Team:US AFRL CarrollHS/Measurement
Bronze Medal Criterion
To fulfill the Measurement criterion, we decided to measure one of our past team’s composite parts from 2017, PLas_sfGFP Part BBa_K2522001 which produces sfGFP in the presence of N-3-oxododecanoyl homoserine lactone (3OC12). Prior to measuring our part, we had to create our standard curves using the iGEM Measurement Plate Reader protocol which are graphed below.
We created six overnights that contained 3 mL of LB Broth, 3 μL of the antibiotic Chloramphenicol, E. coli DH5α cells transformed with PLas_sfGFP, and either 3 μL of 10 mM 3OC12 (three overnights) or 3 μL of DMSO (three overnights) to confirm the functionality of the plasmid and one 3 mL overnight that contained only LB Broth to serve as our blank.
Through these overnights, we were able to confirm the functionality of PLas_sfGFP 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:
Fluorescence (MEFL/Particle)
|
With 3OC12 (final concentration 10 μM) |
With DMSO |
|
1290000.00 |
7810.00 |
|
1310000.00 |
1580.00 |
|
1270000.00 |
8140.00 |
Since we were able to confirm the functionality of the PLas_sfGFP circuit, we also decided to create a Dose Response Curve for PLas_sfGFP by making overnight triplicates of the PLas_sfGFP induced at different concentrations of 3OC12.
First, we performed serial dilutions of the 10 mM stock solution of 3OC12 with DMSO. Then, we created overnight in triplicate of cells transformed with PLas_sfGFP that were induced by the different 3OC12 concentrations. Afterward, we diluted and measured the fluorescence and absorbance of each overnight as explained above. Using our 100 μL Standard Curve, we converted our raw measurements and graphed them below:
Fluorescence (MEFL/Particle)
|
Final Concentration (μM) |
Triplicate #1 |
Triplicate #2 |
Triplicate #3 |
|
5 |
1581451 |
1501519 |
1605886 |
|
1 |
1564336 |
1418952 |
1550982 |
|
0.5 |
1612388 |
1396958 |
1600123 |
|
0.1 |
1421045 |
1461045 |
1416788 |
|
0.05 |
1389708 |
1407451 |
1417182 |
|
0.01 |
1289253 |
1275768 |
1222314 |
|
0.005 |
1108580 |
1006372 |
1255536 |
|
0.001 |
642864 |
563153 |
734513 |
|
0.0005 |
362993 |
348382 |
305982 |
|
0.0001 |
101434 |
108762 |
98193 |
|
0.00005 |
58325 |
63723 |
68752 |
|
0 |
32018 |
34156 |
32305 |
The circuit responded to 3OC12 in a dose-dependent manner. We began seeing signal at 10-4 μM and the signal saturated at 10-1 μM. The sensor showed a 60-fold increase in signal upon activation based on the non-linear fit analysis. Analysis was done on GraphPad Prism 5.
We have submitted this characterization in order to fulfill our Bronze Medal Criterion, although we have also used these measurement protocols for the remainder of our experiments. However, our standard curves differ based on factors such as volume and plate type. We’ve graphed all of the other standard curves below.
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.