Team:ITB INDONESIA/results/lab
RhamCOLIpid
Engineered E. coli producing rhamnolipid for green enhanced oil recovery
Lab Results
We utilise the Biosurfactor biobrick (BBa_K653000) designed by Panama 2011 that encodes rhlA and rhlB, two enzymes for rhamnolipid production. We put the biobrick under T7lac promoter with strong RBS (BBa_K613010), and double terminator BBa_B0015. We order our parts to be chemically synthesised via BioBasic.
We transformed the part in BioBasic plasmid into E. coli DH5a. The confirmed plasmid was then used for transformation into E. coli BL21 (DE3). The true transformant was used for expression.
We assembled out part in the BioBasic plasmid into pSB1C3 encoding RFP and then transformed it into E. coli DH5a. We used two ligation composition, and transformed each mix. Utilising red-white colony selection, we selected four white colonies candidate for colony PCR from each plate. Four of our selected colonies showed positive result: having a band around 2500-3000 kb.
It was further confirmed by plasmid isolation and PCR, showing that we have successfully clone our part into pSB1C3
We designed our Rhamcolipid to express reporter protein when producing rhamnolipid. We tried to find the best reporter for our system that has fast turnover and does not have significant fitness cost to the bacteria. We tested two reporters, RFP and aeBlue, with each reporter has different tags, either not tagged, LAA-tagged, or LVA-tagged. We also designed our control device which encodes LacI under different promoter strength, strong, medium, or even without the control. Unfortunately we run out of time to assemble these devices.
Rhamnolipid production
We transformed E. coli BL21 (DE3) with rhamnolipid production module. After IPTG induction, the culture was centrifuged. The cell was collected and lysed for protein analysis by SDS-PAGE. It showed that there is a band that we assumed to be our rhlA and rhlB due to their size around 30 and 40 kDa respectively. The low concentration of putative rhlA and rhlB might be caused by the codon usage. But it needs more confirmation and further studies.
The supernatant was collected and tested for surfactant activity.
Rhamnolipid test
We tested our produced rhamnolipid for its characteristics and surfactant activites. Here we describe for the very first time the application of the Biosurfactor biobrick (BBa_K653000). The emulsification result showed that our supernatant has similar feature with positive control, Tween 20%, while our LB media (not from transformant’s supernatant) had similar property with negative control, water.
For make it sure, we tried to test our sample again in different size using test tube for emulsification test. After 24H we can saw that our supernatant had higher activity to decrease tension between oil and water, it showed with a higher emulsification zone (brown foam in the bottom of test tube) from uninduced and induced transformant than positive and negative control.
Emulsification result
Using oil drop test, it was showed that supernatant from induced transformant (TI) has surfactant activity, almost similar to synthetic surfactant, Tween 20%. The uninduced transformant still created a clear zone. This maybe due to the leaky nature of lac operator. The negative control, LB media, had no clear zone. We used 40uL of the tested supernatant in 20 uL of crude oil.
Oil Drop Test Result
We tested the supernatant in Lemigas, Indonesia’s national research and development centre for oil and gas technology. We tested the supernatant’s interfacial tension (IFT) in 60oC and 70oC. It was showed that the supernatant from induced transformant has lower IFT (which is better) in 60oC. This IFT was even lower in higher temperature, 70oC. The negative control (null) failed to separate the oil at 70oC so it does not have IFT value.
The IFT was within the range of good quality surfactant. Therefore, Lemigas was interested to do more test, but we did not have enough time to receive the result before Wiki Freeze.
