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

Summary

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.

Abstract

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.

Introduction

Cyanobacteria are autotrophic bacteria that can be used for the biosynthesis of a wide variety of natural and heterologous high value metabolic products^1,2,3,4,5,6. 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 syn....

Protocol

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 systems^12,15.

1. 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). Alternatively, sequences can be amplified from a source template (e.g., a vector or purified genomic DNA). Here, how to generate a new Level 0 part f....

Results

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 P_cpc560 (pC0.005), the coding sequence for eYFP (pC0.008) and the double terminator T_rrnB (pC0.082)¹². Following transformation of the assembly reaction, successful assemblies were identified using.......

Discussion

Golden Gate assembly has several advantages compared to other vector assembly methods, particularly in terms of scalability^20,21. 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.......

Materials

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