CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli

Oct 19, 2015 - †Molecular and Cellular Biology and Biochemistry, ‡Biological Design Center (BDC), ∥Department of Electrical and Computer Enginee...
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CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enables Rapid Combinatorial Design for Synthetic and Traditional Biology Sonya Iverson, Traci L. Haddock, Jacob Beal, and Douglas Densmore ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00124 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enables Rapid Combinatorial Design for Synthetic and Traditional Biology Sonya V. Iverson1,2 Traci L. Haddock2, Jacob Beal3, Douglas M. Densmore2,4 1

Molecular and Cellular Biology and Biochemistry, Boston University, Boston, Massachusetts, USA. Biological Design Center (BDC), Boston University, Boston, Massachusetts, USA. 3 Raytheon BBN Technologies, Cambridge, Massachusetts, USA. 4 Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts, USA. 2

Correspondence should be directed to D.M.D ([email protected]) Keywords: assembly, modular, part library, Type IIS, multiplex ABSTRACT Multipart and modular DNA part libraries and assembly standards have become common tools in synthetic biology since the publication of the Gibson and Golden Gate assembly methods, yet no multipart modular library exists for use in bacterial systems. Building upon the existing MoClo assembly framework, we have developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design and expression tuning in E. coli. The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols.

Despite improvements in DNA assembly technologies, high throughput iterative designs and combinatorial methods are still cost prohibitive in a synthesis-based assembly due to the lack of reusable parts. Multipart modular assembly part libraries have been developed for use in yeast4 and mammalian systems1, however no such library exists for E. coli. Modular Cloning, or MoClo,1 is a one-pot digestion and ligation multipart assembly method, derived from Golden Gate,2 in which user-defined overhangs specific to each part type, such as a promoter or a coding sequence (CDS), create interchangeable DNA modules in the form of plasmids. This format allows for simple library propagation and combinatorial assembly from reusable parts with reliable ligation of up to six DNA fragments in a one-pot reaction. Three MoClo part libraries are currently available providing reusable parts and vectors for plant transformation constructs3 (Addgene, #1000000047), general eukaryotic multigene construct assembly1 (Addgene, #1000000044), and yeast4 (Addgene, #1000000061). Recent publications have also detailed the development of yeast Golden Gate (yGG)5 and BASIC assembly6 methods both of which employ a similar digestion and ligation reaction, though neither offer a part library to accompany the methods. To address this lack of publically available standardize parts for bacterial systems, we have constructed and characterized a library of commonly used genetic parts and the necessary vectors in MoClo format for use in E. coli (Addgene, #1000000059). MoClo relies upon Type IIS restriction enzymes (BbsI and BsaI) which each recognize a 6 base pair non-palindromic sequence and cut at a specified distance from that recognition sequence resulting in modular overhanging 4 bp fusion sites (Fig. 1). For example, coding sequence (CDS) parts are flanked with ‘-AATG-’ (abbreviated “C”) at the 5’ end and ‘-ATTG-’ (abbreviated “D”) on the 3’ end making all CDS parts interchangeable. Plasmid names include the fusion sites designated by single letter abbreviations (ex. E0030m_CD) as in Figure 1. The restriction recognition sites are placed and oriented such that the digest product ends in these specific four base pair overhangs and no longer contains the restriction enzyme recognition sequence. Once ligated it cannot be recut allowing for a hierarchical multipart assembly (Fig 1). The original MoClo protocol allowed for the reliable assembly of up to six parts with a five hour digestion-ligation reaction with large reagent volumes and rotating use of three antibiotics and two color ACS Paragon Plus Environment

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selection modules.6 Although this provided a substantial improvement in modularity compared to Golden Gate, the reaction conditions were still time and cost-prohibitive for most applications. Here we introduce a modified protocol (Supplemental Table 1) that, while maintaining the same capacity for assembling multiple modules and a >95% cloning efficiency, also reduces reaction time from five hours to 90 minutes and lowers reaction costs by 85% while simplifying the hierarchical assembly standard. CIDAR MoClo uses an alternating series of vectors (Fig 1). Basic parts are provided in a Destination Vector - Ampicillin (DVA) derived from BBa_pSB1A2, with BsaI sites flanking the part and fusion sites. These basic parts combine to create a simple transcriptional unit (TU) consisting of promoter:ribosome binding site (RBS):CDS:terminator in a Kanamycin vector (DVK) derived from BBa_pSB1K3 with BbsI sites flanking the newly constructed TU. These units can be further combined into multi-TU devices in the same manner (Fig 1). We created a standard four-part structure which allows users to easily assemble a genetic device consisting of a promoter, RBS, CDS, and terminator. This basic four part format provides a solid foundation for high throughput assembly while remaining adaptable to addition of new parts (i.e. new fusion site combinations) and part types. Most of the parts contained in the CIDAR E. coli MoClo Library are derived from the BioBricks Registry (http://parts.igem.org/) and were selected for their functional reliability and utility in synthetic biological designs (see Supplemental Table 2 for full list of parts). To further enable rational design, three of the basic parts were selected from the BIOFAB collection of bicistronic design (BCD) translational elements which have been shown to enable more rational design in terms of protein expression.7 These BCD parts contain a leader peptide followed by a secondary ribosome binding site (RBS) in order to physically separate transcriptional and translational regulation. Destination vectors are included in the library to allow for simple cloning of new parts with any of the standard fusion site pairs. Additionally, a set of fluorescent expression plasmids are also included to be used as standards with the TASBE flow cytometry analysis tools8 (Supplemental Table 2). Sequence and part information for all plasmids is available in the CIDAR Inventory of Composible Elements (ICE) registry9 (www.cidar-ice.org). Other plasmids for use in E. coli not included in this library are available through the CIDAR ICE registry upon request. To demonstrate the utility of this system, we used the library and protocols to perform a series of comparisons with red and green fluorescent proteins in an assay designed to provide a basis for expression tuning of complex devices. By normalizing flow cytometry fluorescence data to SpheroTech RCP-30-5-A beads10 as a fluorescein standard, we created a color model with which to correlate RFP and GFP expression as molecules of equivalent fluorescein (MEFL) counts rather than arbitrary units. This color model was validated by comparing MEFL counts of 28 pairs of plasmids with various promoter, RBS, and fusion site combinations (mean square error = 1.80 fold) in which each pair differed only in the coding sequence (E0040m_CD GFP or E1010m_CD RFP) (Fig. 2a). An array of GFP expressing TUs was made to further characterize a set of constitutive promoters to determine the extent of rational design capabilities. Using 16 promoters from the J23100 Anderson series and 6 RBS parts (3 Weiss RBS, 3 BCD), 96 iterations of a simple GFP expression plasmid in a DVK_AE vector were analyzed using flow cytometry (Supplemental Fig. 1). Two-way ANOVA analysis identified 36.3% of expression variation as being due to the promoter, 43.9% due to RBS part, and 19.3% due to the interaction of the two factors. A subset of these promoters are included in the library and were selected to provide a full coverage of expression levels as defined by GFP expression ranging from 102 – 105 MEFLs. To further assess the capability for rational design under commonly varied genetic contexts, further pairwise comparisons were performed to evaluate the effects of gene order, variation in vector, and use of specific fusion sites flanking the transcriptional unit. Expression of a single TU was shown to remain constant when expressed in a plasmid with a second TU up- or downstream (Fig. 2b) (mean sq. error = 1.53 fold). Changing the vector from DVA to DVK while maintaining the same transcriptional unit showed a higher variability yet retained a nearly linear relationship (mean sq. error = 2.61 fold) (Fig. 2c). Modifying the four base fusion sites did have an effect on expression (p95% cloning efficiency. Examples of this methodology include screening of variant sequences, or modulating expression levels of a single transcriptional unit by multiplexing the promoter and/or RBS part (Supplemental Figure 2). This library, especially when combined with a related design tool,11 allows for rapid assembly of synthetic gene networks and cost efficient combinatorial assembly and is ideal for academic research and educational uses in teaching laboratory settings. Though formatted for cloning in E. coli, iterative design of eukaryotic circuits is also possible through the introduction of species specific parts. Bio-design automation activities are further promoted with the included Eugene design files that capture not only the parts in the library but also initial design guidelines and data (Supplemental Data).The CIDAR MoClo Library can be requested whole through Addgene (www.addgene.org/cloning/moclo/densmore) and additional individual plasmids are available for distribution through the CIDAR ICE Public Registry (www.cidar-ice.org) along with functional information and DNA sequence data for all parts described above. CONTRIBUTIONS S.V.I., T.L.H., and D.M.D. conceived of the project. S.V.I., T.L.H., and J.B. designed the experiments; S.V.I built the library, performed the experiments, analyzed data, and wrote the manuscript. J.B. performed computational analysis of flow cytometry data. D.M.D and T.L.H created the Eugene code. All authors edited the manuscript. ACKNOWLEDGEMENTS Thanks to Dr. Mike Smanski for assistance with designing the MoClo library, the 2012 and 2013 iGEM teams for constructing various parts, Evan Appleton and Swati Carr for assistance in the lab, Dr. Swapnil Bhatia for continued support of Pigeon12 (www.pigeoncad.org) used to make figures, Prashant Vaidyanathan for the configuration of the ICE repository, and the labs of Dr. James Collins, Dr. Wilson Wong, and Dr. Ahmad Khalil for use of miscellaneous equipment. This work was supported by NSF Grant # 1253856. COMPETING FINANCIAL INTERESTS The S.V.I., T.L.H., J.B. declare no competing interests. D.M.D. is the co-founder of Lattice Automation, Inc. Lattice creates bio-design automation software solutions. METHODS MoClo cloning protocols: In developing optimal protocols, various reaction conditions were tested. The following components were added to a 0.2 mL tube: 10-60 fmol of each DNA component with up to six components total, equimolar, (PCR product or previously made MoClo DNA parts, and the appropriate Destination Vector), 10-50 U of BsaI or BbsI (NEB), 5-50 U of T4 DNA ligase (Cat #M1794, Promega, Madison, WI, USA or #M0202 NEB), 1 X T4 DNA Ligase Buffer (Promega or NEB), and sterile, deionized water to at total volume of 10-60 µL.. Reactions were performed using the following parameters: 15-40 cycles (37°C 1.5-3’, 16°C 3-5’), followed by 50°C for 5’ and 80°C for 10’ then held at 4°C or -20°C until transformed. Optimized protocols are found in Supplemental Table 1 using 10 fmol each DNA component, 10 U of BsaI or BbsI, 20 U of T4 Ligase, 1x T4 DNA Ligase Buffer (Promega), final volume of 10-20uL. Of this, 2-5 uL are used to transform cells as described above. Multiplex MoClo cloning protocol: MoClo reactions were prepared as above with the following differences: The multiplex part type(s) was added such that the total concentration of that type was equimolar to the other part types. When multiplexing less than 6 of any one part type, samples were pipetted individually. To accommodate accurate measurements, multiplex reactions were performed in 20 uL volumes with 60 fmol of ACS Paragon Plus Environment

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each part type. For example, in a reaction using 6 different promoters, 10 fmol each of these promoters was used along with 60 fmol of each other part. For larger multiplex examples all iterations of a given part type are mixed in equimolar ratio prior and added as one mixed part. Statistical Analysis Methods: Flow cytometry data was converted from arbitrary units to compensated MEFL (Molecules of Equivalent Fluorescein) using the TASBE characterization method13. An affine compensation matrix is computed from single color and blank controls: RFP alone (J23102:BCD2:E1010m:B0015, abbreviated as pJ02B2Rm, in _AE and EF DVAs), GFP alone (J23102:BCD2:E0040m:B0015 abbreviated as pJ02B2Gm, in _AE and EF DVAs), RFP:GFP (pJ02B2Rm:J02B2Gm in _AF DVAs) as well as the reciprocal GFP:RFP (pJ02B2Gm:J02B2Rm in _AF DVAs) together and untransformed DH5 Alpha Select E. coli cells (Bioline), respectively. FITC measurements (for GFP) are calibrated to MEFL using SpheroTech RCP-30-5-A beads16. An estimated mapping from RFP measured in the PE-Texas Red channel to equivalent FITC is computed from transformation of constitutive co-expression of RFP and GFP expressed together (RFP:GFP as pJ02B2Rm:J02B2Gm as _AF in DVA, GFP:RFP as pJ02B2Gm:J02B2Rm as _AF in DVA); RFP measurements are translated to MEFL by first mapping to estimated equivalent FITC. Geometric statistics are then computed over data in MEFL units. FIGURE LEGENDS Figure 1 | CIDAR MoClo overview, basic assembly strategy Left: The CIDAR MoClo Library is provided on a 96-well plate (Addgene, #1000000059). A standard CIDAR MoClo assembly is based on four part transcription units comprised of a promoter, ribosome binding site, coding sequence and terminator assembled into a DVK (Kanamycin-resistance destination vector). Destination vectors alternate antibiotic resistance at each assembly level and use lacZα blue-white selection. Right: Assembly strategy for MoClo Type IIS enzyme one-pot digestion ligation reactions. Basic parts shown with green backbones are prepared in DVAs with part specific fusion sites, indicated with single capital letter codes. The 3’ fusion site of the upstream part must match the 5’ fusion site of the following part in order to be correctly assembled. Digesting parts and a DVK with the BsaI and simultaneous ligation with T4 DNA ligase results in a transcriptional unit (TU) shown here with the orange backbone. Multiple TUs can be combined into a complex device using BbsI in place of BsaI with the appropriate DVA. The library is described in more detail in the Supplemental Table 2. Figure 2 | Molecules of Equivalent Fluorescein (MEFL) conversion and predictable expression. (a) SpheroTech RCP-30-5-A beads were used to provide a MEFL normalization of red and green fluorescence data in order to directly relate RFP to GFP in a rational unit. This normalization was validated by comparing 28 pairs of vectors in which each pair differed only in the choice of coding sequence, either GFP or RFP, in a transcriptional unit with a given promoters and RBS part. (b) Single TU expression compared to the same TU when expressed in the same plasmid with another TU. Expression of a single TU is consistent when assembled into a larger device. (c) Changing vector from DVA to DVK shows a high level of variability, suggesting all expression tuning should be done in the same vector. (d) Changing the 4 base pair fusion sites flanking a given TU influences expression likely due to the proximity of the 5’ fusion site to the minimal 35 bp promoter used in this study. The colored circles containing capital letters indicate the relevant 4 base pair fusion sites, the sequences of which are noted in Figure 1.

SUPPORTING INFORMATION Additional methods, CIDAR_MoClo_Eugene_Final.eug (a design file using the Eugene programming language), MEFL normalized data and plasmid descriptions for all samples represented in Figure 2, other supporting figures, and all GenBank files for the CIDAR MoClo Library are available free of charge via the Internet at http://pubs.acs.org.

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References [1] Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. (2011) A modular cloning system for standardized assembly of multigene constructs, PloS one 6, e16765. [2] Engler, C., Gruetzner, R., Kandzia, R., and Marillonnet, S. (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes, PloS one 4, e5553. [3] Engler, C., Youles, M., Gruetzner, R., Ehnert, T. M., Werner, S., Jones, J. D., Patron, N. J., and Marillonnet, S. (2014) A Golden Gate Modular Cloning Toolbox for Plants, ACS Synth Biol. [4] Lee, M. E., DeLoache, W. C., Cervantes, B., and Dueber, J. E. (2015) A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly, ACS Synth Biol. [5] Agmon, N., Mitchell, L. A., Cai, Y., Ikushima, S., Chuang, J., Zheng, A., Choi, W. J., Martin, J. A., Caravelli, K., Stracquadanio, G., and Boeke, J. D. (2015) Yeast Golden Gate (yGG) for efficient assembly of S. cerevisiae transcription units, ACS Synth Biol. [6] Storch, M., Casini, A., Mackrow, B., Fleming, T., Trewhitt, H., Ellis, T., and Baldwin, G. S. (2015) BASIC: a new Biopart Assembly Standard for Idempotent Cloning provides accurate, single-tier DNA assembly for synthetic biology, ACS Synth Biol. [7] Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q. A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., and Endy, D. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements, Nature methods 10, 354-360. [8] Beal, J., Weiss, R., Densmore, D., Adler, A., Appleton, E., Babb, J., Bhatia, S., Davidsohn, N., Haddock, T., and Loyall, J. (2012) An End-to-End Workflow for Engineering of Biological Networks from High-Level Specifications, ACS Synthetic Biology. [9] Ham, T. S., Dmytriv, Z., Plahar, H., Chen, J., Hillson, N. J., and Keasling, J. D. (2012) Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools, Nucleic acids research 40, e141. [10] Spherotech, I. (2001) Measuring molecules of equivalent florochrome (MEF) using SpheroTM rainbow and ultra rainbow calibration particles, SPHEROTM Technical Note. [11] Appleton, E., Tao, J., Haddock, T., and Densmore, D. (2014) Interactive assembly algorithms for molecular cloning, Nature methods 11, 657-662. [12] Bhatia, S., and Densmore, D. (2013) Pigeon: A Design Visualizer for Synthetic Biology, ACS Synth Biol. [13] Beal, J., Weiss, R., Densmore, D., Adler, A., Appleton, E., Babb, J., Bhatia, S., Davidsohn, N., Haddock, T., Loyall, J., Schantz, R., Vasilev, V., and Yaman, F. (2012) An end-to-end workflow for engineering of biological networks from high-level specifications, ACS synthetic biology 1, 317-331.

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Page 7E.ofcoli 8 Modular

CIDAR MoClo Assembly Format Parts and VectorsACS Synthetic Biology BsaI

GGTCTCNGAGG CGAGAGNCTCC

TACTNGAGACC ATGANCTCTGG

Fusion Sites A GGAG

1 BsaI B TACT 2 + GGTCTCNTACT AATGNGAGACC 3 C AATG CGAGAGNATGA TTACNCTCTGG 4 D AGGT + 5 GCTT E GGTCTCNAATG AGGTNGAGACC 6 CGAGAGNTTAC TCCANCTCTGG F CGCT 7 + G TGCC 8 GGTCTCNAGGT GCTTNGAGACC H ACTA CGAGAGNTCCA CGAANCTCTGG 9 + 10 BbsI BsaI Promoter RBS CDS Terminator DVK GAAGACNNGAGGNGAGACC GGTCTCNGCTTNNGTCTTC 11 lacZ CTTCTGNNCTCCNCTCTGG CGAGAGNCGAANNCAGAAG lacZ 12 BbsI BsaI Basic Parts Vector 13 BsaI T4 Ligase 14 + BsaI Reaction Time 15 + T4 Ligase ≤ 90 min 16 Reduced Volume Reduced From 5-Hour Less Enzyme & DNA Published Protocol 17 Transcriptional Unit (TU) 18 19 20 Destination Vectors 21 + Basic Part vector BsaI BbsI lacZ 22 GGTCTCNGAGGNNGTCTTC GAAGACNNACTANGAGACC ampicillin resistance lacZ CTTCTGNCTCCNNCAGAAG CTTCTGNNTGATNCTCTGG 23 BsaI BbsI Transcription Unit vector lacZ 24 kanamycin resistance BbsI 25 T4 Ligase Device vector 26 lacZ ampicillin resistance 27 28 Device or Circuit 29 30 31CIDAR Workflow 32 Test Share Specification Design Build 33 34 Programming Software tool for Collection of DNA part Software tool to Inventory of 35 language for creating DNA libraries based on convert arbitrary Composable 36 describing synthetic assembly plans in CIDAR MoClo fluorescent units to Elements (ICE), 37 biology devices common assembly assembly. Methyl Equivilant online database of 38 formats Fluorescein (MEFL) sequences and 39 physical units. characterization data. 40 41 TASBE 42 Tools Hummingbird 43 ACS Paragon Plus Environment 44 www.eugenecad.org www.ravencad.org www.cidarlab.org/moclo synbiotools.bbn.com www.cidar-ice.org 45 46

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