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 designed our Rhamncolipid to express reporter protein when producing rhamnolipid. We tried to find best reporter for our system that has fast turnover and doesn’t 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.

Reporter Selection

We transformed E. coli BL21(DE3) with different reporter proteins (RFP and aeBlue) with different tags (none, LAA, LVA). We induced the transformants using IPTG and observe the OD600 for bacteria growth and measure each reporter.

The results showed that [to be continued]. Therefore, we selected [to be continued] as our reporter device.

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. The supernatant was collected and tested for surfactant activity.

Rhamnolipid test

We tested our produced rhamnolipid for its characteristics and surfactant activites.

The results showed that supernatant from empty cells, induced or uninduced, and uninduced transformants had no or little surfactant activity. Supernatant from induced transformant has surfactant activity, comparable to synthetic surfactant, Tween 20%.