Team:ITB INDONESIA/pre-wetlab
RhamCOLIpid
Engineered E. coli producing rhamnolipid for green enhanced oil recovery
Pre Wetlab Modelling
Model
ITB_Indonesia 2015 initially wanted to developed a system where rhamnolipid production is controlled under quorum sensing. Quorum sensing proteins, LuxI and LuxR, would be expressed under trp promoter. Inoculum would be grown in minimal media containgi glycerol and tryptophan. When grown in media without tryptophan, quoring sensing protein will be expressed. LuxI will produce co-activator AHL. When AHL passes a threshold concentration, it will bind effectively with LuxR and activated enzyme’s expression and eventually rhamnolipid production. We wanted to make nitrogen as limiting factor, therefore putting a nitrogen sensor that will repress rhamnolipid production under nitrogen absence.
Model was adapted from Henkel et. al, 2014. In that paper, Pseudomonas aeruginosa PAO 1 was utilised as inoculum, sunflower oil as carbon & energy source, and nitrate (NO3) as nitrogen source. In the model, sunflower will be metabolised into glycerol and fatty acid, which will be used for productions of biomass, N-butyryl-homososerine (HSL) needed for quorum sensing, and rhamnolipid. The presence of 2 carbon sources will cause diauxic growth resulted from carbon preference. Fatty acid is expected to inhibit cell’s growth. In Henkel’s model, nitrate is assumed to produce biomass as well as lipase enzyme needed for sunflower oil metabolism. Rhamnolipid concentration is related to produced HSL concentration.
There are differences from initial system designed by ITB_Indonesia 2015 with Henkel’s. We would utilise E. coli as inoculum, glycerol as carbon source, and ammonia (NH4) as nitrogen source. Therefore, there are modifications done to the model:
1. There will be no growth inhibition nor carbon preference resulted from carbon utilisasion.
2. The bacteria will not need to produce lipase to metabolize sunflower oil, therefore nitrogen will only be used for biomass production.
3. Polysaccharide nor di-rhamnolipid will be produced.
Visually, modified model will be as the figure below.
Equations
Following are equations after simplification based on points above. Values used for calculation were based on Henkel’s paper unchanged (Tabel 1).
We tested our model using the starting concentration of 100 g/L glycerol and 3 g/L NH4. It showed that our rhamnolipid production started just when the nitrogen is exhausted. If this is the case, our nitrogen system would turn on and suppress the xpression of rhlAB before production, so there would be no rhamnolipid production.
Therefore, we developed a new system which we used throughout the summer.
Legend:
X = biomass;
Gly: glycerol conc;
NH4: NH4 conc;
C4-HSL: HSL conc;
Acyl: acyl conc;
RLMono: mono-rhamnolipid conc;
CPS: cellular polysaccharide conc;