Team:Valencia/cultures

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Cultures

Genetic engineering:

In first place, we cloned our biobricks: sintethiced psbA, CI lambda inverter, RBS, luxI, and terminator; and we load them in iGEM Registry parts.

The next step was the ligation to plasmid for Escherichia coli and Synechococcus elongatus, either WT as cscB. Later, we started the characterization of psbA: psbA-RBS-luxAB-ter (with plasmid lent by S. Golden) in E. coli by means of transformation; and we did a luminescence test with decanal.The transformation of E. coli, WT and cscB S. elongatus was done with our construct.
(fig.)

The ligation of (IPTG sensitive promoter)-RBS-luxI—ter (all iGEM Registry Parts) was used to transform E. coli for mass production of AHL to test bioluminescent response in A. fischeri.

Chassis growth and metabolism:

Growth media experimental array:

Media: COMPO, BG11, BG11+NaCl (200,250,300mM), BG11+NaCl(200,250,300mM)+20g/l Sucrose, TCBS, marine broth/agar, BG11+NaCl250mM+20gl Sucrose+10g/l Peptone+5g/l Yeast extract.
Swirling broth/agar plates.
Chassis: S. elongatus WT, S. elongatus cscB, Aliivibrio fischeri.

Coculture:

Once found the ideal medium where to grow both cscB S. elongatus with A. fischeri, we’d set an array of coculture dispositions:

• Mixed populations: Agar plates and broth


• Biphasic broth (membrane separated): a dialysis semipermeable membrane (only sucrose, water, ion and gas exchange; no AHL) of 0.45microns pore (minimal to retain cells, full exchange of molecules including AHL)


• Future design development: Biphasic coculture device with turning valve of different membrane pore size, to allow or not the diffusion of AHL (in a system with a knocked-out A. fischeri for the luxI gene, uncapable of isolated autoinduction – Further synthetic ecology development), enabling an element of human control rather than just a diel switch.
  We had to analyze in each case (chassis, medium, disposition) a temporal series for growth curves using a photometer at different wavelengths of optical densities.


• For S. elongatus cscB we analized the export of sucrose (saccharase and Fehling’s reagent, analysis in colorimeter; or clinical glucometer) with IPTG induction at different salt stress and growth phases. Night production of AHL in the transformed strain at different sucrose export rates and growth phases analysising by coelution in HPLC. Residence times in the liquid medium (light controls).


• For A. fischeri we analized the consumption rate of sucrose (saccharase and Fehling’s reagent, analysis in colorimeter; or clinical glucometer) at different growth phases. In addition, we wanted to measure the luminescence of Aliivibrio fischeri (in a luminometer) at different conditions of initial sucrose concentrations, growth phase and external input of AHL (synthesized by our transformed E. coli).
  The qualitative luminescence test under ‘natural’ conditions, “Marine Snow Experiment”, consists of placing a colony growing on a small lump of agar, in a sea water solution, emulating a marine snow particle colonized by A. fischeri. Mechanical stimulation is known to trigger bioluminescence in this kind of aggregates at the marine environment, which we imitate by shaking.

Tests on bioluminescence of algal cells:

• Test of luminescence of Synechococcus elongatus Wylde Type with psbA-luxAB as grown broth biolamp.


• Test to check the transformation of Chlamydomonas reinhardtii’s chloroplast with the lux cassette under regulation of psbA promoter (ligation including psbA-RBS-luxABCDE-Ter). These eukaryotic microalgae have a large chloroplast with prokaryote-like genetic system. This opens the way for further research on chloroplast transformation, so that we can achieve Cambridge 2011 iGEM latest aim: bioluminescent trees lighting the way in boulevards.

Advanced continuous coculture system:

This idea consist in separate two culture modules, a flat wide one for Synechococcus elongatus cscB as a solar module and a smaller compact one for Aliivibrio fischeri as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO₂ sink), but with a Pasteurian design opening to avoid contamination from deposition. And Set tubing connecting both cultures.

Development on a continuous

[Further developments: Auxotrophy artificial symbiosis knockouts in S. elongatus and A. fischeri, by extraction of genes responsible for the synthesis of aminoacids, where each partner synthesizes the one that the other lacks].

[Further developments: Mechanical standariztion of the S. elongatus cscB module as a Powercell (Brown-Stanford 2011 iGEM), with application to feed other heterotrophic cultures of any kind of biotechnological industry, which normally use Saccharromyces cerevisiae or E. coli, which grow well with sucrose as a carbon source].