Team:Valencia/cultures

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<br>Cultures<br>
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Cultures: Chassis growth and metabolism:
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<h2><b>Genetic engineering:</b></h2><br>
 
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In first place, we cloned our biobricks: synthesised psbA, CI lambda inverter, RBS, luxI, and terminator; and we load them in iGEM Registry parts.<br><br>
 
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The next step was the ligation to plasmid for Escherichia coli and Synechococcus elongatus, either WT as cscB.
 
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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. <br>(fig.)<br><br>
 
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The ligation of (IPTG sensitive promoter)-RBS-luxI—ter (all iGEM Registry Parts) was used to transform <i>E. coli</i> for mass production of AHL to test bioluminescent response in <i>A. fischeri</i>.<br><br>
 
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<h2><b>Chassis growth and metabolism:</b></h2><br>
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<h4>Growth media experimental array:</h4><br>
<h4>Growth media experimental array:</h4><br>
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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.<br>
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Original media for our chassis:
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Swirling broth/agar plates.<br>
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-BG11: Classical medium for freshwater cyanobacteria, without organic carbon neither nitrogen sources, rich in micronutrients for the development of photosynthetic apparatus.
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Chassis: <i>S. elongatus</i> WT, <i>S. elongatus</i> cscB, <i>Aliivibrio fischeri</i>.<br><br>
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-TCBS: Thio-Citrate-Bile-Salts, selective medium for Vibrionacean gamma-proteobacteria, rich in organic nitrogen sources such as peptone, and sucrose as carbon source. Ox bile and other reagents prevent the growth of enteric bacteria.
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<h4>Coculture:</h4>
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Media to test:  
-
<br>
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-COMPO (commercial plant fertilizer diluted in water)
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Once found the ideal medium where to grow both cscB S. elongatus with <i>A. fischeri</i>, we’d set an array of coculture dispositions:<br><br>
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-BG11
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<ul style="list-style-type: square">
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-BG11+NaCl (200,250,300mM): Salt is an essential component for the growth of A. fischeri, and necessary to activate the cscB transporter protein.
-
<li>Mixed populations: Agar plates and broth</li><br>
+
-BG11+NaCl(200,250,300mM)+20g/l Sucrose: A basal quantity of sucrose can be useful to start off growth of A. fischeri, and can be interesting in terms of osmotic response of cscB sucrose export.
-
<li>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)</li><br>
+
-TCBS
-
<li>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 <i>A. fischeri</i> for the luxI gene, incapable of isolated selfinduction – Further synthetic ecology development), enabling an element of human control rather than just a diel switch.<br>
+
-Marine broth/agar: Basic medium for heterotrophic marine bacteria, such as A. fischeri
-
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.</li><br>
+
-Marine broth/agar + micronutrients: Could be a solution to sustain both autotrophic and heterotrophic cultures, in salty conditions to induce sucrose export.
-
<li>For <i>S. elongatus</i> 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 analysing by coelution in HPLC. Residence times in the liquid medium (light controls).</li><br>
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-BG11+NaCl250mM+20gl Sucrose+10g/l Peptone+5g/l Yeast extract: Includes not only carbon but also nitrogen sources to help A. fischeri grow, besides BG11 micronutrients for cyanobacterial development.                                    
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<li>For <i>A. fischeri</i> 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 <i>Aliivibrio fischeri</i> (in a luminometer) at different conditions of initial sucrose concentrations, growth phase and external input of AHL (synthesized by our transformed <i>E. coli</i>).<br>
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The qualitative luminescence test under ‘natural’ conditions, “Marine Snow Experiment”, consists in placing a colony growing on a small lump of agar, in a sea water solution, emulating a marine snow particle colonized by <i>A. fischeri</i>. Mechanical stimulation is known to trigger bioluminescence in this kind of aggregates at the marine environment, which we imitate by shaking.</li>
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<br><br>
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<h2><b>Tests on bioluminescence of algal cells:</b></h2>
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Conditions: Swirling broth/agar plates for each medium
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Chassis to test: S. elongatus WT, S. elongatus cscB, S. elongatus cscB + psbA-cI-luxI construct, Aliivibrio fischeri.
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<ul style="list-style-type: square">
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<li>Test of luminescence of <i>Synechococcus elongatus</i> Wylde Type with psbA-luxAB as grown broth biolamp.</li><br>
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<li>Test to check the transformation of <i>Chlamydomonas reinhardtii</i>’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.</li></ul>
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<br><br>
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<h2><b>Advanced continuous coculture system:</b></h2>
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Basic coculture:
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This idea is to separate two culture modules, a flat wide one for <i>Synechococcus elongatus</i> cscB as a solar module and a smaller compact one for <i>Aliivibrio fischeri</i> 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 CO<sub>2</sub> sink), but with a Pasteurian design opening to avoid contamination from deposition. And Set  tubing connecting both cultures.
+
 
-
<br><br>
+
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): 0.45microns pore (minimal to retain cells, full exchange of molecules including AHL), dialysis semipermeable membrane (only sucrose, water, ion and gas exchange; no AHL).
 +
 
 +
Analyze in each case (for chassis, medium, and disposition) a temporal series for:
 +
 
 +
-Growth curves using a photometer at different wavelengths of optical densities
 +
-For S. elongatus cscB:
 +
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 (analysis by co-elution in HPLC). Residence time of AHL in the liquid medium and degradation half-life (24h light controls).
 +
-For A. fischeri:
 +
Consumption rate of sucrose (saccharase and Fehling’s reagent, analysis in colorimeter; or clinical glucometer) at different growth phases.
 +
Luminescence of A. fischeri (in a luminometer) at different conditions of initial sucrose concentrations, growth phase and external inputs of AHL (synthesized by our transformed E. coli).
 +
Qualitative luminescence test under ‘natural’ conditions: “Marine Snow Experiment”, 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.
 +
 
 +
[Further developments: As an idea for improving our system’s biosafety, we could induce auxotrophic artificial symbiosis knockouts in S. elongatus and A. fischeri, by extraction of genes responsible for the synthesis of determined aminoacids, where each partner synthesizes the one that the other lacks. This reduces the chances of contamination of the natural environment, as hardly ever organisms from both culture modules could escape simultaneously and be able to stay together to survive].
 +
 
 +
[Further developments: We realized our photosynthetic sucrose exporter bioreactor can have a wide spectrum utility by itself, when connected to a diffusion membrane system. Mechanical standardization of the S. elongatus cscB module as a Powercell (Brown-Stanford 2011 iGEM), would have application as a solar-powered energy donor to feed other heterotrophic cultures from any kind of biotechnological industry, which normally use Saccharromyces cerevisiae or E. coli, which grow well with sucrose as a carbon source].
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RESULTS:
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Key:
 +
+=Grown
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-=Not grown
 +
(Note: 2 replicates were done to assure the result obtained.)
 +
 
 +
Growth of cscB S. elongatus:
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-BG11 broth: ++
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-BG11 agar: --
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-COMPO broth: -+
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-COMPO agar: ++
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-BG11+200mM NaCl broth: +-
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-BG11+250mM NaCl broth: ++
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-BG11+300mM NaCl broth: +-
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-BG11+250mM NaCl agar: --
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-BG11+250mM NaCl+20g/l Sucrose+10g/l Peptone+5g/l Yeast extract broth: --
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-BG11+250mM NaCl+20g/l Sucrose+10g/l Peptone+5g/l Yeast extract agar: --
 +
 
 +
Coculture: Construction of functional bi-vessel sealed device with a central 0.45micron pore membrane (built from a clarification syringe filter).
 +
 
 +
Growth curve of S. elongatus cscB (growing on BG11+250mM NaCl – pH 8.9) and S. elongatus WT (growing on BG11 – pH 7.4), linear regression on OD750, OD660, and OD630 from spectrophotometric readings:
 +
 
 +
(fig 1, 2)
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<h2><b>Development on a continuous</b></h2>
 
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<br>
 
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[<u>Further developments:</u> Auxotrophy artificial symbiosis knockouts in <i>S. elongatus</i> and <i>A. fischeri</i>, by extraction of genes responsible for the synthesis of aminoacids, where each partner synthesizes the one that the other lacks].
 
-
<br><br>
 
-
[<u>Further developments:</u> Mechanical standarization 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 Saccharomyces cerevisiae or <i>E. coli</i>, which grows well with sucrose as carbon source].
 
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Revision as of 12:35, 26 September 2012


Cultures: Chassis growth and metabolism:

Growth media experimental array:


Original media for our chassis: -BG11: Classical medium for freshwater cyanobacteria, without organic carbon neither nitrogen sources, rich in micronutrients for the development of photosynthetic apparatus. -TCBS: Thio-Citrate-Bile-Salts, selective medium for Vibrionacean gamma-proteobacteria, rich in organic nitrogen sources such as peptone, and sucrose as carbon source. Ox bile and other reagents prevent the growth of enteric bacteria. Media to test: -COMPO (commercial plant fertilizer diluted in water) -BG11 -BG11+NaCl (200,250,300mM): Salt is an essential component for the growth of A. fischeri, and necessary to activate the cscB transporter protein. -BG11+NaCl(200,250,300mM)+20g/l Sucrose: A basal quantity of sucrose can be useful to start off growth of A. fischeri, and can be interesting in terms of osmotic response of cscB sucrose export. -TCBS -Marine broth/agar: Basic medium for heterotrophic marine bacteria, such as A. fischeri -Marine broth/agar + micronutrients: Could be a solution to sustain both autotrophic and heterotrophic cultures, in salty conditions to induce sucrose export. -BG11+NaCl250mM+20gl Sucrose+10g/l Peptone+5g/l Yeast extract: Includes not only carbon but also nitrogen sources to help A. fischeri grow, besides BG11 micronutrients for cyanobacterial development. Conditions: Swirling broth/agar plates for each medium Chassis to test: S. elongatus WT, S. elongatus cscB, S. elongatus cscB + psbA-cI-luxI construct, Aliivibrio fischeri. Basic 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): 0.45microns pore (minimal to retain cells, full exchange of molecules including AHL), dialysis semipermeable membrane (only sucrose, water, ion and gas exchange; no AHL). Analyze in each case (for chassis, medium, and disposition) a temporal series for: -Growth curves using a photometer at different wavelengths of optical densities -For S. elongatus cscB: 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 (analysis by co-elution in HPLC). Residence time of AHL in the liquid medium and degradation half-life (24h light controls). -For A. fischeri: Consumption rate of sucrose (saccharase and Fehling’s reagent, analysis in colorimeter; or clinical glucometer) at different growth phases. Luminescence of A. fischeri (in a luminometer) at different conditions of initial sucrose concentrations, growth phase and external inputs of AHL (synthesized by our transformed E. coli). Qualitative luminescence test under ‘natural’ conditions: “Marine Snow Experiment”, 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. [Further developments: As an idea for improving our system’s biosafety, we could induce auxotrophic artificial symbiosis knockouts in S. elongatus and A. fischeri, by extraction of genes responsible for the synthesis of determined aminoacids, where each partner synthesizes the one that the other lacks. This reduces the chances of contamination of the natural environment, as hardly ever organisms from both culture modules could escape simultaneously and be able to stay together to survive]. [Further developments: We realized our photosynthetic sucrose exporter bioreactor can have a wide spectrum utility by itself, when connected to a diffusion membrane system. Mechanical standardization of the S. elongatus cscB module as a Powercell (Brown-Stanford 2011 iGEM), would have application as a solar-powered energy donor to feed other heterotrophic cultures from any kind of biotechnological industry, which normally use Saccharromyces cerevisiae or E. coli, which grow well with sucrose as a carbon source]. RESULTS: Key: +=Grown -=Not grown (Note: 2 replicates were done to assure the result obtained.) Growth of cscB S. elongatus: -BG11 broth: ++ -BG11 agar: -- -COMPO broth: -+ -COMPO agar: ++ -BG11+200mM NaCl broth: +- -BG11+250mM NaCl broth: ++ -BG11+300mM NaCl broth: +- -BG11+250mM NaCl agar: -- -BG11+250mM NaCl+20g/l Sucrose+10g/l Peptone+5g/l Yeast extract broth: -- -BG11+250mM NaCl+20g/l Sucrose+10g/l Peptone+5g/l Yeast extract agar: -- Coculture: Construction of functional bi-vessel sealed device with a central 0.45micron pore membrane (built from a clarification syringe filter). Growth curve of S. elongatus cscB (growing on BG11+250mM NaCl – pH 8.9) and S. elongatus WT (growing on BG11 – pH 7.4), linear regression on OD750, OD660, and OD630 from spectrophotometric readings: (fig 1, 2)

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