The Improvement of the F2620 Receiver Vector:
This year's team conducted multiple experiments to imrpove the characterization of F2620. The improved part itself can also be viewed here on the iGEM registry page.
There are 3 types of experiments used to characterize this improved receiver: F2620 Induced by Synthetic AHLs, F2620 Induced by Sender Supernatants, and F2620 induced by Sender cells on Agar.
The first experiment, F2620 Induced by Synthetic AHLs, included F2620 receiver cells being induced by synthetic AHL chemicals under different concentrations. The maximum GFP expression was analyzed to understand the effect AHL concentration had on the receiver expressing GFP.
The following experiment, F2620 Induced by Sender Supernatants, used F2620 receiver cells being induced by a variety of combinations of senders in supernatant form. The maximum GFP expression was analyzed to understand the effect different percentages of sender supernatant had on the receiver expressing GFP.
The final experiment included F2620 receiver cells that were spread on agar plates and induced by spread sender cells. Images are then taken at different time intervals of the agar plate to analyze the induction rates and diffusion of F2620 induction.
F2620 Induced by Synthetic AHLs
The graphs below depict the GFP production from the F2620 receiver to be higher at a higher concentration of the synthetic AHL of Las. The second graph, depicts the same nature however at a higher concentration of the synthetic AHL of Lux. It is interesting to note that the Las sender, at a lower concentration, promotes a higher GFP expression than the Lux. Our group hypothesized that it would be opposite considering the LuxI sender comes from the same system as F2620. These results show differently.
F2620 Induced by Sender Supernatants
Test #1 with LuxR: Sender A: LasI, B: EsaI, C: RpaI
The receiver being used for the below results is the Lux receiver. The second set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #1 with LuxR:
In this set of tests with the Lux receiver the results showed that the LasI expressed the highest, EsaI 2nd highest and RpaI 3rd highest. The graphs concluded that the higher the Las and EsaI combination, the higher the overall GFP expression. No combinations pushed the GFP expression higher than any 50% sender alone.
Test #2 with LuxR: Sender A: RhlI, B: CerI, C: AubI
The second set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #2 with LuxR:
This test showed some notable results. As seen clearly in the last graph, the AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 40% AubI mixed with 10% negative sender and the 40% AubI mixed with 10% RhlI both expressed higher than the 50% AubI by itself. This result was confirmed in another test where 40% Aub mixed with 10% EsaI and 40% AubI mixed with 10% CerI both expressed higher than the 50% AubI alone.
Test #3 with LuxR: Sender A: LasI, B: EsaI, C: RpaI
The third set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #3 with LuxR:
In this set of tests with the Lux receiver the results showed that the RpaI expressed the highest, LasI 2nd highest and EsaI 3rd highest. The graphs concluded that the higher the RpaI and LasI combination, the higher the overall GFP expression. No combinations pushed the GFP expression higher than any 50% sender alone.
Test #4 with LuxR: Sender A: AubI, B: EsaI, C: CerI
The fourth set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #4 with LuxR:
This test showed some notable results. As seen clearly in the last graph, the AubI showed a higher expression when mixed with 10% of a second sender (even when that sender was a negative control sender). The 40% AubI mixed with 10% negative sender and the 40% AubI mixed with 10% EsaI both expressed higher than the 50% AubI by itself. This result was confirmed in the previous test #2 with the LuxR (40% Aub mixed with 10% EsaI and 40% AubI mixed with 10% CerI both expressed higher than the 50% AubI alone).
Test #5 with LuxR: Sender A: LuxI, B: BraI, C: RpaI
The fifth set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #5 with LuxR:
In this test there were some results that were not able to be replicated, meaning that there may have been some contamination or other unaccounted for error. The 40% LuxI + 10% negative sender expressed higher than the 50% LuxI alone, this result was not able to be replicated. Again the 40% LuxI + 10% RpaI expressed higher than the 50% LuxI alone but the result was unable to be replicated and lastly the 40% LuxI + 10% BraI expressed higher than the 50% LuxI and again, the result was unable to be replicated.
Test #6 with LuxR: Sender A: LuxI, B: RhlI, C: LasI
The sixth set of senders that was tested is shown below, these are all the combinations and percentages of the AHLs for the test including the controls. Each data point was tested in triplicate. The colors will coordinate with the graphs for each set of tests. The graphs for each set of data will include the overall average GFP signal, the average OD 600 and the normalization of the GFP over the OD 600. The number of data points used made adding individual error bars ineffective as the data was not able to be read. Error was calculated on the controls and added as separate bar graphs below the full data set. There was also Hill curve (trans equations) made that include error/ standard deviation if more information is needed for any notable results.
Results from test #6 with LuxR:
In this set of tests with the Lux receiver the results showed that the LuxI expressed the highest, RhlI 2nd highest and LasI 3rd highest. The graphs concluded that the higher the LuxI combination, the higher the overall GFP expression. No combinations pushed the GFP expression higher than any 50% sender alone.
F2620 Induced by Sender Cells on Agar
