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Biology

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Published: May 29th, 2016

DOI:

10.3791/54001

1Department of Biochemistry, University of Cambridge

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Cyanobacteria are an evolutionarily ancient and diverse phylum of bacteria found in nearly every natural environment on Earth. In marine ecosystems they are particularly abundant and play a key role in many nutrient cycles, accounting for approximately half of carbon fixation1, the majority of nitrogen fixation2 and hundreds of millions of tons of hydrocarbon production3 in the oceans annually. Chloroplasts, the organelle responsible for photosynthesis in eukaryotic algae and plants, are likely to have evolved from a cyanobacterium that was engulfed by a host organism4. Cyanobacteria have proved useful model organisms for th....

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1. Preparation of Culture Media

  1. Prepare BG11 medium according to Castenholz, 198817.
    1. Prepare stock solutions of 100x BG11, trace elements and iron stock (Table 1).
    2. Prepare separate solutions of phosphate stock, Na2CO3 stock, N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) buffer and NaHCO3 (Table 1).
    3. Autoclave the phosphate and Na2CO3 stocks. Filter-ster.......

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Plasmid design is critical for successful generation of both marked and unmarked mutants. Figure 1 gives an example of plasmid A and B used to generate a deletion mutant in the Synechocystis genes cpcC1 and cpcC213. In each case the 5' and 3' flanking regions are approximately 900-1,000 bp. Reduced flanking regions can be used although the smallest we have successfully trialed has been approximately 500 bp. Plasmid B can also .......

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The most critical steps in generation of unmarked mutants are: 1) careful plasmid design to ensure only the targeted region is altered; 2) ensuring that samples remain axenic, especially when cultured on sucrose; 3) plating transformed cells for marked mutant generation initially on BG11 agar plates lacking antibiotics, followed by addition of agar plus antibiotics 24 hr later; 4) culturing marked mutants for 4 full days prior to plating on BG11 plus sucrose agar plates: 5) ensuring that marked mutants are fully segregat.......

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We are grateful to the Environmental Services Association Education Trust, the Synthetic Biology in Cambridge SynBio fund and the Ministry of Social Justice and Empowerment, Government of India, for financial support.

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Name Company Catalog Number Comments
NaNO3 Sigma S5506
MgSO4.7H2O Sigma 230391
CaCl2 Sigma C1016
citric acid Sigma C0759
Na2EDTA Fisher EDT002
H3BO3 Sigma 339067
MnCl2.4H2O Sigma M3634
ZnSO4.7H2O Sigma Z4750
Na2MoO4.2H2O Sigma 331058
CuSO4.5H2O Sigma 209198
Co(NO3)2.6H2O Sigma 239267
Ferric ammonium citrate Sigma F5879
K2HPO4 Sigma P3786
Na2CO3 Fisher SODC001
TES Sigma T1375
NaHCO3 Fisher SODH001
HEPES Sigma H3375
cyanocobalamin Sigma 47869
Na2S2O3 Sigma 72049
Bacto agar BD 214010
Sucrose Fisher SUC001
Petri dish 90 mm triple vented Greiner 633185
0.2 µm filters Sartorius 16534
100 mL conical flasks Pyrex CON004
Parafilm M 100 mm x38 m Bemis FIL003
Phusion high fidelity DNA polymerase  Phusion F-530
Agarose Melford MB1200
DNA purification kit  MoBio 12100-300
Restriction endonucleases NEB
T4 ligase Thermo Scientific EL0011
Luria Bertani broth Invitrogen 12795-027
MES Sigma M8250
Kanamycin sulfate Sigma 60615
Ampicillin Sigma A9518
GeneJET plasmid miniprep kit Thermo Scientific K0503
14 mL round-bottom tube BD falcon 352059
GoTaq G2 Flexi DNA polymerase Promega M7805
425-600 µm glass beads Sigma G8772
Glycerol Sigma G5516
DMSO Sigma D8418
Fluorescent bulbs Gro-Lux 69
HT multitron photobioreactor Infors

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