Team:Valencia/cultures

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Cultures:

Chassis growth and metabolism

Here we aim to experiment with the chemical conditions of growth media for our chassis organisms, in order to assess the best conditions for maximum growth and best metabolic response of their biomachine functions.

Growth media experimental array:

Original media for our chassis:

For every strain we used an specific culture medium for each one:


Fig 1



• BG11 (Fig 1): for S. elongatus. Classical medium for freshwater cyanobacteria, without organic carbon neither nitrogen sources, rich in micronutrients for the development of photosynthetic apparatus.





Fig 2


• TCBS (Fig 2): for Vibrio. 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:

We made different media cultures changing different components, to test which was better to grow.

• 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).

Click HERE and have a quick look in "Advanced continuous coculture system"

Analyze, in each case (for chassis, medium, and disposition), a temporal series for growth curves using a photometer at different wavelengths of optical densities (figures 6&7):

• 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.

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: --

Fig 3: Initial Sunechococcus culture Fig 4: Synechococcus culture pasted two weeks Fig 5: Definitive Synechococcus culture


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 (wavelenght wich Chlorophyl A conteined in bacterial membranes takes up light) and OD630 (wavelenght which measure celular density) from spectrophotometric readings:

Fig 6: At ʎ=750 nm, the constant increasing OD obtained in S.elongatus
prove the population growth, because Chlorophyl A concentration increase when
bacterial population grow. Nevertheless, S.elongatus WT OD went down
because cells died.



Fig 7 : At ʎ=630nm, the behaviour of every strain is seemed to ʎ=750 nm,
but not so pronounced like that. High OD symbolize high population density,
and low OD a low population density.



FIg 8: Different cultures in different growth moments of Synechococcus elongatus

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