Synechocystis PCC6803

Introduction

Synechocystis PCC6803 is a freshwater cyanobacterial species. It is a model organism for photosynthetic bacteria. It is capable of both autotrophic (light-dependent) and heterotrophic (organic carbon source-dependent) lifestyles.

Why?

We chose Synechocystis for the same reason it is so difficult to work with in the lab – it has a 1-2 day doubling time. This means it is unlikely to overwhelm any mammalian cell it is inhabiting. Additionally, it has been previously shown [1] to survive stably inside the cytoplasmic environment. We were also inspired by the sci-fi connotations of having photosynthesis-capable organisms living in our cells – could we eventually create human chloroplasts and solve the world’s food shortage? Finally, the strain we used did not produce any toxic secondary metabolites and was completely non-pathogenic. Synechocystis has actually been shown to be part of the healthy human microbiome .

Design:

• We decided to use the same invasion device used for E. coli (i.e. invasion and listeriolysin), as the unmodified genes were previously used and seemed to work for mammalian cell invasion [1]. Therefore, the bulk of our work was in designing a biobrick compatible integrative plasmid.
• Using the open source plasmid registry Addgene, we were able to source previously tested integrative plasmids, psbA2-PHLS (b) and cpc-PHLS (g) (Addgene accession numbers: 52307 and 52311, respectively) [2].
• Using long primers containing annealing regions and tails for extension containing biobrick prefixes and suffixes, we were able to tailor these plasmids for use with Biobrick standards. The plasmids were originally designed to test PHLS (beta-phellandrene) under endogenous promoters in Synechocystis. Our modified version of the plasmids allow integration of any Biobrick compatible gene construct, assembled through 3A assembly.
• The two 500 bp recombination sites with sequence homology enable double recombination, and allow the gene of interest into the chromosome.
• The plasmids carry an ampicillin resistance gene, while the inserted gene must also carry a chloramphenicol resistance gene (such as chloramphenical acyl transferase). Ampicillin is used for selection during cloning of plasmid in E. coli, while chloramphenicol is used for selection during integrative transformation of Synechocystis.
• The terminator following the first recombination site prevents read-through translation of the protein the insert gene is interrupting.

Challenges

Despite being a model organism, there is a lack of well-characterised genetic parts for use in Synechocystis. It is very different functionally and phylogenetically to E. coli, and standard parts often do not function in Synechocystis. For example, there are currently no effective inducible promoters, and no effective terminators. This made it challenging to effectively implement our biosafety strategy. Next, plasmids do not exist stably inside Synechocystis cells - transformants are created through use of integrative plasmids. These homologously recombine into the chromosomes. Synechocystis has 12 chromosomes, therefore gradually increasing antibiotic selection pressure is required to ensure integration into all chromosome copies. In the lab, the 2-4 week transformation time (due to slow doubling and gradual application of antibiotics) was also prohibitive, especially in the relatively short timeframe of the competition. Finally, Synechocystis PCC6803 is generally naturally competent – transformations involve simply exposing the organism to high concentrations of desired plasmid. However, we found out halfway through the competition that the strain we were using was most likely non-competent and therefore fundamentally not transformable. We then had to source a transformable strain.

References

1. Agapakis, C. M., Niederholtmeyer, H., Noche, R. R., Lieberman, T. D., Megason, S. G., Way, J. C., & Silver, P. A. (2011). Towards a synthetic chloroplast.PLoS One, 6(4), e18877.
2. Formighieri, C., & Melis, A. (2014). Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants. Planta, 240(2), 309-324.