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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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-sterilize TES buffer and NaHCO3 with 0.2 µm filters.
    4. Prepare BG11 by combining 976 ml of water, 10 ml of 100x BG11, 1 ml of tra....

Representative Results

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 .......

Discussion

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.......

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
NaNO3SigmaS5506
MgSO4.7H2OSigma230391
CaCl2SigmaC1016
citric acidSigmaC0759
Na2EDTAFisherEDT002
H3BO3Sigma339067
MnCl2.4H2OSigmaM3634
ZnSO4.7H2OSigmaZ4750
Na2MoO4.2H2OSigma331058
CuSO4.5H2OSigma209198
Co(NO3)2.6H2OSigma239267
Ferric ammonium citrateSigmaF5879
K2HPO4SigmaP3786
Na2CO3FisherSODC001
TESSigmaT1375
NaHCO3FisherSODH001
HEPESSigmaH3375
cyanocobalaminSigma47869
Na2S2O3Sigma72049
Bacto agarBD214010
SucroseFisherSUC001
Petri dish 90 mm triple ventedGreiner633185
0.2 µm filtersSartorius16534
100 mL conical flasksPyrexCON004
Parafilm M 100 mm x38 mBemisFIL003
Phusion high fidelity DNA polymerase PhusionF-530
AgaroseMelfordMB1200
DNA purification kit MoBio12100-300
Restriction endonucleasesNEB
T4 ligaseThermo ScientificEL0011
Luria Bertani brothInvitrogen12795-027
MESSigmaM8250
Kanamycin sulfateSigma60615
AmpicillinSigmaA9518
GeneJET plasmid miniprep kitThermo ScientificK0503
14 mL round-bottom tubeBD falcon352059
GoTaq G2 Flexi DNA polymerasePromegaM7805
425-600 µm glass beadsSigmaG8772
GlycerolSigmaG5516
DMSOSigmaD8418
Fluorescent bulbsGro-Lux69
HT multitron photobioreactorInfors

References

  1. Zwirglmaier, K., et al. Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes. Environ Microbiol. 10, 147-161 (2008).
  2. Galloway, J. N., et al.

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CyanobacteriaSynechocystisSynechococcusUnmarked MutantsMarked MutantsAntibiotic ResistanceNegative Selectable MarkerGenetic ManipulationBG11 MediumTransformationKanamycinPCRKnockout

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