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C-Brick: A New Standard for Assembly of Biological Parts using Cpf1 Shiyuan Li, Guoping Zhao, and Jin Wang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00114 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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ACS Synthetic Biology

C-Brick: A New Standard for Assembly of Biological Parts using Cpf1 Shi-Yuan Li†,‡, Guo-Ping Zhao†,‡,§,∥,⊥,* and Jin Wang†,‡,* † Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 20032, China ‡ University of Chinese Academy of Sciences § Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China ∥ State Key Lab of Genetic Engineering & Center for Synthetic Biology; Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200032, China ⊥ Department of Microbiology and Li KaShing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China; * To whom correspondence should be addressed. Mailing address: Jin Wang ([email protected]) and Guo-Ping Zhao ([email protected]), 300 Fenglin Road, Shanghai 200032, China (Tel: +86-21-54924002 (O); Fax: +86-21-54924015)

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ABSTRACT So far, several DNA assembly standards have been developed, enabling scientists to conveniently share and modify characterized DNA parts. However, majority of the restriction endonucleases used in these standards bear short recognition sites (e.g. 6 bps in BioBrick standard), which are widely distributed and need to be removed before further construction, causing much inconvenience. Although homing endonucleases, which recognize long DNA sequences, can be used for DNA assembly (e.g. iBrick standard), long scars will be left between parts, limiting their application. Here, we introduce a new DNA assembly standard, namely C-Brick, which employs the newly identified class 2 type V CRISPR-Cas systems protein Cpf1 endonuclease. C-Brick integrates both advantages of long recognition sites and short scars. With C-Brick standard, three chromoprotein cassettes were assembled and further expressed in Escherichia coli, producing colorful pigments. Moreover, C-Brick standard is also partially compatible with the BglBrick and BioBrick standards.

KEYWORDS Synthetic biology, DNA assembly, Cpf1, C-Brick, BioBrick, CRISPR

Since the characterization of restriction enzymes and DNA ligases, dozens of DNA assembly technologies have been developed, which have greatly promoted the research progress in molecular biology1–6. At present, popular DNA assembly methods are mainly based on three disciplines, i.e. the restriction endonuclease (RE)-based assembly technology, site-specific 4

recombination and long-overlap-based assembly strategies , among which the RE-based method is the most frequently used technology. However, as there exist dozens of different REs, their combinations would be astronomical, bringing much complexity and inconvenience. To address this problem, BioBrick standard (BBF RFC 10) was proposed early this century 7,8

(http://hdl.handle.net/1721.1/21168) , which employs EcoRI and XbaI cutting sites as the prefix sequence and SpeI and PstI as the suffix sequence. Because XbaI and SpeI have complementary coheisive ends, BioBrick DNA parts that are cut with XbaI and SpeI can be joined together, producing a new BioBrick part for further hierarchical assembly. However, several defects have been found for BioBrick standard. For example, it produces an 8-bp scar between parts and thus does not support the construction of in-fusion proteins. To solve this problem, several modified assembly standards have been developed, including the BglBrick standard, which employs BglII, BamHI, EcoRI, and XhoI sites and produces a 6-bp scar of “GGATCT”. As the 6-bp scar could be translated into Gly-Ser, BglBrick standard can therefore be used to construct a fusion of multiple protein domains without changing the 9

reading frame or introducing stop codons . Besides, these standards use the 6-bp cutting REs, whose cutting sites are widely distributed, especially in long DNA sequences. Because these REs sites are forbidden within DNA parts, they have to be removed before the parts construction, which can be a huge cost of time and money. Under this background, iBrick was then developed, which employs the homing endonucleases (HEs) that recognize long DNA 10

sequences . As the HEs recognition sites rarely exist in natrual DNA sequences, basically there is no need to modify the internal DNA sequences when using the iBrick standard.

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However, because iBrick standard leaves a 21-bp scar, it is sometimes unpopular, especially when small DNA parts such as promoters, ribosome binding sites (RBS) and terminator sequences are used for DNA assembly. The clustered regularly interspaced short palindromic repeats (CRISPR) system has developed rapidly in recent years11,12. Among the CRISPR-associated (Cas) proteins, Cas9 endonuclease from Streptococcus pyogenes is now widely used, which mostly introduces double-strand DNA breaks with blunt endings

13–16

. In 2015, Zhang and his co-workers firstly

characterized the class 2 type V CRISPR-Cas systems protein Cpf1, which is a crRNA-guided endonuclease that introduces a DNA double-strand break with a 4 or 5-nt 5’ overhang17 (Figure 1). In this study, Cpf1 is employed to develop a new DNA assembly standard, namely C-Brick standard. It is of particular importance that C-Brick standard takes advantage of both long target sequences and short scars. The 6-bp scar (GGATCC) generated by C-Brick encodes Gly-Ser, which therefore allows for the construction of fusion proteins. To test the C-Brick standard, three chromoprotein expression cassettes were assembled and further expressed in Escherichia coli. As the cleavage sites of Cpf1 endonucleases were found to be inaccurate, which may leave 17

4 or 5-nt 5’ overhangs depending on the target DNA sequences , two distinct Cpf1s (i.e. FnCpf1 and AsCpf1) were tested by Cpf1 digestion. Five different target DNA sequences and their corresponding crRNAs were employed for assays, and the Cpf1-digested target DNA sequences were then directly sequenced to identify the exact Cpf1 cleavage sites (Figure S1). th

Both FnCpf1 and AsCpf1 mainly cut at the 18 base site on the non-complementary strand rd

and the 23

base site on the complementary strand downstream of the PAM sequence,

introducing a 5-nt 5’ overhang, which was named as the “18-23” cleavage pattern. While, for some target sequences, there were a small portion of the cleavage sites at the 17th base on the non-complementary strand and 22nd or 24th base on the complementary strand. Because FnCpf1 showed more specificity than AsCpf1 in most DNA target sequences, it was chosen for further DNA assembly. Meanwhile, to test the influence of reaction time and FnCpf1/crRNA concentration on the cleavage sites, different concentrations of FnCpf1 enzyme (i.e. 50 nM, 250 nM and 1250 nM) and crRNA (i.e. 100 nM, 200 nM, 1000nM) were used and digestion reactions were stopped at different time points (i.e. 20 min, 1 h and 2 h), followed by cleavage site detection. According to the sequencing results, no obvious difference was found among the above tested conditions (Figure S2). Usually, four restriction sites (two in the prefix sequence and two in the suffix sequence) are used in a DNA assembly standard, e.g. the BioBrick, BglBrick, Biofusion and Freiburg standard7,9,18,19. Similarly, four Cpf1 target sequences, including two prefix targets (T1 and T2) and two suffix targets (T3 and T4), were designed for a C-Brick standard part (Figures 2a and 2b). All four target sequences together with their paired crRNAs were tested by FnCpf1 digestion, and the sequencing results showed that all four target sequences were mainly cleaved with the “18-23” cleavage pattern (Figure 2c), enabling these target sequences to be applied in further C-Brick assembly. In the C-Brick standard, the FnCpf1-digested T2 and T3 target sequences produce complementary cohesive ends (i.e. “GGATC” coheisive end for DNA parts with T2 target sequence and “GATCC” for T3) and can be ligated together, which generates a scar and meanwhile eliminates both target sites, supporting interative assembly in 3



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a similar way to that of BioBrick. As shown in Figure 3, a C-Brick DNA part of a protein coding gene can be released by Cpf1 through cutting at the T1 and T3 sites. Meanwhile, another C-Brick standard plasmid containing the terminator part can be cut by Cpf1 at the T1 and T2 sites. As the two T1-digested sites produce the same coheisive ends and the T2- and T3-digested sites produce complementary coheisive sites, the coding gene and the terminator can then be ligated, producing a 6-bp scar of “GGATCC”. The newly generated brick once again contains the two prefix sites (T1 and T2) and the two suffix sites (T3 and T4), and can therefore be used for further assembly (Figure 3). In addition, several restriction sites (e.g. EcoRI, XbaI, BamHI, SpeI, XhoI and PstI) were designed in the interface of the C-Brick standard, making C-Brick standard partially compatible with the present standards (i.e. BglBrick and BioBrick) (Figures S3 and S4; for details, please ref to supplementary “Materials and Methods”). In 2012, an iGEM team from Uppsala University created a collection of chromoproteins that are visible with naked eyes (http://parts.igem.org/Protein_coding_sequences/Reporters)20. Among them, cjBlue (BBa_K592011), eforRed (BBa_K592012) and amilGFP (BBa_K592010) produce colors similar to cyan, magenta and yellow, respectively. According to the CMY (cyan-magenta-yellow) Color Model, all colors can be produced through mixing three colors of 21

cyan, magenta and yellow . Therefore, the above three genes were assembled in different combinations to produce kinds of colors, using the C-Brick standard. Firstly, the coding sequences of the three genes and terminators were individually cloned into a C-Brick standard vector (pCB1A2_2) (Table S1). When strong promoters were cloned into pCB1A2_2, mutations were found in the promoter sequences of all obtained clones. Probably, strong promoters may cause instabilities of the plasmid. To solve this problem, a terminator (CBE_T000001) was added to the downstream of the suffix sequence in pCB1A2_2, obtaining pCB1A2_1, which was then used for successful cloning of the promoter sequences. In future work, pCB1A2_1 is recommended for cloning of all kinds of biological parts. Then, all DNA parts were assembled in the same procedure as shown in Figure 3 and Figure 4a. Detailed information for the obtained constructs could be found in Table S1, and the nomenclature of a C-Brick standard part was described in the supplementary “Materials and Methods”. C-Brick assembly standard was compared with the traditional Type II RE-based assembly method, which used the same REs as those in BioBrick standard. We found the assembly efficiency of C-Brick standard was much lower than the Type II RE-based method (i.e. less than a half) (Table 1). However, the accuracy of C-Brick standard assembled clones, which was measured by DNA sequencing (Table 2), was found to be more than 90% for the most frequently adopted T2-T3 and T4-T4 types of ligation. The lower assembly efficiency and incorrect assembly might be caused by the inaccuracy of Cpf1 cleavage. Correct constructs were transformed into E. coli and three beautiful colors could be visible. Subsequently, two color expression cassettes were further assembled with C-Brick standard to create more colors, i.e. red color from amilGFP plus eforRed, green color from amilGFP plus cjBlue and light purple color from eforRed plus cjBlue (Figure 4b). The C-Brick logo was then drawn with E. coli cells expressing different colorful pigments (Figure 4c). Although Cpf1 mainly cleaves target DNA sequences with the “18-23” cleavage pattern, for 4


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some target sequences, cleavage nearby the two sites could also be detected. Moreover, it is quite possible that the inaccuracy of the cutting sites reduced the assembly efficiency and caused the mutations after DNA assembly. Therefore, it is very important to find new Cpf1s or perform protein engineering of the present Cpf1s to enhance the accuracy of Cpf1 digestion in future. METHODS Synthesis of crRNAs To prepare the templates for in vitro transcription, ssDNA oligos (Table S3) were annealed to either complementary ssDNA oligos or a short T7 priming sequence (T7-crRNA-F). The synthesis of crRNAs was performed using the T7 High Yield Transcription Kit (Thermo Fisher Scientific). T7 in vitro transcription was performed over night (about 16 hr), and then RNA was TM

purified using the RNA Clean & Concentrator -5 (Zymo Research) and quantified with NanoDrop 2000C (Thermo Fisher Scientific). Purification of Cpf1 Proteins Protein coding sequences of FnCpf1 and AsCpf1 were firstly synthesized with codon optimization by GenScript Company (China) and then cloned into a bacterial expression vector (pET28a-TEV) (DNA coding sequences of FnCpf1 and AsCpf1 were shown in the supplementary Sequence S1). Cpf1 expression plamsids were transformed into E. coli BL21(DE3) (Invitrogen) and the transformants were used for production of recombinant Cpf1. 17

Purification of Cpf1 protein was similar to that described by Zhang et al. , but with a little modification. Briefly, harvested cells were resuspended in the Lysis Buffer (50 mM Tris-HCl [pH 7.4], 200 mM NaCl, 2 mM DTT and 5% glycerol) supplemented with protease inhibitors (Roche, EDTA-free), and then lysed with French press. Cpf1 protein was firstly purified with Nickel column (HisTrap FF), then in order purified with HiTrap Q HP, HiTrap SP HP and HiLoad 16/600 Superdex 200 pg via FPLC (AKTA Pure). Fractions from gel filtration were analyzed by SDS-PAGE and fractions containing homogeneous Cpf1 were concentrated by ultrafiltration. Protein concentration was determined by the Bradford method. Cpf1 in vitro cleavage assay Cpf1 in vitro cleavage reaction was performed at 37 °C in cleavage buffer (NEB Buffer 3) for 2 h, employing 250 nM Cpf1, 1000 nM synthesized crRNA, 1 µg target DNA and 20 U RNase inhibitor (Takara) in a RNase-free reaction system of 20 µl volume. Reactions were stopped by heating at 75 °C for 10 min, which can be directly used for DNA sequencing or gel electrophoresis. If needed, reaction products can be cleaned up using the Wizard SV Gel and PCR Clean-Up System (Promega). Construction and assembly of C-Brick parts Three chomoprotein sequences, i.e. cjBlue (BBa_K592011), eforRed (BBa_K592012) and amilGFP (BBa_K592010), were firstly de novo synthesized by Tolo Biotech. (China), and then cloned into the C-Brick standard vector. Their detailed DNA sequences can be downloaded from the iGEM website (http://parts.igem.org/Protein_coding_sequences/Reporters). Short DNA parts, such as promoters and terminators, were introduced into the C-Brick standard 5



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vector by PCR, followed by the self-ligation method using T4 PNK (NEB) and T4 DNA ligase (Tolo Biotech.). To assemble DNA parts in the C-Brick standard, C-Brick standard plasmids were treated by Cpf1 digestion in the same method as described in the in vitro cleavage assay, generating linearized vectors and released DNA parts, which were puried through gel electrophoresis and gel purification. DNA parts and the vector were ligated with T4 DNA ligase (Tolo Biotech.) and then transfered into E. coli DH10B for further analysis. Comparison of the C-Brick assembly efficiency with the Type II RE-based method Fragments of pCB1A2_1-CBP_X000035 and pCB1A2_1-CBP_X000036 were used for assembly efficiency test, which harbored the eforRed and the amilGFP expressing cassettes, respectively. Using C-Brick, plasmid pCB1A2_1-CBP_X000035 was cut by FnCpf1 at T1/T3 sites to produce the foreign fragment, while pCB1A2_1-CBP_X000036 was cut at T1/T2 sites sites to produce the vector, leading to the T3-T2 type ligation. Alternatively, plasmid pCB1A2_1-CBP_X000035 could be cut at T2/T4 sites and pCB1A2_1-CBP_X000036 was cut at T3/T4 sites, leading to the T2-T3 type ligation. When the Type II RE-based method was employed, plasmid pCB1A2_1-CBP_X000035 was digested with EcoRI/SpeI to generate the foreign fragment and pCB1A2_1-CBP_X000036 was digested with EcoRI/XbaI to produce the vector, leading to the SpeI-XbaI type ligation. Alternatively, plasmid pCB1A2_1-CBP_X000035 was digested with XbaI/PstI and pCB1A2_1-CBP_X000036 was digested with SpeI/PstI, leading to the XbaI-SpeI type ligation. DNA ligation was performed with the usage of T4 DNA ligase (Tolo Biotech.). Successfully assembled clones produced red colors, otherwise the clones were yellow. The assembly efficiency was counted on the basis of the colors of the clones. AUTHOR INFORMATION Jin Wang and Guo-Ping Zhao designed the experiments. Shi-Yuan Li performed all the experiments. All authors wrote and revised the manuscript. ACKNOWLEDGEMENT We thank Chao Lei for his valuable comments on the C-Brick standard and Shanghai Tolo Biotech. (China) for their assistance in plasmid construction. FUNDING This work was supported by the National Basic Research Program of China [2012CB721102]. REFERENCES (1) Zimmerman, S. B., Little, J. W., Oshinsky, C. K., and Gellert, M. (1967) Enzymatic joining of DNA strands: a novel reaction of diphosphopyridine nucleotide. Proc. Natl. Acad. Sci. U. S. A. 57, 1841–1848. (2) Lobban, P. E., and Kaiser, A. D. (1973) Enzymatic end-to-end joining of DNA molecules. J. Mol. Biol. 78, 453–471. (3) Cohen, S. N., Chang, A. C., Boyer, H. W., and Helling, R. B. (1973) Construction of 6


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biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. U. S. A. 70, 3240–4. (4) Casini, A., Storch, M., Baldwin, G. S., and Ellis, T. (2015) Bricks and blueprints: methods and standards for DNA assembly. Nat. Rev. Mol. Cell Biol. 16, 568–576. (5) Ellis, T., Adie, T., and Baldwin, G. S. (2011) DNA assembly for synthetic biology: from parts to pathways and beyond. Integr. Biol. (Camb). 3, 109–118. (6) Cobb, R. E., Ning, J. C., and Zhao, H. (2014) DNA assembly techniques for next-generation combinatorial biosynthesis of natural products. J. Ind. Microbiol. Biotechnol. 41, 469–477. (7) Knight, T. (2003) Idempotent Vector Design for Standard Assembly of Biobricks. Dspace http://hdl. handle. net/1721.1/21168. (8) Shetty, R. P., Endy, D., and Knight, T. F. (2008) Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5. (9) Anderson, J. C., Dueber, J. E., Leguia, M., Wu, G. C., Goler, J. a, Arkin, A. P., and Keasling, J. D. (2010) BglBricks: A flexible standard for biological part assembly. J. Biol. Eng. 4, 1. (10) Liu, J.-K., Chen, W.-H., Ren, S.-X., Zhao, G.-P., and Wang, J. (2014) iBrick: A New Standard for Iterative Assembly of Biological Parts with Homing Endonucleases. PLoS One 9, e110852. (11) Sternberg, S. H., and Doudna, J. A. (2015) Expanding the Biologist’s Toolkit with CRISPR-Cas9. Mol. Cell 58, 568–574. (12) Hsu, P. D., Lander, E. S., and Zhang, F. (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278. (13) Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012) A Programmable Dual-RNA – Guided DNA Endonuclease in Adaptice Bacterial Immunity. Science 337, 816–822. (14) Jiang, F., Zhou, K., Ma, L., Gressel, S., and Doudna, J. A. (2015) A Cas9-guide RNA complex preorganized for target DNA recognition. Science (80-. ). 348, 1477–1481. (15) Garneau, J. E., Dupuis, M.-È., Villion, M., Romero, D. a, Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magadán, A. H., and Moineau, S. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71. (16) Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. 109, E2579–E2586. (17) Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 1–13. (18) Phillips, I., and Silver, P. (2006) A new biobrick assembly strategy designed for facile 7



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protein engineering. Dspace http://hdl.handle.net/1721.1/32535. (19) Müller, K. M., Arndt, K. M., Freiburg, T., and Grünberg, R. (2009) BBF RFC 25: Fusion Protein (Freiburg) Biobrick assembly standard. Dspace http://hdl.handle.net/1721.1/45140. (20) Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., Miller, D. J., Wiedenmann, J., Salih, A., and Matz, M. V. (2008) Diversity and evolution of coral fluorescent proteins. PLoS One 3. (21) Ibraheem, N. a, Hasan, M. M., Khan, R. Z., and Mishra, P. K. (2012) Understanding Color Models : A Review. ARPN J. Sci. Technol. 2, 265–275.

TABLES AND FIGURES LEGENDS Table 1. Comparison of the C-Brick assembly efficiency with the Type II RE-based method Ligation Type

a

Efficiency (positive/total clones)b

T3-T2

T2-T3

SpeI-XbaI

XbaI-SpeI

29.4±4.3%

25.5±1.7%

67.1±5.2%

79.1±4.5%

a, Details of the ligation type could be found in “Materials and Methods”. b, The efficiency was obtained from four independent experiments. The total number of clones obtained from four experiments was 731 for the T3-T2 type of ligation, 934 for the T2-T3 type, 470 for the SpeI-XbaI type and 605 for the XbaI-SpeI type, respectively. Table 2. Accuracy of the DNA constructs assembled with C-Brick standard Ligation Type

T1-T1

Correct /Tested Clones

a

10/12

b

Accuracy

83.3%

T2-T3 44/47

c

93.6%

T4-T4 d

38/42

90.5%

a, The obtained clones were verified through direct DNA sequencing. b, One incorrect clone contained a 7-bp deletion mutation, while the other contained 1-bp mismatch mutation. c, Mutations contained a 1-bp deletion mutation, a 1-bp mismatch mutation and a 30-bp deletion mutation. d, Mutations contained a 1-bp deletion mutation, a 2-bp deletion mutation, and two 12-bp deletion mutations. Figure 1. Schematic diagram of the Cpf1/crRNA/DNA target complex. In the presence of protospacer adjacent motif (PAM) sequence (FnCpf1's PAM is TTN), a specific crRNA guides FnCpf1 to cut the target DNA sequence, generating a double-stranded DNA break. FnCpf1 mainly cuts after the 18th (non-complementary strand) and 23rd (complementary strand) bases downstream of the PAM site, which are indicated with red solid triangles.

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Figure 2. C-Brick standard vector and its interface with cleavage sites. (a) DNA sequence of a C-Brick standard interface (T1, T2, T3 and T4). These sites can be recognized by corresponding crRNAs and a 5-nt 5’ overhang can then be generated by FnCpf1 digestion. Complementary overhangs could be generated for T2 and T3 target sequences after FnCpf1 digestion, i.e. “GGATC” for T2 target and “GATCC” for T3 target. (b) Plasmid map for a C-Brick standard vector. (c) Characterization of the FnCpf1 cleavage sites in the C-Brick standard interface sequence. The cleavage sites were indicated with red solid triangles, where the size of the triangle showed the frequency of FnCpf1 digestion. Figure 3. Workflow of assembling a typical functional expression cassette (a promoter with RBS, protein coding sequences and a terminator sequences) in C-Brick standard. FnCpf1-digested T2 and T3 target sites produce complementary cohesive ends of “GGATC” and “GATCC”, respectively, which produces a short scar of “GGATCC” after ligation. Assembly of the three chromoprotein expression cassettes was performed following this workflow. Figure 4. Colorful bacterial pigments produced by E. coli harboring constructs assembled in C-Brick standard. (a) A schematic chart for assembly of amilGFP and eforRed co-expression cassettes. Similarly, other dual chromoproteins co-expression plasmids were constructed, following the flowchart in Figure 4a. Detailed information could be found in the supplementary “Materials and Methods”. (b) Three chromoproteins and three assembled dual chromoproteins were expressed in E. coli grown in liquid LB medium. From 1 to 6, constructs expressed eforRed (pCB1A2_1-CBP_X000021), amilGFP (pCB1A2_1-CBP_X000022), cjBlue (pCB1A2_1-CBP_X000023), amilGFP plus eforRed (pCB1A2_1-CBP_X000033), amilGFP plus cjBlue (pCB1A2_1-CBP_X000034) and eforRed plus cjBlue (pCB1A2_1-CBP_X000032), respectively. (c) The C-Brick logo was drawn with E. coli cells expressing different colorful pigments.

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Supplementary Materials and Methods: Assembly of DNA parts using C-Brick standard in this study; Transformation of BglBrick and BioBrick parts to C-Brick standard DNA parts; Nomenclature of the C-Brick standard ; Table S1: Vectors, plasmids and strains used in this study ; Table S2: Parts, elements and interface sequences used in this study; Table S3: The crRNA sequences used in this study; Table S4: Oligoes used in this study; Table S5: Primers used in this study; Table S6: Letter allocations for parts in the C-Brick standard; Table S7: Letter allocations for elements in C-Brick standard; Table S8: Letter abbreviations for antibiotic resistance markers in C-Brick; Figure S1: Identification of the cleavage sites of Cpf1 enzymes; Figure S2: Test of the influence of both the reaction time and FnCpf1 concentrations on the cleavage sites of T1 target sequence; Figure S3: Test of the cleavage efficiency with both the reaction time and FnCpf1/crRNA concentrations; Figure S4: C-Brick standard interface and the backup T2' and T3' target DNA sequences; Figure S5: Workflow of the DNA assembly in C-Brick standard using DNA parts originated from the BioBrick standard; Sequence S1: The DNA coding sequence of FnCpf1 and AsCpf1 in this study.

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C-Brick: A New Standard for Assembly of Biological Parts using Cpf1 †,‡

†,‡,§,∥,⊥,

Shi-Yuan Li , Guo-Ping Zhao

†,‡,

* and Jin Wang

For Table of Contents Use Only

10


*

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


FnCpf1 PAM

DNA target

18 non-complementary strand

……

……

complementary strand

23 crRNA


a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 BamHI (2153) BamHI (5) 21 C-Brick prefix 22 23 24 25 26 27 C-Brick Standard Vector 28 2157 bp 29 30 31 32 Amp 33 34 35 36 37 38 39 40 41 42


b

c C-Brick suffix

rep（ pMB1）


Page 12 of 14

Page 13 of 14


T1 T2

T1 T2

T1 T2

T3 T4

T3 T4

protein coding sequence

terminator

resistance

resistance

T3 T4

digest

digest Cpf1-T1/T2

Cpf1-T1/T3

CTTAA GGATCC G GAATT

promoter with RBS resistance

digest

T3 T4

T2 terminator Cpf1-T3/T4 CCTAG

T1 T2

T1

CCTAG

Mix and ligase

TCAAG

T1 T2

T4

GGATCC G

AGTTC

T3

Scar

promoter with RBS resistance

digest

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

resistance Scar

T3 T4

protein coding sequence resistance

Cpf1-T2/T4 Mix and ligase T1 T2

Scar

Scar

T3 T4

functional expression cassette resistance

The scar sequence is “GGATCC”.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

a

Page 14 of 14

b 1 4

5

2

3 6

c


C-Brick: A New Standard for Assembly of ... - ACS Publications

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