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Team:UNIK Copenhagen/Green

Description of the Green Lab

What is the purpose of the Green lab?

The green lab is responsible for the development of the actual strains to be used in the co-culture and the media used to grow them at optimal rates, as well as the mechanical container for the co-culture.

With the central aim of the project defined as a modular platform for bioproduction with plastic as a proof-of-concept, usable in a 3D-printer such as that on the ISS, the green lab identified a suitable bioplastic that had been well-researched.  The metabolic pathway and genes to manufacture this chemical were to be transformed into the appropriate organism and the applicability of the platform proven with growth tests and various configurations for the co-culture’s container.

In addition, the green lab members participate in many of the outreach programmes and provide technical advice to other sub-teams where appropriate.

Introduction of the organisms, and why we chose those strains of cyano and bacillus we are using

This project uses resources that should be available in many extraterrestrial environments: light (such as that from a star) and CO2 (from a planetary atmosphere or produced by respiring organisms).  Photosynthesis is a logical way to harness both of these, and cyanobacteria are convenient microorganisms capable of such, specifically in their production of sucrose.

The sucrose can then be metabolised into relevant end-products by specialised organisms.  The benefit of easily switching from one product-producing strain to another is a leading reason behind using a co-culture.  However, the non-continuous use of the second organism means a system of storage, over long periods of space travel and with minimal maintenance from a crew, is optimal.  Therefore, a sporulating bacterium amenable to genetic manipulation was chosen.

Synechococcus elongatus PCC 7942 was chosen as the cyanobacterium.  It is a commonly used research species which, then known as Anacystis nidulans R2, was the first cyanobacterium proven to transform with exogenous DNA [1].  Besides its photosynthetic capability, it demonstrates good sucrose overproduction in high-salt environments to balance the external osmotic pressure.  The exact strain used (provided by Prof. Pamela Silver, see acknowledgements) has been transformed with cscB sucrose permease, a sucrose/proton symporter.  The internal proton concentration produced by photosynthesis enables export of sucrose, though it will progressively acidify its environment [2].

Bacillus subtilis was selected as the second species.  It is both capable of sporulation when nutritionally stressed and widely used in research, being generally regarded as safe (GRAS) [3].  Previous P(LA-co-3HB) production in Escherichia coli could be transferred to B. subtilis via shuttle vectors common to both, and there is little codon bias between these species [4].  The vector pHT254 was used, conferring Ampicillin resistance on the E. coli and then transformed into B. subtilis.

Short description of the co-culture

The mechanical container of the co-culture uses common laboratory items: a large conical flask and dialysis bags with clips.  The flask contains the cyanobacteria free in the media and the sealed dialysis bag, which contains the appropriate B. subtilis strain.  This arrangement puts the cyanobacteria on the outer layer, allowing for better light exposure, and the B.subtilis bag is removable and replaceable when a new product is required.

The dialysis tubing (threshold = 2kDa) allows the sucrose and necessary components of the media to pass between species, but does not mix the species themselves.  It is kept sterile with ethanol prior to use, and sealed with regular bag clips at either end.

The co-culture, supplied with the precursors identified above, is exposed to light and begins the synthesis of sucrose.  The transport of the sucrose to the B. subtilis is facilitated by a high salt concentration, and is then used as a feedstock in B. subtilis’s further processing.

Extensive experiments on the mutual survivability of the two species were necessary, to ensure that a careful balance of nutrients maintains both evenly and that products of one (waste or otherwise) do not harm the other.  These tests included using ‘spent’ cyanobacterial medium to grow B. subtilis, mixed forms of media and, eventually, live tests of the two species together.

Short description of the metabolic engineering

With cscB already inserted into the cyanobacteria, no further alterations were made to this organism.

B. subtilis is capable of using sucrose as an energy source, breaking it into lactate.  Previous research on PLA synthesis in E. coli suggested the production of PLA itself was impractical, and so the P(LA-co-3HB) plastic, a copolymer of lactate with acetyl-CoA was decided upon.

This required the insertion of four genes into B. subtilis [5]:

  • Propionyl-CoA transferases (PCT)
  • Lactate polymeriseing enzyme (LPE)
  • β-ketothiolase (PhaA)
  • Acetoacetyl-CoA reductase (PhaB)

Due to the constraints of time, only PhaA and PhaB were eventually inserted into B. subtilis, but PCT and LPE remain an option to complete the genetic circuit.  No further alterations to other potential avenues of sucrose utilisation were made; for these purposes, the overabundance of sucrose is hoped to be sufficient to give some bioplastic.

References

[1] Shestakov SV, Khyen NT. 1970. Evidence for genetic transformation in blue-green alga Anacystis nidulans R2. Molec. Gen. Genet. 107: 372-5

[2] Ducat DC, Avelar-Rivas JA, Way JC, Silver PA. 2012. Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol. 78(8):2660-8

[3] Higgins D, Dworkin J. 2012. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev. 36(1):161-48

[4] Hershberg R, Petrov DA. 2009. General Rules for Optimal Codon Choice. PLoS Genet 5(7): e1000556. doi:10.1371/journal.pgen.1000556

[5] Jung YK, Kim TY, Park SJ, Lee SY. 2010. Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers. Biotechnol Bioeng. 105(1):161-71

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