Part:BBa_K3145003:Experience
Applications of BBa_K3145003
After we confirmed through sequencing that we removed the XbaI cut site, we created three overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO and three overnights that contained 2.4 mL of LB Broth, 600 µL of Tryptophan (a precursor to indole), 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.
To measure indigo production, we performed an extraction protocol on these overnights to extract indigo using DMSO and measured the absorbance on the plate reader. To convert the raw data, we made an indigo standard curve (shown below) and graphed the data below:
As shown in the graph above, there was a greater yield of indigo in the overnights that contained additional tryptophan.
Cell-Free Experiments
After we measured the overnights, we moved on to optimizing our system in cell-free. We set up cell-free reactions and graphed the results below.
Unfortunately, the only “response” we saw in cell-free were reactions with indole, which we believe was due to the indole crashing out of solution. Although we saw indigo production in cells, we did not see similar results in cell-free which will require further optimization.
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