Part:BBa_J18912:Experience
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Applications of BBa_J18912
2019 US AFRL CarrollHS
This part was characterized as part of composite parts "J18912-sfGFP"and "J18912-bFMO"
J18912-sfGFP
We created six overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and either E.coli DH5α cells containing J18912-sfGFP (three overnights) or either E.coli DH5α cells containing pY71-sfGFP(three overnights) 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.
Through these overnights, we were able to confirm the functionality of both plasmids, and we measured the fluorescence and absorbance of each overnight by performing an endpoint read of each overnight in a 96-well flat bottomed plate. In order to obtain data in the range of our standard curve, we diluted the overnights two-fold by mixing 50 µL of each overnight with 50 µL of LB.
We then used the 100 µL standard curve we created using the iGEM Measurement protocols and converted our data from arbitrary absorbance and fluorescence units to MEFL/particle which we graphed below
Cell-Free Experiments
After confirming functionality in cells, we moved on to optimizing our system in cell-free. We set up cell-free reactions and also Although, in cells, there seemed to be no difference in signal of our two plasmids, in cell-free, there was a difference, as shown in the graph below. We believe that this most likely resulted from the difference in DNA concentrations which affects the cell-free system.
We also lyophilized our cell-free reactions, and rehydrated them with 25 µL of water. After reading them on the plate reader for four hours, and graphing the data, we were able to confirm functionality of both plasmids, although there was a lower signal in the lyophilization.
Paper-Based Sensor
Since both of our plasmids functioned in cell-free, we moved on to applying our plasmids on our paper-based sensor. After one hour of incubation at 30 °C, we were able to confirm functionality and see signal for both plasmids.
J18912-bFMO
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. 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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