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Recursive DNA assembly using Protected Oligonucleotide Duplex Assisted Cloning (PODAC) Bob Van Hove, Chiara Guidi, Lien De Wannemaeker, Jo Maertens, and Marjan De Mey ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00017 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017
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Recursive DNA assembly using Protected Oligonucleotide Duplex Assisted Cloning (PODAC) Bob Van Hove, Chiara Guidi, Lien De Wannemaeker, Jo Maertens, and Marjan De Mey∗ Centre for Synthetic Biology (CSB), Department of Biochemical and Microbial Technology, Ghent University, Ghent, Belgium E-mail:
[email protected]
Abstract A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness towards sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.
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Keywords molecular cloning, DNA assembly, recursive cloning, pathway assembly, CRISPR, Golden Gate cloning
1
Introduction
Molecular cloning has come a long way since the creation of the first recombinant plasmids. The rapidly advancing field of synthetic biology has spurred the development of new DNA assembly techniques in order to meet increasing demands for speed, efficiency and accuracy(1 ). These methods can, with some exceptions, be classified into two broad categories: (1) assembly based on the action of restriction endonucleases and DNA ligase that cut and paste the desired fragments; (2) homology-directed methods that make use of DNA polymerase to join fragments by extending partially annealed regions (2 , 3 ). Homology-directed methods such as Gibson isothermal assembly impose few constraints on the genetic sequence of the designed plasmid, and therefore are very well suited to bespoke designs. However, reliance on PCR to introduce overlapping terminal regions compromises fidelity and limits the length of the parts to be assembled. Restriction based methods, on the other hand, require parts to be flanked by restriction sites that can lead to assembly scars in the final construct. Assembly scars may be problematic depending on sequence and location, but this can be avoided by using Type IIS restriction enzymes, which cleave outside of their recognition site. In addition, this allows for restriction and ligation to be performed simultaneously in a one-pot reaction. This property is exploited by the Golden Gate technique and its derivatives, increasing efficiency and significantly shortening the assembly workflow(4 ). Moreover, because the sequence of the cohesive ends can be specified in advance, it becomes possible to assemble multiple parts in a defined order. Besides ease of cloning, a criterion that is increasingly taken into account by the Synthetic Biology community is the capability to operate in a standardized framework(5 , 6 ). In 2
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this context, Storch et al. put forward the following six key concepts: “standard reusable parts; single-tier format . . . ; idempotent cloning; parallel (multipart) DNA assembly; size independence; automatability” (7 ). A standardized genetic assembly framework inherently implies standardized part junctions, and these should not just be seen as sequence scars but rather as linkers. While homology-directed techniques require linkers of 20-50 bps(8 , 9 ), most restriction-based methods require complementary ends of only 4 bps. An often overlooked criterium is the capacity for iterative cloning, i.e., how to introduce additional parts in the created construct without resorting to inverse PCR. The BioBrick™ platform, for example, adopts a four-enzyme approach whereby pairs of endonucleases recognise different sites that generate compatible cohesive ends. After ligation, non-cleavable scars are formed in-between parts while active cloning sites continue to flank the assembled construct (10 ). However, this is not a one-pot reaction and the choice of linker sequence is limited by the available restriction enzymes, each of which imposes an additional sequence constraint on the genetic part library. Golden Gate cloning can also be adapted to iterative cloning as exemplified by MoClo and GoldenBraid cloning (11 –13 ). Both methods implement a multi-level system that alternates between two different Type IIS restriction enzymes to enable further subcloning of assembled constructs. MoClo and derivatives such as the recently published EcoFlex(14 ) follow a hierarchical topology where vectors at each level serve a defined purpose. The cloning of basic parts from level-0 storage vectors into level-1 expression vectors is performed using one restriction enzyme. Afterwards, a second enzyme can be used to subclone the resulting level-1 constructs into a level-2 vector. Workflows of more than three levels are made possible by including a genetic part that contains a cloning site from the previous level. This idea of indefinite assembly expansion was further explored by the GoldenBraid methodology, which adopts a double loop topology of only two vector levels: alpha and omega. One restriction enzyme is used to combine two alpha vectors into one omega vector, subsequently two omega vectors can be subcloned into an alpha vector using the other restriction enzyme. As such, complex assemblies can be created
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by alternating back and forth between alpha and omega vectors (12 ). Although the aforementioned methods offer a form of iterative cloning, the use of multiple restriction enzymes imposes significant limitations on the design of the storage vectors and parts themselves. One could use PCR to introduce the required restriction sites or linker sequences ad hoc, but this becomes impractical for designs containing sequence repeats and can introduce unwanted mutations. Alternatively, overlapping linker sequences can be added using restriction and ligation, as is the case for the BASIC assembly method, for example (7 ). Still, this requires processing steps each time a part is to be used in a new design. In response, we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), the first one-pot recursive cloning strategy that only makes use of a single restriction enzyme to iteratively create complex assemblies. PODAC eschews multi-level vector designs in favor of universal donor vectors that can be reused at each iteration, simplifying the cloning workflow and enabling further standardisation of Golden Gate cloning. The versatility of the method is demonstrated using two case studies: the assembly of a heterologous biosynthetic pathway and the construction of artificial CRISPR guide RNA arrays.
2 2.1
Results and discussion Protected Oligonucleotide Duplex Assisted Cloning
PODAC is an extension upon Golden Gate cloning, and as such adheres to the same basic principle: by using a Type IIS endonuclease such as BsaI, which cleaves outside of a nonpalindromic recognition site, restriction and ligation can be performed simultaneously. The genetic parts to be joined can be supplied either in the form of plasmids or PCR products. In both cases they are designed to be flanked by two copies of the recognition site in opposite orientation, such that digestion detaches each site from the original part, leaving behind two cohesive ends of choice. Linear fragments with complementary cohesive ends are subsequently joined together by DNA ligase. The reaction mixture is gradually enriched for the desired 4
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ligation product because incorrect assemblies, e.g., reconstituted supply vectors, result in repeated digestion (4 ). From this stems the efficiency and accuracy of the Golden Gate method, but it also results in an inherent drawback of Golden Gate cloning, i.e., the created construct can no longer be used for further cloning using the same enzyme. PODAC aims to solve this predicament by including a pair of diverging BsaI sites as one of the parts to be assembled. A PODAC reaction involves three different cohesive ends (CE) of choice, which will be henceforth be referred to as CEX , CEY and CEZ . Digestion of an initial acceptor vector results in a backbone with cohesive ends CEX and CEZ (Figure 1.1). The part to be cloned, however, is supplied by a donor vector as an insert flanked by CEX and CEY (Figure 1.2). With this in mind, a synthetic oligonucleotide duplex is designed with two BsaI sites such that digestion of the duplex yields a product with cohesive ends CEY and CEZ , each carrying the 5'-phosphate required for efficient ligation. In addition, this synthetic duplex carries two protected internal BsaI sites each containing a 5-methyl deoxycytosine base modification (Figure 1.3). These are vital to the functioning of PODAC because they prevent the part itself from being cut during the Golden Gate reaction. Storch et al. demonstrated that methylation of either one of the cytosines in the GGTCTC recognition site confers protection from BsaI digestion in much the same way that organisms that naturally produce restriction enzymes protect their own genome via concomitant expression of a site-specific methyltransferase (7 ). By design, transformation into Escherichia coli activates the protected BsaI sites because the cytosine methylation pattern is not maintained during DNA replication. Moreover, the PODAC pattern is unaffected by the endogenous McrBC (modified cytosine restriction) system, which requires a distance of at least 30 bp between consecutive methylcytosine sites for digestion to take place (15 ). As shown in Figure 1.5, the resulting plasmid contains two active BsaI sites with the same cut sites CEX and CEZ as the original acceptor vector. It is this property that enables recursive cloning and the reuse of donor plasmids. Contrary to
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Figure 1: PODAC mechanism. An initial acceptor vector (1) receives a genetic part carried on a standardized donor plasmid (2i=1 ) and an oligonucleotide duplex (3) that contains two methylated BsaI sites in a one-pot reaction. The methylation pattern protects the BsaI sites from digestion during the assembly reaction and is removed upon replication in E. coli (4). This exposes a new cloning site on the assembled construct (5), which can accept another genetic part (2i=2 ) during another PODAC iteration (6). classical Golden Gate cloning, the order of the parts is not determined by the sequence of their cohesive ends, but by the order in which they are introduced.
2.2
Case study 1: iterative assembly of a biosynthetic pathway
As a first proof of principle for the PODAC technique, we assembled an operon encoding the biosynthesis pathway of the indole derivative violacein (16 ). The complete pathway consists of 5 enzymes (VioABCDE) and because consecutive reactions result in intermediates of various colours, sequential incorporation of vioC, vioD and vioE could be monitored visually based on the colour of the E. coli colonies (Figure 2d)(17 ). Initially, the acceptor vector contained a standard transcription and translation initiation element (i.e., a constitutive promoter and bicistronic ribosome binding site [RBS]) (18 ) upstream of the vioA and vioB coding sequences and a PODAC cloning site in the form of the aeBlue chromoprotein marker gene flanked by BsaI sites. To sequentially complete 6
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the violacein pathway, three donor plasmids carrying either vioC, vioD or vioE were constructed as well, with each coding sequence positioned downstream of a ribozyme insulator with synthetic RBS and surrounded by BsaI sites (19 , 20 ) (Figure 2b). First vioE was introduced, resulting in colonies that initially were colourless due to the excision of the aeBlue reporter, but turned greenish brown after prolonged incubation at lower temperatures due to accumulation of prodeoxyviolacein and its oxidation product deoxychromoviridans (21 ). The presence of vioE was confirmed by colony PCR and Sanger sequencing showed that the oligonucleotide duplex was inserted successfully. Next, vioC and vioD were each introduced separately, leading to the constructs vioABEC and vioABED, respectively. The vioABEC construct resulted in the synthesis of the purple pigment deoxyviolacein, colonies expressing vioABED on the other hand, turned a green colour due to the accumulation of oxidation products of proviolacein. Finally, the pathway was completed by a third cycle of PODAC, which resulted in vioABECD and vioABEDC, both of which gave rise to violet colonies. The corresponding genotype for each phenotype was confirmed by colony PCR and sequencing. An ethanolic extract of pelletised vioABEDC liquid cultures displayed the UV-vis absorption spectrum corresponding to that of violacein reported in the literature (Figure S1c) (22 ). Each PODAC cycle, the number of colonies obtained after transformation, as well as their phenotype, was recorded as an indication of assembly efficiency and accuracy (Table S1). To demonstrate that the oligonucleotide duplex was protected by the methylation pattern, each experiment was accompanied by a control reaction with an unprotected oligonucleotide duplex, all other conditions remaining the same. These controls did yield viable colonies, but at a much lower assembly efficiency and accuracy. We therefore concluded that the duplex methylation pattern indeed confers protection from digestion and demonstrated that PODAC can be used to efficiently assemble a biosynthetic pathway from donor vectors with identical restriction sites.
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Figure 2: Assembly of the violacein pathway using PODAC. (a) Initial acceptor vector containing the vioAB operon. (b) Donor vectors carry vioC /D/E preceded by a ribozyme insulator and ribosome binding site. (c) Various constructs created by iteratively combining donor with acceptor vectors. (d) These constructs result in colonies of various colours, confirming assembly success (order as in (c)). Regular Synthetic Biology Open Language (SBOL) symbols were used for all parts other than the PODAC cloning site (23 ).
2.3
Case study 2: cloning of constructs containing sequence repeats
Modern homology-based assembly techniques often fail on designs containing extensive repeated sequences (24 ). On that account, we applied PODAC to one such case: the construction of artificial CRISPR arrays for use with a catalytically dead Streptococcus pyogenes Cas9 variant (dCas9) that is incapable of cleaving DNA, but instead remains bound to its target, repressing transcription (25 ). In contrast to the CRISPathBrick cloning method(26 ), we made use of chimeric guide RNAs known as single guide RNAs (sgRNA) that do not require RNA processing. We targeted the malT, lacZ and araB genes of E. coli, which are essential to the catabolism of maltose, lactose and L-arabinose, respectively. To this end, three donor vectors were constructed, each carrying a single guide RNA with promoter and terminator (Figure 3a). Their sequence was identical, except for the 20 bps protospacers designed to target the promoter of malT, lacZ or araB. Using PODAC, we successfully created vari-
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ous sgRNA combinations and transformed these into a strain expressing dCas9. Successful disruption of the expression of these carbohydrate catabolic genes was visualised using a variant of MacConkey agar (27 ). Figure 3c-e demonstrates the various discolouration patterns observed after growth on three media, each with a different sugar. If cells are able to metabolize the sugar, medium acidification causes the pH indicator to turn pink and bile salts to precipitate. Whereas the absence of a pink halo indicates that the expression of the catabolic gene(s) in question is repressed and that the cells rely on peptides and free amino acids as carbon sources instead.
Figure 3: Construction of artificial CRISPR guide RNA arrays using PODAC. (a) Donor vectors with a sgRNA targetted towards malT, lacZ or araB. (b) Iterative cloning into an acceptor vector results in constructs containing 1 to 3 sgRNAs. (c) CRISPR/dCas9 repression (targets indicated on empty plate) visualized through medium acidification, or lack thereof. The absence of a pink halo indicates that catabolism of the added carbohydrate (shown above the plate) is inhibited. (d) Constructs containing two sgRNAs repress both targets (m, malT ; l,lacZ ; a,araB ). (e) Even when adding a third sgRNA, PODAC does not suffer from sequence repeats.
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Based on this visual assay we concluded that the resulting constructs indeed encode multiple sgRNAs. Colony PCR demonstrated that oligonucleotide protection greatly impacts assembly accuracy, as shown in Supporting Information, Figure S2. The unprotected control reactions resulted in constructs carying inserts of varying size, but only very rarely the desired sgRNA insert. Finally, Sanger sequencing was used to verify the exact sequence of the sgRNA arrays. Unlike natural S. pyogenes CRISPR arrays, each sgRNA is transcribed from its own promoter. This design necessitated the cloning of tandem repeats of 180 bps, which would preclude the use of molecular techniques that depend on PCR and sequence homology. PODAC, on the other hand, is not hindered by sequence repeats and was successfully used to assemble synthetic CRISPR arrays. Both case studies demonstrate that PODAC is a reliable and efficient method for the assembly of multi-part constructs. PODAC does introduce an 8 bps scar sequence in-between parts, i.e. the combined length of two cohesive ends. While this may be seen as a limitation compared to classical Golden Gate cloning, assembly scars are an inherent consequence of the trade-off between customizability and universal applicability. Moreover, the scar sequence can be chosen at the time of standardisation to minimize the potential biological impact. One limitation of PODAC is the fact that, by design, it sacrifices the speed of simultaneous multi-part assembly in favor of an iterative approach. It would be ill-suited for the construction of a TAL effector array, for example, as few of the construction intermediates would be useful by themselves. Similarly, the need to create donor plasmids can be a hassle for one-off cloning tasks where the part will not be reused again. In our laboratory, we use a modified PODAC variant that uses PCR fragments for such cases. As illustrated in Supporting Information, Figure S5b, using PCR fragments directly has the added benefit of enabling simultaneous multi-part assembly. However, this does come at the cost of greatly reduced reusability and an increased risk of mutations. Moreover, it is our opinion that the reuse of part donors and a single restriction enzyme, requiring neither PCR nor adapter ligation, is precisely that which makes PODAC a useful technique.
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In theory, other Type IIS endonucleases could be used as well if a suitable protection pattern is known. Methylation need not be the only way of protecting an oligonucleotide duplex. Photolabile backbone modifications, for example, might be used to create a photocaged duplex that is activated not by DNA replication but by irradiation at 365 nm (28 ). In comparison to techniques such as BASIC, the arrangement of genetic parts in a multipart PODAC construct is not determined by the sequence of single stranded overhangs, but by the order in which parts are cloned during individual iterations. Like PODAC, BASIC also avoids the use of PCR to create compatible linear fragments; but unlike PODAC, it requires ligation of specific phosphorylated oligonucleotides to create individual overhangs (7 ).
2.4
Conclusion
We have developed a new cloning method called PODAC that enables iterative Golden Gate cloning using a single restriction enzyme. New parts can easily be added to the resulting constructs without performing inverse PCR amplification of the entire plasmid, improving robustness towards sequence repeats and reducing the risk of PCR-derived mutations. To this end, PODAC makes use of a single synthetic oligonucleotide duplex to introduce a future cloning site together with the cloned part. During assembly, methylated bases confer protection to the duplex against digestion by BsaI; subsequent replication of the assembled construct in E. coli removes this methylation pattern, resulting in the formation of a new BsaI cloning site identical in sequence to the original cloning site. This recursive cloning workflow maximises construct reuse and, in contrast to techniques such as MoClo and GoldenBraid, is constrained by only one ‘forbidden sequence’, i.e. the BsaI recognition site. A first case study demonstrated that PODAC can be used to efficiently assemble metabolic pathways starting from standardized storage vectors without first having to introduce homology regions through PCR or ligation of adapter sequences. Three donor vectors, each containing a different gene but identical in other respects, were used to iteratively complete 11
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the violacein biosynthesis pathway. A second case study highlighted the robustness of PODAC towards sequence repeats, which often complicate the use of homology-based assembly techniques. We created artificial CRISPR arrays that contain sequence repeats of 180 bps and used these to repress expression of three catabolic operons in E. coli. While other Golden Gate based techniques have been used to create sgRNA arrays, these make use of two restriction enzymes rather than one as is the case for PODAC (29 ). Both case studies confirmed that PODAC is a reliable and efficient method for the assembly of multi-part constructs and we think these characteristics make it a valuable addition to the synthetic biologist’s toolbox.
3 3.1
Methods Chemicals, oligonucleotides and molecular reagents
Tryptone and yeast extract were procured from Becton Dickinson (Erembodegem, Belgium). All other chemicals were purchased from Sigma-Aldrich (Diegem, Belgium), unless mentioned otherwise. Oligonucleotides were purchased from Integrated DNA Technologies (Leuven, Belgium). Restriction enzymes, T4 DNA ligase, Bovine Serum Albumin, Taq and Q5® DNA polymerase were purchased from New England BioLabs (Ipswich, USA), PrimeSTAR HS DNA polymerase was supplied by TaKaRa Bio (Saint-Germain-en-Laye, France). Deoxynucleotides, agarose and ethidium bromide were purchased from Thermo Fisher Scientific (Erembodegem, Belgium). Analytik Jena kits (Jena, Germany) were used for all DNA preparations.
3.2
Plasmids and genes
Intermediary plasmids used in this study were constructed using a combination of circular polymerase extension cloning (CPEC) and Golden Gate cloning (3 , 4 ). DNA sequences of 12
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the resulting plasmids are given in Supporting Information. For the violacein case study, a part storage vector pD-riboJ_blue was built using a high-copy plasmid from a previous study (30 ) and the BBa_K1073020 BioBrick part (31 ). This storage vector was then used to create donor plasmids pD-riboJ-vioC, pD-riboJ-vioD and pD-riboJ-vioE. The initial acceptor vector pA-vioAB_blue was constructed using parts from pSC101-mutP3-BCD9-RFP-neo, which was sourced from our lab and contains a BIOFAB expression operating unit (18 ). The Chromobacterium violaceum genes vioA, vioB, vioD and vioE were obtained codon optimised for E. coli from BBa_K598019 (31 ). The Pseudoalteromonas luteoviolacea gene vioC was isolated from pETM6-E12-vioABECD, which was a gift from Mattheos Koffas (Addgene plasmid # 66537) (32 ). For the CRISPR case study, three individual donor plasmids were constructed using the single strand assembly method (SSA)(30 ) with pD-sgRNA_blue as template, which itself was derived from pgRNA-bacteria (25 ). The acceptor vector pA-sgRNA_blue and the dCas9 expression plasmid pBR322-kan-dCas9 were built from parts of pdCas9 (25 ) and pBR322 (New England Biolabs). Both pgRNA-bacteria and pdCas9 were gifts from Stanley Qi (Addgene plasmid # 44251 and 44249, respectively).
3.3
PODAC
Two oligonucleotides were designed to form a duplex containing 4 BsaI sites of which two are inactivated by a 5-methyl-cytosine base modification as demonstrated in Figure 1 and listed in Supporting Information. The cohesive ends CEX (TCCG), CEY (CATC) and CEZ (CAAT) were designed to the specifications by Engler et al. (4 ) and spacer sequences of 5 bps were added to the edges of the duplex and in-between the internal BsaI sites. These spacers were designed using the NUPACK web application in order to promote correct hybridisation of the oligonucleotides by suppressing the formation of internal secondary structures and increasing the melting temperature of the duplex (33 ). Dried oligonucleotides were individually dissolved to a concentration of 100 µM in Duplex Buffer (100 mM Potassium 13
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Acetate, 30 mM HEPES, pH 7.5), as per manufacturer’s instructions. 15 µL aliquots of each oligonucleotide solution were mixed and incubated in a thermocycler for 2 min at 94 °C, followed by a gradual cooling down to room temperature over 30 to 45 min. For practical reasons, the solution was then diluted 100-fold in Duplex Buffer to a concentration of 500 nM hybridised duplex. This solution can be frozen and thawed repeatedly without an apparent loss of efficiency. The cloning of a part from a donor vector into an acceptor, together with the protected oligonucleotide duplex, entails a normal Golden Gate assembly reaction and as such is subject to the user’s preference. The reaction mixture used in this work comprises: 100 ng of acceptor plasmid, an equimolar amount of donor plasmid, a 5-fold molar excess of duplex solution, 1.5 µL of T4 DNA ligase buffer, 0.15 µL of Bovine Serum Albumin (10 mg/µL stock), 0.75 µL of BsaI-HF® (15 units), 0.75 µL of T4 DNA ligase (300 units), and (nuclease-free) water to a total volume of 15 µL. Reaction mixes were incubated under the following conditions: 50 cycles of 2 min at 37 °C and 3 min at 16 °C, followed by 10 min at 50 °C and 10 min at 80 °C.
3.4
Strains
E. coli Subcloning Efficiency™ DH5α™ chemically competent cells (Thermo Fisher Scientific) were used during the construction of various intermediary plasmids. For the violacein case study, 1 µL of each PODAC reaction mixture was used to transform 25 µL aliquots of TransforMax™ EC100™ electrocompetent E. coli (Epicentre, Wisconsin, USA) as per manufacturer’s instructions. The strain used for the CRISPR case study is E. coli MDS™ 42 LowMut ΔrecA (Scarab Genomics, Wisconsin, USA) carrying the plasmid pdCas9_2.1. This strain was transformed using the protocol described by Warren(34 ).
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3.5
Media and culture conditions
Lysogeny Broth (LB), consisting of 1% tryptone, 0.5% yeast extract and 0.5% sodium chloride, was used during cloning work. The composition of solid culture medium, Lysogeny Agar (LA), was identical, except for the addition of 12 g/L agar. If required, media were supplemented with the antibiotics ampicillin (100 µg/mL), kanamycin (50 µg/mL) or chloramphenicol (34 µg/mL). For the violacein case study, pigment production was greatest when cultures were first incubated overnight at 30 °C, followed by 24 h at 4 °C. During other experiments, cultures were incubated at 37 °C. Liquid cultures were agitated at 200 rpm for all experiments. The indicator media used during the CRISPR study consisted of 2% peptone, 0.5% sodium chloride, 1.5 g/L bile salts, 30 mg/L neutral red. This broth was set to pH 7.1 using HCl or NaOH and 12 g/L agar was added. After autoclaving, antibiotics and the inducer anhydrotetracycline (100 nM) were added, as well as a sterile solution of maltose, lactose or L-arabinose (10 g/L). Single colonies were transferred from LB agar and streaked onto three indicator plates. Medium discolouration and bile salt precipitation was readily visible after 9 h incubation at 37 °C. Prolonged incubation is detrimental to the assay as it results in gradual acidification of the culture medium, irrespective of dCas9 mediated transcriptional repression.
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Author contributions
B.V.H. conceived the PODAC strategy, designed the experiments and wrote the manuscript. C.G. built the constructs used during the violacein case study, L.D.W performed molecular work for the CRISPR case study and optimized the visual assay. J.M. and M.D.M. supervised the work and J.M. edited the manuscript. All authors report no conflict of interest.
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Acknowledgement The authors thank the Fund for Scientific Research-Flanders (FWO) for support in the form of the projects G.0321.13N and a PhD grant for the first author, as well as the Ghent University Special Research Fund (BOF) for the IOP project BOF16/IOP/040.
Supporting Information Available • Supporting_information.pdf :(Figure S1) Biosynthesis and properties of violacein and intermediates; (Figure S2) colony PCR results of sgRNA array assembly; (Figure S3) oligonucleotide duplex design; (Figure S4) SSA strategy used to construct pD-sgRNA plasmids; (Figure S5) PODAC system variants: subcloning mode using a second enzyme and fast mode using PCR fragments; (Table S1) efficiency and accuracy of pathway assembly case study. • Oligonucleotides.csv: (Table S2) Oligonucleotide duplex sequences; (Table S3) Primer sequences. • Plasmid_maps.zip: annotated sequence files of the plasmids described in this publication. This material is available free of charge via the Internet at http://pubs.acs.org/.
References 1. Ellis, T., Adie, T., and Baldwin, G. S. (2011) DNA assembly for synthetic biology: from parts to pathways and beyond. Integrative Biology 3, 109–10. 2. Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345. 16
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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 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Synthetic Biology
3. Quan, J., and Tian, J. (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4, e6441. 4. Engler, C., Kandzia, R., and Marillonnet, S. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3, 3647. 5. Canton, B., Labno, A., and Endy, D. (2008) Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26, 787–793. 6. Casini, A., Storch, M., Baldwin, G. S., and Ellis, T. (2015) Bricks and blueprints: methods and standards for DNA assembly. Nature Reviews Molecular Cell Biology 16, 568– 576. 7. 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. 4, 781–787. 8. Casini, A., MacDonald, J. T., Jonghe, J. D., Christodoulou, G., Freemont, P. S., Baldwin, G. S., and Ellis, T. (2013) One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy. Nucleic Acids Research 42, e7–e7. 9. Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., and Silver, P. A. (2013) Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research 42, 681–689. 10. Shetty, R. P., Endy, D., and Knight, T. F. (2008) Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering 2, 5–12. 11. 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. 17
ACS Paragon Plus Environment
ACS Synthetic Biology
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 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
12. Sarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del Carmen, A., Granell, A., and Orzaez, D. (2011) GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE 6, e21622. 13. Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., and Orzaez, D. (2013) GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY 162, 1618–1631. 14. Moore, S. J., Lai, H.-E., Kelwick, R., Chee, S. M., Bell, D., Polizzi, K. M., and Freemont, P. S. (2016) EcoFlex - A Multifunctional MoClo Kit for E. coli Synthetic Biology. ACS Synthetic Biology 15. Pieper, U., Groll, D. H., Wünsch, S., Gast, F.-U., Speck, C., Mücke, N., and Pingoud, A. (2002) The GTP-Dependent Restriction Enzyme McrBC from Escherichia coli Forms High-Molecular Mass Complexes with DNA and Produces a Cleavage Pattern with a Characteristic 10-Base Pair Repeat. Biochemistry 41, 5245–5254. 16. Durán, N., Justo, G. Z., Ferreira, C. V., Melo, P. S., Cordi, L., and Martins, D. (2007) Violacein: properties and biological activities. Biotechnology and Applied Biochemistry 48, 127–7. 17. Colloms, S. D., Merrick, C. A., Olorunniji, F. J., Stark, W. M., Smith, M. C. M., Osbourn, A., Keasling, J. D., and Rosser, S. J. (2014) Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Research 42, e23–e23. 18. 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 1–15. 18
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Page 18 of 21
Page 19 of 21
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 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Synthetic Biology
19. Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., and Voigt, C. A. (2012) Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology 30, 1137–1142. 20. Borujeni, A. E., Channarasappa, A. S., and Salis, H. M. (2014) Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Research 42, 2646–2659. 21. Balibar, C. J., and Walsh, C. T. (2006) In Vitro Biosynthesis of Violacein from lTryptophan by the Enzymes VioA−E from Chromobacterium violaceum. Biochemistry 45, 15444–15457. 22. Sánchez, C., Braña, A. F., Méndez, C., and Salas, J. A. (2006) Reevaluation of the Violacein Biosynthetic Pathway and its Relationship to Indolocarbazole Biosynthesis. ChemBioChem 7, 1231–1240. 23. Galdzicki, M. et al. (2014) The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology. Nature Biotechnology 32, 545–550. 24. Hommelsheim, C. M., Frantzeskakis, L., Huang, M., and Ülker, B. (2014) PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications. Scientific reports 4, 1–13. 25. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., and Lim, W. A. (2013) Repurposing CRISPR as an RNA-Guided Platform for SequenceSpecific Control of Gene Expression. Cell 152, 1173–1183. 26. Cress, B. F., Toparlak, Ö. D., Guleria, S., Lebovich, M., Stieglitz, J. T., Englaender, J. A., Jones, J. A., Linhardt, R. J., and Koffas, M. A. G. (2015) CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. 4, 987–1000. 19
ACS Paragon Plus Environment
ACS Synthetic Biology
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 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
27. Macconkey, A. (1905) Lactose-Fermenting Bacteria in Faeces. The Journal of Hygiene 5, 333–379. 28. Wang, X., Feng, M., Xiao, L., Tong, A., and Xiang, Y. (2015) Postsynthetic Modification of DNA Phosphodiester Backbone for Photocaged DNAzyme. ACS Chemical Biology acschembio.5b00867–8. 29. Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernández-del Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., and Orzaez, D. (2016) A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods 1– 12. 30. Coussement, P., Maertens, J., Beauprez, J., Van Bellegem, W., and De Mey, M. (2014) One step DNA assembly for combinatorial metabolic engineering. Metabolic Engineering 23, 70–77. 31. Knight, T. F. Registry of Standard Biological Parts. 2003; http://www.partsregistry. org. 32. Jones, J. A., Vernacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., Schultz, V. L., Cress, B., Linhardt, R. J., and Koffas, M. A. G. (2015) ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Scientific reports 5, 11301. 33. Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., Dirks, R. M., and Pierce, N. A. (2010) NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry 32, 170–173. 34. Warren, D. J. (2011) Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation. Analytical Biochemistry 413, 206– 207.
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