is not required. (v) More than 95% of DNA products 16. Nielsen,A.A.K., Der,B.S., Shin,J., Vaidyanathan,P., Paralanov,V., in the largest product after PCR were accurately assembled as Strychalski,E.A., Ross,D., Densmore,D., Voigt,A. et al. (2016) Genetic circuit design automation. Science, 352, aac7341. expected. (vi) Quick and easy replacement of each solution after each step dramatically reduced the time required to complete 17. Gibson,D.G. (2011) Enzymatic assembly of overlapping DNA the whole assembly. (vii) Entire steps in QGA can be achieved in fragments. Methods Enzymol., 498, 349–361. vitro, without the need for an in vivo clone screening process. 18. , Young,L., Chuang,R.Y., Venter,J.C., Hutchison,C.A. 3rd and Smith,H.O. (2009) Enzymatic assembly of DNA molecules Consequently, we believe the QGA system is amenable to auto- mation because of its interactive workflow and its reliance on up to several hundred kilobases. Nat. Methods, 6, 343–345. magnetic beads for purification of fragments. The system 19. Horton,R.M., Hunt,H.D., Ho,S.N., Pullen,J.K. and Pease,L.R. could also be used for the construction of unbiased focused (1989) Engineering hybrid genes without the use of restric- libraries. tion enzymes: gene splicing by overlap extension. Gene, 77, 61–68. Conflict of interest statement. None declared. 20. Quan,J. and Tian,J. (2009) Circular polymerase extension clon- ing of complex gene libraries and pathways. PLoS One,4, e6441. References 21. and (2011) Circular polymerase extension cloning 1. Chao,R., Yuan,Y. and Zhao,H. (2014) Recent advances in DNA for high-throughput cloning of complex and combinatorial assembly technologies. FEMS Yeast Res., 15, 1–9. DNA libraries. Nat. Protoc., 6, 242–251. 2. 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BioBrick-based ‘Quick Gene Assembly’ in vitro

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© The Author 2017. Published by Oxford University Press.
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10.1093/synbio/ysx003
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Abstract

Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: ‘Quick Gene Assembly (QGA)’. The time required for finishing a se- quential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for ‘gene construction based on engineering principles’ at the present time, and is also a method suitable for automation in the near future. Key words: BioBrick; engineering principle; gene assembly; gene designing; magnetic beads. fragments with others. To overcome these limitations, new effi- 1. Introduction cient and reliable biochemical gene assembly methods are still Recent advances in genomics have allowed a great deal of ge- required. nome and cDNAs information in public databases to be re- Recent common DNA assembly methods can be categorized trieved, catalogued, and accessed by all whom require this data. into two major groups based on the molecular mechanisms em- Such public databases are widely used among molecular and ployed (1). The first group of common methods relies on the use synthetic biologists as fundamental open sources to obtain ge- of a type II restriction enzyme digestion of DNA fragments fol- netic information. In order to obtain genetic samples until now, lowed by ligation. This group includes the three-antibiotic (3A) researchers requested DNA clones after sending a material assembly method (2), the BASIC method (3), the Golden Gate as- transfer agreement, before being sent biological materials from sembly method (4,5) employing type IIS restriction enzyme, stock centers. However, recent improvements in de novo DNA BsaI, the Golden Braid system (6), the Modular cloning system synthesis have contributed to the removal of these steps, and (7), the VEGAS method (8) that were developed by improving the dramatically reduced the time required to obtain DNA con- Golden Gate assembly method, the MASTER ligation method (9) structs. Nevertheless, current systems for de novo DNA synthe- employing type IIM restriction enzyme, MspJI (10–13). The 3A as- sis still have technological limitations. For example, they are sembly method is one of the typical and rational protocols for not widely applicable for (i) making tandemly repeated DNA se- gene construction that supports the biological standardized ge- quence units, (ii) for constructing unbiased DNA libraries con- netic parts that are flanked between a common upstream and taining DNA fragments with sequence multiplicity in a downstream nucleotide sequences in an appropriate plasmid specific region of target genes, or (iii) for replacement of DNA DNA vector (BioBrick)-based gene designs. BioBricks, biological Submitted: 27 December 2016; Received (in revised form): 13 April 2017; Accepted: 13 April 2017 V C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1 2| Synthetic Biology, 2017, Vol. 2, No. 1 standardized genetic parts, were previously proposed by Knight a novel BioBrick-based DNA assembly method, ‘Quick Gene (14). All BioBrick parts are flanked between a common upstream Assembly (QGA)’, for overcoming the limitations in the tech- nucleotide sequence (called a ‘prefix’) and a common down- niques described above. stream nucleotide sequence (called a ‘suffix’) in an appropriate plasmid DNA vector (15). Knight also proposed and realized a 2. Materials and methods unified concept of BioBrick, in which each genetic fragment can be fused upstream or downstream by a simple ligation reaction 2.1 DNA primers, reagents, magnetic beads, and other after restriction digestion of BioBricks, using only a limited tools number of restriction enzymes. This assembly method relies on a combination of positive and negative selection to achieve a Nucleotide sequences of DNA oligomers prepared for this study high frequency of correctly assembled clones. The greatest ad- were as follows, QGA adaptor antisense strand: 5’-Phosphate- vantage of this method is the avoidance of DNA fragment purifi- CTAGAAGCGGCCGCGAATTC-(dA) -3’, QGA adaptor sense cation using column and agarose gel in all steps. However, in strand: 5’-TTGAATTCGCGGCCGCTT-3’, 100-bp upstream primer: order to perform the 3A assembly method, the destination vec- 5’-AACCTATAAAAATAGGCGTATCAC-3’, and 200-bp down- tor, into which two BioBrick parts will be assembled, must have stream primer: 5’-CCCCTGATTCTGTGGATAACCGTATTACCG-3’. a different antibiotic resistance marker from the plasmids Restriction endonucleases, XbaI (20 U/ll), SpeI-HF (20 U/ll), SpeI encoding the two input parts. Therefore, it requires time and ef- (10 U/ll), EcoRI (20 U/ll), and solution for digestion (CutSmart) fort to construct a large set of BioBricks fused to plasmid DNA were purchased from New England Biolabs Japan Inc (Tokyo, vectors with distinct drug resistant markers. Moreover, the Japan). DNA polymerase, KOD-Plus-Neo, and the solution for number of BioBricks assembled in one cycle of the experiment PCR were purchased from TOYOBO Co Ltd (Osaka, Japan). The li- is limited to two parts. Another typical protocol for gene con- gation kit, Mighty Mix, was purchased from TaKaRa BIO INC struction in the first group is the Golden Gate assembly method, (Shiga, Japan). Triton X-100 was purchased from Sigma-Aldrich which employs the type IIS restriction enzyme, BsaI. This en- Japan (Tokyo, Japan). Trehalose dehydrate was purchased from zyme can cut outside of its recognition site and generates a Wako Pure chemical Industries Ltd (Osaka, Japan). The mag- unique 5’ overhang structure composed of four complementary netic beads, SiMAG-Oligo-dT, were purchased from Chemicell nucleotides. By using this type of enzyme, digested fragments (Berlin, Germany). The neodymium magnet, ND-8R, was pur- can be ligated to generate expected products lacking recognition chased from Magna Co Ltd (Tokyo, Japan). The kit for purifica- sites of the restriction enzyme between each genetic part. This tion of DNA fragments, FastGene Gel/PCR Extraction Kit, was assembly method can be achieved by adding all DNA fragments purchased from NIPPON Genetics Co Ltd (Tokyo, Japan). and reagents required into one tube. Successful assembly of up to 12 DNA fragments into a destination vector in one reaction 2.2 Preparation of DNA fragments containing BioBricks was demonstrated (16). However, this method requires manipu- lations for design and synthesis of various DNA primers con- One microliter of template DNA (1 ng/ll) containing BioBrick, taining a BsaI recognition site and different cutting motif at 1 llof 10 lM 100-bp upstream primer (10 pmol), 1 llof 10 lM 200- both ends of each DNA fragment. Cutting sequences at the ends bp downstream primer (10 pmol), 11.3 ll of Distilled Water (DW), of DNA fragments must be sufficiently different to avoid unex- 2 ll of 10xPCR solution, 2 ll of 2 mM deoxyribonucleotide tri- pected insertion, deletion, duplication or reversal of genetic phosphate (dNTPs), 1.2 ll of 25 mM MgSO , and 0.5 ll of 1 U/50 ll parts (5). This method employs a one-pot/one-step protocol. DNA polymerase (KOD-Plus-Neo) were mixed for amplification However, it requires much more time and effort to check the fi- of DNA fragments by PCR. Amplified DNA fragments were puri- delity of assembly and to select the correct assembly clone. fied by using a FastGene PCR Extraction Kit. Amplified DNA frag- The second group of common assembly methods is based on ments were digested by XbaI, separated by agarose gel (1%) sequence homology at the ends of each DNA fragment and in- electrophoresis, and purified by using a FastGene Gel Extraction cludes techniques such as the in vitro Gibson assembly method Kit. Concentration of each DNA fragment solution was adjusted (17,18), the overlapping extension polymerase chain reaction to 0.1 pmol/ll by adding 10 mM Tris-HCl (pH ¼ 8.0) containing (OE-PCR) method (19), the circular polymerase extension clon- 1 mM EDTA (TE). ing method (20,21), the sequence and ligation-independent cloning method (22), the seamless ligation cloning extract 2.3 Preparation of adapter DNA solution method (23), the Urasil-Specific Excision Reagent (USER) method (24) and the in vivo DNA assembler-yeast method (25), and so on Five microliters of 100 lM QGA adaptor antisense strand (26–30). In the Gibson assembly method, T5 exonuclease chews (500 pmol), 5 ll of 100 lM QGA adaptor sense strand (500 pmol), back 5’-ends to generate single-strand complementary cohesive and 40 ll of TE containing 0.4 M NaCl were mixed and heated at ends. After specific annealing among DNA fragments, Phusion 80 C for 3 min. The mixture was left for 10 min until tempera- DNA polymerase fills in the gaps and Taq DNA ligase seals the ture went down to 25 C and diluted 10 times by adding TE con- nicks covalently (17). This assembly method is suitable for mak- taining 0.4 M NaCl to make ‘Adapter Solution’. ing large constructs from long DNA fragments. It was reported that full-length genome (583 kbp) derived from Mycoplasma geni- 2.4 Ligation of adapter to first BioBrick talium was assembled from separate fragments by this method (18). However, this protocol is not suitable for the assembly of Two microliters of 0.1 pmol/ll 1st BioBrick solution (0.2 pmol), short DNA fragments of less than 100 bp in length or tandemly 0.2 ll of 1 pmol/ll ‘Adapter Solution’ (0.2 pmol), 2.8 ll of deion- repeated DNA fragments, as T5 exonuclease removes dozens of ized water, and 5 ll of Mighty Mix, ligation mixture containing nucleotides from 5’-ends. polyethylene glycol for elevation of ligation efficiency (31,32), As described above, each common assembly method is were mixed and incubated at 16 C for 15 min followed by mix- widely used and has significant advantages, but each technique ing by pipetting up and down and additional incubation at 16 C also has its limitations. Here, we will show the development of for 15 min. K. Yamazaki et al. | 3 2.5 Preparation of magnetic beads 2.11 Amplification of assembled DNA fragment by PCR Two microliters of magnetic beads (SiMAG-Oligo-dT) were sus- Twenty microliters of TE was added to the beads and the beads pended in TE (50 ll) containing 0.4 M NaCl by agitating the tube were suspended by agitation. A total of 0.2 ll of the suspension, by touching it to the head of a sonic toothbrush (OMRON 1 ll (10 pmol) of 10 lM QGA adaptor sense strand, 1 ll (10 pmol) HEALTHCARE Co., Ltd, Kyoto Japan), the beads were collected by of 10 lM 200-bp downstream primer, 11.3 ll of deionized water, attracting them to one side of a 0.2 ml polypropylene tube using 2 ll of 10xPCR solution, 2 ll of 2 mM dNTPs, 1.2 llof25mM a neodymium magnet (4 mm in diameter), and the supernatant MgSO , and 0.5 ll of KOD-Plus-Neo (1 U/50 ll) were mixed. At was removed. first, the assembled DNA fragments were dissociated from the beads by incubating them at 94 C for 2 min, then the assembled the DNA fragments were amplified through step-down PCR (5 2.6 Fixation of adapter-conjugated first BioBrick to cycles of incubation at 98 C for10 s followed by incubation at magnetic beads 74 C for 1 min, 5 cycles of incubation at 98 C for 10 s followed by incubation at 72 C for 1 min, 5 cycles of incubation at 98 C Ten microliters of adapter-conjugated first BioBrick solution, for 10 s followed by incubation at 70 C for 1 min, 10 cycles of in- 10 ll of TE containing 0.8 M NaCl, and the beads previously cubation at 98 C for 10 s followed by incubation at 68 C for rinsed by the above protocol were mixed by agitation. The sus- 1 min). The amplified DNA fragments were purified by using a pension was incubated at 60 C for 5 min and left for 35 min until FastGene PCR Extraction Kit. The DNA fragments were digested by the temperature decreased to 25 C at a rate of 1 C/min. The EcoRI and SpeI, digested DNA fragments were separated on 1% aga- beads were resuspended by agitation and incubated for a fur- rose gel, then purified by using a FastGene Gel Extraction Kit. A ther 25 min at 25 C. The beads were collected using the magnet, total of 0.1 pmol of EcoRI/SpeI digested linear assembled DNA frag- and the supernatant containing excess-free DNA fragments was ments were ligated with 0.035 pmol of EcoRI/SpeIdigestedlinear removed. plasmid DNA. The ligated DNA constructs were transformed into Escherichia coli DH5a (HIT competent cells, RBC Bioscience), and 2.7 Beads rinsing cells were spread on an Luria-Bertani Broth (LB)-plate containing the appropriate antibiotic for screening transformed clones. Twenty microliters of TE containing 0.1% Triton X-100 was added to the beads and the beads were suspended by agitation. The beads were collected using a magnet to remove superna- 2.12 Notes tant by pipetting. Then, the beads were rinsed by repeating the (1) Usage of primer set (100-bp upstream primer and 200-bp process of suspending them in 20 ll of TE by agitation, and col- downstream primer) for gene amplification is recommended to lection by magnet to remove the supernatant. increase the length of the DNA fragment, since recovery of short DNA fragments using affinity columns (for preparation of DNA fragment) is inefficient when fusion of a short BioBrick DNA 2.8 Removal of 200-bp downstream DNA fragment fragments are required. This process is also helpful to make ‘Digestion Premix’ containing 1 llof 10 RE solution, 3 llof2M identification of completely digested DNA fragments easier. trehalose, 5.5 ll of DW, and 0.5 ll of restriction endonuclease, (2) All DNA fragments used in this experiment should be SpeI-HF, was added to the beads, and suspended by pipetting up highly purified. Usage of glycogen for DNA purification should and down. The suspension was incubated at 37 C for 15 min, be avoided (33). followed by mixing by pipetting up and down and further incu- (3) All steps for beads rinsing should be performed by agita- bation at 37 C for 15 min. Then, the beads were collected by tion of the tube by touching to the head of sonic toothbrush to magnet to remove the supernatant. The beads were rinsed avoid loss of beads by adsorption onto the inner surface of plas- twice, as described in the ‘Beads-rinsing’ section above. ticware during pipetting up and down. (4) All steps for mixing enzyme solutions should be per- formed by pipetting up and down to avoid reduction of enzyme 2.9 Fusion of second BioBrick to first BioBrick activity. ‘Ligation Premix’ containing 2 ll of second BioBrick solution (5) The beads should always be fixed on one side of the inner (0.2 pmol), 3 ll of deionized water, and 5 ll of Mighty Mix was surface of the polypropylene tube by a magnet when a superna- added to the beads and mixed by pipetting up and down. The tant is removed by pipetting. mixture was incubated at 16 C for 15 min, followed by mixing (6) We used a very small neodymium magnet (4 mm in diam- by pipetting up and down and further incubation at 16 C for eter) to avoid magnetization of magnetic beads. 15 min. The beads were rinsed twice, as described in ‘Beads- rinsing’. The 200-bp downstream DNA fragment was removed by SpeI restriction enzyme, as described in the ‘Removal of 200- 3. Results bp downstream DNA fragment’, section, followed by ‘Beads- 3.1 Schematic representation of QGA rinsing’ twice. First, (i) all DNA fragments containing BioBricks were prepared by PCR amplification using a common primer set consisting of 2.10 Fusion of more downstream BioBricks 100-bp upstream and 200-bp downstream DNA primers. (ii) Downstream BioBricks were fused by repeating the protocol de- Second, all DNA fragments were digested by restriction enzyme scribed in the ‘Fusion of second BioBrick to first BioBrick’ sec- (XbaI) to unmask the upstream end of each fragment and make tion. The final round of BioBrick fusion does not contain a step them competent for ligation to their upstream fragment, as for the removal of the 200-bp downstream DNA fragment, since shown in Figure 1a. (iii) The upstream end of the first BioBrick the downstream fragment is required for amplification of as- should be immobilized by fixing it to the origin of gene assem- sembled gene by PCR as a primer annealing site. bly, and its downstream end should be kept masked 4| Synthetic Biology, 2017, Vol. 2, No. 1 dT (Chemicell), we succeeded in achieving a dramatic reduction of the time required for the sequential precipitation and rinsing steps. Since oligo-dT conjugated to the beads can hybridize to oligo-dA, we made oligo-dA the central part of the adapter DNA fragment required to fix the first BioBrick to the beads. Utilizing the magnetic properties of the beads allows for much faster iso- lation and purification of the assembled DNA fragments with- out the need for multiple sequential centrifugation steps. Replacing the precipitation and rinsing steps by magnetic bead isolation using a magnet largely contributed to the simplifica- tion of the whole protocol. 3.2 Possible number of cycles for accurate assembly A short DNA fragment containing a promoter-BioBrick, P tetR (BBa_R0040, 54 bp) was used as a building block to test the possi- ble number of cycles for accurate assembly. As seen in Figure 2a, the sizes of PCR products derived from the amplification of tem- plates generated after each cycle of assembly was expected to in- crease stepwise with an increase in the cycle number of assembly. The PCR product size after each cycle should be 200-bp longer than the DNA fragments composed of only fused BioBricks after each DNA fragment is fused to a common 200-bp down- stream fragment. All BioBricks were connected by a 6-bp inter- vening scar site sequence (ACTAGA). This scar site occurs when the ends resulting from a 5’-overhang restriction digestion by SpeI and XbaI are ligated. The reduction of efficiency of the complete assembly was evaluated by measuring the rate of light intensity associated with the largest PCR products on a gel image using software, ‘Gel Analyzer 2010a (http://www.gelanalyzer.com/down load.html)’. Light intensity associated with the largest product in each lane (from lane 1 to lane 4 in Figure 2b)was thehighest among products. The efficiency of the complete assembly reac- tion was calculated by comparing the light intensity of the largest DNA product with other products. The efficiency decreased de- pending on the number of assembly cycles, where observations of 61.8% (after 2 cycles), 44.3% (after 3 cycles), and 31.1% (after 4 Figure 1. Schematic representation of the QGA protocol. (a) Preparation of a cycles) were made. The efficiency of the complete assembly reac- DNA fragment containing a BioBrick part. P: prefix DNA fragment, BB: BioBrick, tion after 5 cycles was calculated as 22.8%. These observations S: suffix DNA fragment, 100 bp UP-F: 100 base pair upstream forward primer, 200 bp DN-R: 200 base pair downstream reverse primer, circled P: phosphate res- suggest that the majority of the products are full length and re- idue, X: XbaI restriction site. (b) Structural change of extending the DNA mole- sult from up to four cycles of assembly. The potential of this as- cule in each step of QGA. MAGB: magnetic beads, E: EcoRI restriction site, S: SpeI sembly method was tested by repeating more than five BioBrick restriction site, S/X: mixed restriction site (scar), TTT...TTT: oligo-dT conjugated part assembly cycles. The largest products, corresponding to to magnetic beads, AAA...AAA: oligo-dA in DNA adaptor. eight BioBrick parts are still visible, even after eight cycles of as- sembly (data not shown). The largest product was isolated from (dephosphorylated) to avoid unexpected fusion with itself when agarose for further ligation with a plasmid vector when only per- fectly assembled constructs were required. sequentially assembling multiple BioBrick parts. (iv) The down- stream 5’-terminus should be removed by restriction enzyme digestion using SpeI to unmask its downstream end for ligation 3.3 Conditions affect higher yield of correctly assembled to the second BioBrick. (v) The second BioBrick is ligated down- product stream of the first BioBrick. The number of BioBricks fused to the downstream end can be increased by repeating this diges- To evaluate each factor affecting the yield of the largest product tion/ligation cycle, as is illustrated in Figure 1b. among all PCR products, only one factor was changed from the The protocol described above requires frequent replacement optimum condition described in Materials and Methods section. of reaction solutions for the treatment of both ends of all The assembled products generated under optimum condition BioBricks. The most common method for replacement was eth- are shown in lane 1 in Figure 3 as a positive control. In lane 2, anol precipitation of the DNA fragments. In this case, at least 10 the high-fidelity restriction enzyme SpeI(SpeI-HF) in digestion precipitation by ethanol addition steps, 10 rinsing by 70% etha- premix was replaced by a standard SpeI enzyme. The yield of nol steps, 20 centrifugation steps, and 10 drying-up steps are re- the largest product was reduced to 78.6% when the yield ob- quired, just to complete the assembly of 5 BioBricks. These tained in the optimum condition was set as 100%. The yield was protocols require a long period of time, and DNA sample yield further reduced to 72.9% when optimum conditions (30 min at tends to decrease dramatically after sequentially repeated etha- 16 C) were changed to incubation for 10 min at 25 C, as shown nol precipitation and rinsing steps. By taking advantage of the in lane 3. The yield was reduced to 66.3% when a rinsing by TE usage of ‘magnetic beads’ conjugated to oligo-dT, SiMAG-Oligo- containing 0.1% Triton X-100 was replaced by rinsing with TE, K. Yamazaki et al. | 5 Figure 4. Negative effects of enzyme solution mixing from pipetting up and down compared to the agitation method. Distribution of amplified DNA frag- ments by PCR when assembled DNA fragments were used as a template after each cycle. Cycle numbers of QGA are indicated above each lane. Copy numbers of PtetR fragments are indicated at the right side of each band. 3.4 Optimization of mixing steps Mixing enzyme solutions by pipetting up and down is generally considered to be one of the best methods to avoid enzyme de- naturation. However, a considerable reduction in the number of Figure 2. Size analysis of PCR products after an increase in the number of QGA magnetic beads and a decrease in the yield of the DNA fixed to cycles. (a) Predicted assembled structures of products after increase of cycles of them were observed through repetition of this manipulation. To QGA. Cycle numbers of QGA and predicted sizes of assembled DNA fragments address this problem, we tested agitating the test tube to avoid after each cycle are indicated at the left of each construct. PtetR: promotor BioBrick of tetracycline resistance gene. (b) Distribution of amplified DNA frag- the loss of beads by adsorption onto the inner surface of the ments by PCR when assembled DNA fragments after each cycle were used as a plastic pipette tips, and the shear forces associated with re- template. QGA cycle numbers are indicated above each lane. Copy numbers of peated pipetting up and down. Complete replacement of the PtetR fragments are indicated at the right side of each band. pipetting up and down manipulation by the agitation method considerably reduced the yield of the largest assembled product after assembly, as shown in Figure 4. The efficiency of complete assembly after cycles 1, 2, 3, 4, and 5 were reduced, step by step, from 100% to 57.9%, 38.9%, 21.3%, and 16.2%, respectively. These observations suggest that repeated agitation contributes to avoiding the loss of magnetic beads; however, the manipulation may cause a reduction of enzyme activity. Therefore, the vibra- tion method should be employed for beads rinsing, and the pipetting up and down manipulation should be used for mixing enzyme solutions, such as the ‘Digestion Premix’ and ‘Ligation Premix’ solutions. Figure 3. Negative effects on the QGA process resulting from the change of each condition away from optimum conditions. Lane 1: assembled products under optimum conditions. Lane 2: replacement of the restriction enzyme from SpeI- 3.5 Effects of increase of BioBrick length in QGA HF (NEB) to SpeI (NEB). Lane 3: changing of ligation condition from ‘at 16 C for 30 min’ to ‘at 25 C for 10 min’. Lane 4: removal of 0.1% Triton X-100 from the The results shown in Figures 2–4 indicate that assembly of a rinsing solution. Lane 5: removal of 0.6 M trehalose from the digestion premix. longer DNA fragment from five or more short Biobricks by QGA Lane 6: combination of all changes tested from lanes 2–5. Copy numbers of PtetR was achieved. To test if this protocol is applicable for the gene fragment are indicated at the right side of each band. assembly of longer BioBricks, a DNA fragment (300 bp) contain- ing five tandem repeats of the short BioBrick part BBa_R0040 as shown in lane 4. Moreover, production of immature product, was used as a building block for the assembly. The efficiency of the smallest product, increased 1.35 times. These observations complete assembly from longer BioBricks after five cycles was suggest that the presence of a low concentration of detergent in calculated to be 10.4%, shown in Figure 5, lane 5. Although the the rinsing solution is essential for the optimum removal of so- efficiency of the complete assembly was rather low, the amount lution components used in the previous step. The yield of the of DNA fragment isolated from an agarose gel was enough to be largest product was reduced to 41.4% when 0.6 M trehalose was used for an enzyme digestion followed by ligation and transfor- removed from ‘Digestion Premix’, as shown in lane 5. mation of bacterial cells. These observations suggest that genes Furthermore, production of immature product, the smallest up to 1500 bp in length can be assembled from 5 BioBricks bio- product, increased 2.4 times. Trehalose is a glucose disaccharide chemically in 24 h. synthesized in Saccharomyces cerevisiae and E. coli, and is known to retain the potential to elevate an enzyme reaction in vitro 3.6 Assembly of a functional gene through ligation of a (34,35). Combining all these changes dramatically reduced the promoter, protein coding sequence, and terminator yield to 29.2%, as shown in lane 6. These observations shown in Figure 5 suggest that the addition of 0.1% Triton X-100 to TE for To test the potential of the assembly system to construction rinsing and the addition of 0.6 M trehalose to the digestion pre- functional genes from distinct BioBricks, Green fluorescent pro- mix are essential for successful BioBrick assembly. However, tein (GFP), and Red fluorescent protein (RFP) coding sequences use of the high-fidelity restriction enzyme SpeI-HF and little were used as reporter genes. Schematic representations of DNA change of ligation conditions were not essential for higher yield constructs on the process to finish complete gene assembly are of the largest product. shown in Figure 6a together with their lengths. The efficiency of 6| Synthetic Biology, 2017, Vol. 2, No. 1 encoding transcription units for expression of GFP or RFP were digested by restriction enzymes, EcoRI and SpeI, ligated with plasmid vector, pSB1A3 which has previously been digested by EcoRI and SpeI, and the ligated samples were subjected for trans- formation of bacteria strain DH5a. Images of colonies appeared on agarose LB plates containing ampicillin (100 mg/mL), which are shown in Figure 6c. GFP and RFP expression was monitored under white light. Ratios of Figure 5. Effects of the increase of BioBrick length in QGA. Distribution of ampli- fied DNA fragments by PCR (length of each PCR product containing 5, 10, 15, 20, green colonies to all colonies and red colonies to all colonies and 25 tandem repeats of PtetR is 514 bp, 814 bp, 1114 bp, 1414 bp, and 1714 bp, were calculated by counting the total number of colonies. respectively) when assembled DNA fragments were used as a template after Colonies of 92% or 90% successfully produced GFP or RFP, re- each cycle. Cycle numbers of QGA correspond to lane numbers. Copy numbers spectively. These results suggest that the majority of the largest of PtetR fragments are indicated at the right side of each band. products after the assembly and gene amplification have a po- tential for successful expression. 4. Discussion We have often encountered a requirement to construct hun- dreds of recombinant genes and genetic circuits to investigate the function of various biological systems at the molecular level, or to generate novel biological systems in the field of syn- thetic biology. This process always requires continuous effort, especially given the increasing demand for new constructs in research. Therefore, the reduction of the time required for each design and construction cycle for gene assembly, is a critical is- sue facing the field as a whole. First, the existence of a common engineering principle based gene-design-concept is important to reduce the time required for the design cycle of genes, since the sharing of ideas and fre- quent discussions among researchers and students is crucial to achieve the best designs. The most well-known gene-design- concept based on engineering principles is the ‘standardized ge- netic part (BioBrick)-based gene design protocol’ proposed by Knight (14). To take full advantage of the gene-design-concept, making good use of a highly reliable BioBrick collection is essen- tial. Continuous effort to evaluate the quality of each BioBrick part, accumulation of measurement and characterization infor- mation of each BioBrick part, and the sharing of these data are critical to improve the quality and quantity of the collection. Furthermore, BioBrick parts should be widely used under differ- ent conditions and in different organisms to evaluate the per- formance of each part. The performance of each BioBrick part should be measured under a variety of conditions, and the in- formation should be shared widely and publically. With this accumulated body of knowledge, BioBricks can be used appro- priately under various conditions for researchers to achieve the Figure 6. Assembly of functional genes from distinct BioBricks. (a) Schematic best designs possible. representation of the QGA process from a single DNA construct to the finished Second, the development of an efficient gene assembly complete gene assembly is shown together with their lengths. P: prefix DNA fragment, PtetR: TetR promotor, RBS: ribosome binding site, GFP: DNA fragment method is also important for the construction of hundreds of re- encoding green fluorescent protein (BBa_I13500), RFP: DNA fragment encoding combinant genes, and this development could lead to a reduc- red fluorescent protein (BBa_K093005), dT: double terminator (BBa_B0015), S: suf- tion in the time required for the ‘construction cycle’ of genes. fix DNA fragment. (b) Sizes of PCR products amplified from assembled genes. Furthermore, the method should be based on engineering prin- White arrowheads indicate the expected DNA fragments. The numbers above ciples so that the technique can be applied to automated ge- each lane are corresponding to the numbers shown in panel A. (c) Images of col- netic part assembly. In the introduction section, we described onies on agarose LB plates containing 100 lg/mL ampicillin. Expression of GFP and RFP was monitored under white light. Scale bars are 2 mm. several other gene assembly protocols as well as their advan- tages and limitations. In this study, we succeeded in making a novel BioBrick-based DNA assembly method, and named it the assembly of functional genes from three BioBricks was ‘Quick Gene Assembly (QGA)’. This method enables us to as- monitored by observing a change in the sizes of PCR products semble at least five BioBricks in vitro in 12 h. All steps (including amplified from assembled genes, as shown in Figure 6b. White amplification of DNA fragments by PCR, and assembly of the arrow heads indicate intermediary products and the assembled plasmid DNA vector) can be finished in 24 h. DNA fragments, as we expected. The sizes of the assembled The advantages of QGA are listed as follows: (i) Use of only DNA fragments were successfully increased. The largest PCR one universal DNA primer set is enough to prepare any kind of products (in lanes 3 and 6 in Figure 6b) containing regions BioBrick or other building block for the assembly of genetic K. Yamazaki et al. | 7 14. Knight,T. (2006) Idempotent Vector Design for Standard Assembly parts. (ii) Genetic parts can be fused correctly in as short a time as 12 h. (iii) We can successfully assemble repetitive DNA se- of Biobricks. MIT Synthetic Biology Working Group. quences or short DNA fragments (less than 100 bp). (iv) Filling 15. 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