Part:BBa_J100176:Experience

Purpose: We are testing if fabHAF promoter works in E.coli cells and whether triclosan induces the FabHAF promoter by causing fatty acid deprivation.

Method:

Part 1: Testing the fabHAF in E.coli cells with BsaI restriction enzyme We assembled the sequence of the fabHAF with the sticky ends to make the oligo. Then we prepared our oligo solution by mixing 2 µL of 10X annealing buffer, 1 µL of oligo stock printerT (100µM), 1 µL of oligo stock printerB (100µM), and 16 µL of DI water, then we followed the protocol of Building dsDNA with Oligos to prepare our oligo solution [http://www.bio.davidson.edu/courses/Molbio/Protocols/anneal_oligos.html]. We were provided with two tubes of GGA master mix, which contains plasmid J119137, the BsaI restriction enzyme, the ligase, DI water, buffer, and water. Each tube had a volume of 9 µL. We added 1 µL of the assembled oligos into one tube and labeled it “P” for promoter. For the other tube, we added 1µL of DI water and labeled it “-“ for negative control without the promoter. Then we transformed cells, following the protocol of Zippy Transformation of Z-competent Cells [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html]. At the end, we have 3 plates: a) experimental ligation DNA (with oligos mixture – “P”), b) ligation negative control DNA (with no promoter, only water – “-“), and c) transformative positive control DNA (with pLac promoter). In the following day, we tested whether fabHAF worked in E.coli cells by looking for red fluorescence colonies of E.coli cells in the “P” plate under the UV light. There were few but visible red colonies (circled), meaning that the promoter fabHAF was able to transcribe some of the gene into proteins that are dyed with red fluorescence.

Part 2: Testing the induction of fabHAF promoter by triclosan We collected four E.coli colonies from the experimental plate "P", two green and two red. We also collected two green colonies from the positive control (+) plate. We mixed these abstracted colonies with LB broth and ampicillin. We chose three of the six tubes prepared and added 20 µL of 0.5% triclosan from antimicrobial hand soap to one experimental, one negative, and one positive tube. The other three tubes do not have triclosan. We pipetted triplets of each sample then we used the Synergy machine to measure how much RFP was in each of the six tubes. We also measured the RFP of a blank well, which contained nothing but LBM media. We were able to measure the fluorescence of our data by subtracting the fluorescence and absorbance of LBM media from the fluorescence and absorbance of the triclosan treated and non-treated cells. Then we generated our graph using the ratio of red fluorescence protein to cell density. Our graph illustrates duplicates of our samples rather than triplicates due to extreme outliers exhibited by the results from the Synergy machine.

Figure: fabHAF activity when added BacDown handsoap with 0.5% triclosan

Triclosan are not added into the control groups (red) and added into the experimental group (blue). There are three different treatments for each group: promoter from positive control DNA (J04450), testing promoter from experimental ligation DNA (fabHAF), and negative control DNA.