Name: genetic modification of cyanobacteria by conjugation using the cyanogate modular cloning toolkit
Uploaded: 2019-10-31T15:00:00
Description: Watch this Scientific Journal Video about Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit at JoVE.com

The authors are grateful to the PHYCONET Biotechnology and Biological Sciences Research Council (BBSRC) Network in Industrial Biotechnology and Bioenergy (NIBB) and the Industrial Biotechnology Innovation Centre (IBioIC) for financial support. GARG, AASO and AP acknowledge funding support from the BBSRC EASTBIO CASE PhD program (grant number BB/M010996/1), the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACYT) PhD program, and the IBioIC-BBSRC Collaborative Training Partnership (CTP) PhD program, respectively. We thank Conrad Mullineaux (Queen Mary University of London), and Poul Eric Jensen and Julie Annemarie Zita Zedler (University of Copenhagen) for plasmid vector and protocol contributions and advice.

1. Vector assembly using the Plant MoClo and CyanoGate toolkits
NOTE: Before proceeding with vector assembly, it is strongly recommended that users familiarize themselves with the vector level structures of the Plant and CyanoGate MoClo systems12,15.
<ol>
	<li>Construction of Level 0 parts 
	NOTE: Level 0 parts can be synthesized as complete vectors or as linear sequences for assembly with Level 0 acceptors (e.g., gBlocks, IDT)....

Golden Gate assembly has several advantages compared to other vector assembly methods, particularly in terms of scalability20,21. Nevertheless, setting up the Golden Gate system in a lab requires time to develop a familiarity with the various parts and acceptor vector libraries and overall assembly processes. Careful planning is often needed for more complex assemblies or when performing a large number of complex assemblies in parallel (e.g., making a suite of Le...

Cyanobacteria are autotrophic bacteria that can be used for the biosynthesis of a wide variety of natural and heterologous high value metabolic products1,2,3,4,5,6. Several hurdles still need to be overcome to expand their commercial viability, most notably, the relatively poor yields compared to heterotrophic bio-platforms (e.g., Escherichia coli and yeast)7. The recent expansion of available genetic engineering tools and uptake of the synthetic biology paradigm in cyanobacterial research is helping to overcome such challenges and further develop cyanobacteria as efficient biofactories8,9,10.
The main approaches for introducing DNA into cyanobacteria are transformation, conjugation and electroporation. The vectors transferred to cyanobacteria by transformation or electroporation are &#34;suicide&#34; vectors (i.e., integrative vectors that facilitate homologous recombination), while self-replicating vectors can be transferred to cyanobacteria by transformation, conjugation or electroporation. For the former, a protocol is available for engineering model species amenable to natural transformation11. More recently, a modular cloning (MoClo) toolkit for cyanobacteria called CyanoGate has been developed that employs a standardized Golden Gate vector assembly method for engineering using natural transformation, electroporation or conjugation12.
Golden Gate-type assembly techniques have become increasingly popular in recent years, and assembly standards and part libraries are now available for a variety of organisms13,14,15,16,17. Golden Gate uses type IIS restriction enzymes (e.g., BsaI, BpiI, BsmBI, BtgZI and AarI) and a suit of acceptors and unique overhangs to facilitate directional hierarchical assembly of multiple sequences in a &#34;one pot&#34; assembly reaction. Type IIS restriction enzymes recognize a unique asymmetric sequence and cut a defined distance from their recognition sites to generate a staggered, &#34;sticky end&#34; cut (typically a 4 nucleotide [NT] overhang), which can be subsequently exploited to drive ordered DNA assembly reactions15,18. This has facilitated the development of large libraries of modular Level 0 parts (e.g., promoters, open reading frames and terminators) defined by a common syntax, such as the PhytoBricks standard19. Level 0 parts can then be readily assembled into Level 1 expression cassettes, following which more complex higher order assemblies (e.g., multigene expression constructs) can be built in an acceptor vector of choice12,15. A key advantage of Golden Gate-type assembly techniques is their amenability to automation at high-throughput facilities, such as DNA foundries20,21, which can allow for the testing of complex experimental designs that cannot easily be achieved by manual labor.
CyanoGate builds on the established Plant MoClo system12,15. To incorporate a new part into CyanoGate, the part sequence must first be domesticated, i.e., &#34;illegal&#34; recognition sites for BsaI and BpiI must be removed. In the case of a part coding for an open reading frame (i.e., a coding sequence, CDS), recognition sites can be disrupted by generating synonymous mutations in the sequence (i.e., changing a codon to an alternative that encodes for the same amino acid residue). This can be achieved by a variety of approaches, ranging from DNA synthesis to polymerase chain reaction (PCR) amplification-based strategies such as Gibson assembly22. Depending on the expression host being used, care should be taken to avoid the introduction of rare codons that could inhibit the efficiency of translation23. Removing recognition sites in promoter and terminator sequences is typically a riskier endeavor, as modifications may affect function and the part might not perform as expected. For example, changes to putative transcription factor binding sites or the ribosome binding site within a promoter could alter strength and responsiveness to induction/repression. Likewise, modifications to key terminator structural features (e.g., the GC rich stem, loop and poly-U tail) may change termination efficiency and effect gene expression24,25. Although several online resources are available to predict the activity of promoter and terminator sequences, and inform whether a proposed mutation will impact performance26,27, these tools are often poor predictors of performance in cyanobacteria28,29,30.. As such, in vivo characterization of modified parts is still recommended to confirm activity. To assist with the cloning of recalcitrant sequences, CyanoGate includes a low copy cloning acceptor vector based on the BioBrick vector pSB4K512,16,31. Furthermore, a &#34;Design and Build&#34; portal is available through the Edinburgh Genome Foundry to help with vector design (dab.genomefoundry.org). Lastly, and most importantly, CyanoGate includes two Level T acceptor vector designs (equivalent to Level 2 acceptor vectors)15 for introducing DNA into cyanobacteria using suicide vectors, or broad host-range vectors capable of self-replication in several cyanobacterial species32,33,34.
Here we will focus on describing a protocol for generating Level T self-replicating vectors and the genetic modification of Synechocystis PCC 6803 and Synechococcus elongatus UTEX 2973 (Synechocystis PCC 6803 and S. elongatus UTEX 2973 hereafter) by conjugation (also known as tri-parental mating). Conjugal transfer of DNA between bacterial cells is a well described process and has been previously used for engineering cyanobacterial species, in particular those that are not naturally competent, such as S. elongatus UTEX 297335,36,37,38,39,40,41. In brief, cyanobacterial cultures are incubated with an E. coli strain carrying the vector to be transferred (the &#34;cargo&#34; vector) and vectors (either in the same E. coli strain or in additional strains) to enable conjugation (&#34;mobilizer&#34; and &#34;helper&#34; vectors). Four key conditions are required for conjugal transfer to occur: 1) direct contact between cells involved in DNA transfer, 2) the cargo vector must be compatible with the conjugation system (i.e., it must contain a suitable origin of transfer (oriT), also known as a bom (basis of mobility) site), 3) a DNA nicking protein (e.g., encoded by the mob gene) that nicks DNA at the oriT to initiate single-stranded transfer of the DNA into the cyanobacterium must be present and expressed from either the cargo or helper vectors, and 4) the transferred DNA must not be destroyed in the recipient cyanobacterium (i.e., must be resistant to degradation by, for example, restriction endonuclease activity)35,42. For the cargo vector to persist, the origin of replication must be compatible with the recipient cyanobacterium to allow for self-replication and proliferation into daughter cells post division. To aid with conditions 3 and 4, several helper vectors are available through Addgene and other commercial sources that encode for mob as well as several methylases to protect from native endonucleases in the host cyanobacterium43. In this protocol, conjugation was facilitated by an MC1061 E. coli strain carrying mobilizer and helper vectors pRK24 (www.addgeneorg/51950) and pRL528 (www.addgene.org/58495), respectively. Care must be taken when choosing the vectors to be used for conjugal transfer. For example, in the CyanoGate kit the self-replicating cargo vector pPMQAK1-T encodes for a Mob protein12. However, pSEVA421-T does not44, and as such, mob must be expressed from a suitable helper vector. The vectors used should also be appropriate to the target organism. For example, efficient conjugal transfer in Anabaena sp. PCC 7120 requires a helper vector that protects the mobilizer vector against digestion (e.g., pRL623, which encodes for the three methylases AvaiM, Eco47iiM and Ecot22iM)45,46.
In this protocol we further outline how to characterize the performance of parts (i.e., promoters) with a fluorescent marker using a plate reader or a flow cytometer. Flow cytometers are able to measure fluorescence on a single cell basis for a large population. Furthermore, flow cytometers allow users to &#34;gate&#34; the acquired data and remove background noise (e.g., from particulate matter in the culture or contamination). In contrast, plate readers acquire an aggregate fluorescence measurement of a given volume of culture, typically in several replicate wells. Key advantages of plate readers over cytometers include the lower cost, higher availability and typically no requirement for specialist software for downstream data analyses. The main drawbacks of plate readers are the relatively lower sensitivity compared to cytometers and potential issues with the optical density of measured cultures. For comparative analyses, plate reader samples must be normalised for each well (e.g., to a measurement of culture density, typically taken as the absorbance at the optical density at 750 nm [OD750]), which can lead to inaccuracies for samples that are too dense and/or not well mixed (e.g., when prone to aggregation or flocculation).
As an overview, here we demonstrate in detail the principles of generating Level 0 parts, followed by hierarchical assembly using the CyanoGate kit and cloning into a vector suitable for conjugal transfer. We then demonstrate the conjugal transfer process, selection of axenic transconjugant strains expressing a fluorescent marker, and subsequent acquisition of fluorescence data using a flow cytometer or a plate reader.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0404</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate (ATP) disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>Agar (microbiology tested)</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-500g</td>
			<td>Used in 8.3.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41026</td>
			<td>Used in 6.</td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
			<td>Used in 4.3.1.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>BpiI (BbsI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER1011</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>BsaI (Eco31I)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER0291</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium</td>
			<td>VWR International</td>
			<td>A1491.0005</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated flat-bottom 24 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td>Used in 4.1.3.</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Used in 3.6.2.</td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td>DNA extraction kit. Used in 5.</td>
		</tr>
		<tr>
			<td>FLUOstar Omega Microplate reader</td>
			<td>BMG Labtech</td>
			<td>-</td>
			<td>Used in 4.2.2.</td>
		</tr>
		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K0503</td>
			<td>Plasmid purification kit. Used in step 2.3.2.</td>
		</tr>
		<tr>
			<td>Glass beads (0.5 mm diameter)</td>
			<td>BioSpec Products</td>
			<td>11079105</td>
			<td>Used in 5.2.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>10021083</td>
			<td>Used in 2.3.1, 3.6.2.</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10356553</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11815-024</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Membrane filters (0.45 &#956;m)</td>
			<td>MF-Millipore</td>
			<td>HAWP02500</td>
			<td>Used in 3.3.7</td>
		</tr>
		<tr>
			<td>Microplates, 96-well, flat-bottom (Chimney Well) &#181;CLEAR</td>
			<td>Greiner Bio-One</td>
			<td>655096</td>
			<td>Used in 4.2.1.</td>
		</tr>
		<tr>
			<td>Monarch DNA Gel Extraction Kit</td>
			<td>New England Biolabs</td>
			<td>T1020S</td>
			<td>Used in 1.1.2.2.</td>
		</tr>
		<tr>
			<td>Monarch PCR DNA Cleanup Kit</td>
			<td>New England Biolabs</td>
			<td>T1030</td>
			<td>DNA purification kit. Used in 1.1.2.3.</td>
		</tr>
		<tr>
			<td>Multitron Pro incubator with LEDs</td>
			<td>Infors HT</td>
			<td>-</td>
			<td>Shaking incubator with white LED lights. Used in 4.1.4.</td>
		</tr>
		<tr>
			<td>MyTaq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21108</td>
			<td>Used in 7.1.</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td>Used in 2.3.3.</td>
		</tr>
		<tr>
			<td>One Shot TOP10 chemically competent E. coli</td>
			<td>Thermo Fisher Scientific</td>
			<td>C404010</td>
			<td>Used in 2.1.1.</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS) solution (10X concentrate)</td>
			<td>VWR International</td>
			<td>K813</td>
			<td>Used in 4.3.2.</td>
		</tr>
		<tr>
			<td>Q5High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0491S</td>
			<td>Used in 1.1.2.1.</td>
		</tr>
		<tr>
			<td>Quick-Load 1 kb DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N0468S</td>
			<td>Used in Figure 4.</td>
		</tr>
		<tr>
			<td>Screw-cap tubes (1.5 ml)</td>
			<td>Starstedt</td>
			<td>72.692.210</td>
			<td>Used in 3.6.3</td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>VWR International</td>
			<td>J61820.06</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Sterilin Clear Microtiter round-bottom 96-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>612U96</td>
			<td>Used in 4.3.1.</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0011</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>Bead mill. Used in 5.2.</td>
		</tr>
	</tbody>
</table>

genetic modification of cyanobacteria by conjugation using the cyanogate modular cloning toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

To demonstrate the Golden Gate assembly workflow, an expression cassette was assembled in the Level 1 position 1 (Forward) acceptor vector (pICH47732) containing the following Level 0 parts: the promoter of the C-phycocyanin operon Pcpc560 (pC0.005), the coding sequence for eYFP (pC0.008) and the double terminator TrrnB (pC0.082)12. Following transformation of the assembly reaction, successful assemblies were identified using...

Watch this Scientific Journal Video about Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit at JoVE.com

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

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