Team:BUCT-China/Results
Results
Result prediction
Before the formal verification experiment, we first predicted the experimental results based on the experimental principle, and predicted as follows:
The upper part of the white line drawn by the red line is the arabinose operon, and the red part below is our target operon. Both the arabinose operon and our target operon produce a repressor protein, and the repressor protein binds to the operon. When RNA polymerase is transcribed, it is blocked by the repressor protein, and transcription cannot be performed, thereby inhibiting transcription. Taking the fatty acid operon as an example, when we add arabinose, arabinose binds to the repressor protein on the arabinose operon and causes it to fall off. RNA polymerase is slid through the arabinose operon and slid to the downstream fatty acid operon to be repressed by a repressor protein on the fatty acid operon. At this time, we add fatty acids, and the fatty acids bind to the repressor protein, causing it to fall off. RNA polymerase continues to slide and slides over the fluorescent protein gene downstream of the operon to express the fluorescent protein. Going back to the different conditions of each group before, we predict that if our operons function normally, then the No. 1 tube and No. 4 tube of each group must not change color, and the No. 2 tube must be discolored. By consulting the literature, we found that Arabia The sugar operon has a chance to fail, so the tube 3 may change color.
Qualitative experiment results
- Arabinose: Add 200μL of 5% arabinose solution
- Fatty acid: add 100 μL of 100mmol/L fatty acid solution
- Glyoxylic acid: Add 100 μL of 2mol/L glyoxylic acid solution
Experimental results and analysis
In the experiment, a total of three groups of operons were qualitatively tested. The experimental results are as follows:
Fatty acid ec operon:
Fluorescent protein expression of No. 1 tube and No. 2 tube, no fluorescence of No. 3 tube and No. 4 tube.
analysis:
Combined with the experimental conditions of the fatty acid ec operon validation, we made the following analysis of the experimental results: Based on previous experimental results, we conclude that the No. 1 tube incorporates arabinose, which opens the arabinose-controlled transcriptional pathway, but, according to our experiments, in the presence of our fatty acid ec operon repressor The No. 1 tube does not contain fatty acids to open the transcription channel controlled by the fatty acid ec operon, so transcription cannot proceed normally, and there should be no fluorescent expression, but the experimental results are fluorescent expression, thus indicating our fatty acid ec operon It did not function as expected, so it was judged that the fatty acid ec operon failed. The failure of the fatty acid ec operon can explain the results of the remaining three tubes: the addition of arabinose to tube 2 opens the transcriptional channel controlled by the arabinose operon, while the fatty acid ec operon fails, transcription proceeds normally, and fluorescence is expressed. No. 3 tube. In tube 4, no arabinose was added, and the transcription channel was controlled by the arabinose operon. Transcription was impossible and no fluorescence was expressed. The results were inconsistent with the expectation, indicating that the fatty acid ec operon failed.
Glyoxylic acid operon
No. 1 tube and No. 4 tube have no fluorescence, and No. 2 tube and No. 3 tube have fluorescent expression
analysis:
Combined with experimental predictions, the following conclusions were drawn: No. 1 tube added only arabinose, which opened a transcriptional channel that may be controlled by the arabinose operon, but did not add glyoxylate, so it failed to open the transcription controlled by the glyoxylate operon. Channel, so transcription is not working properly, and tube 1 is not fluorescent. The No. 2 tube was added with arabinose and glyoxylate, which respectively opened the transcription channel controlled by the arabinose operon and the glyoxylate operon, so that transcription was normally carried out and fluorescently expressed. Neither the No. 3 tube nor the No. 4 tube was added with arabinose, and the transcription channel controlled by the arabinose operon could not be opened, so that no fluorescent expression was observed. The results are consistent with the predictions, indicating that the glyoxylic acid operon experiment was successful.
Fatty acid bh operon:
No fluorescent expression was observed in No. 1 tube, No. 2 tube, No. 3 tube, and No. 4 tube.
analysis:
The results of this experiment are very strange. After discussion, our team came to the conclusion that, in view of the addition of arabinose and fatty acids in the No. 2 tube (opening the transcriptional channels controlled by the arabinose operon and the glyoxylate operon, respectively), There was still no fluorescence expression, while the No. 1 tube added only arabinose (opening the transcriptional channel controlled by the arabinose operon). We hypothesized that the fatty acid bh operon has the function of controlling (repressing) the transcriptional pathway, but the substrate (fatty acid) we added is not the correct inducer and therefore cannot open the transcriptional channel controlled by the fatty acid bh operon. We added another substrate to test the fatty acid bh operon and the fatty acid ec operon that had not previously worked.
Supplementary experiment
Change the substrate: fatty acid for hydroxy fatty acid to test fatty acid ec, fatty acid bh operon
Experimental results and analysis
Fatty acid ec operon:
Our main concern is the No. 1 tube, No. 3 tube, No. 5 tube and No. 6 tube (corresponding to the No. 1 tube, No. 2 tube, No. 3 tube, No. 4 tube of the previous experiment). No. 1 tube had fluorescence expression, No. 3 tube had fluorescent expression, and No. 5 tube and No. 6 tube had no fluorescence expression.
analysis:
The experimental results are consistent with the expectation that the No. 1 tube only added arabinose, which opened the transcription channel that may be controlled by the arabinose operon, but did not add glyoxylate, so it failed to open the transcription channel controlled by the glyoxylate operon. Transcription was not normal and No. 1 tube was not fluorescent. The No. 3 tube was added with arabinose and glyoxylate, which respectively opened the transcription channel controlled by the arabinose operon and the glyoxylate operon, so that transcription was normally carried out and fluorescently expressed. Neither the No. 5 tube nor the No. 6 tube was added with arabinose, and the transcription channel controlled by the arabinose operon could not be opened, so that no fluorescent expression was observed. The results are consistent with the expectation that the hydroxy fatty acid is a suitable substrate for the fatty acid ec operon and successfully validated our fatty acid ec operon.
Fatty acid bh operon:
None of the 6 tubes showed fluorescence.
analysis:
Based on an analysis of the previous experimental results for the fatty acid bh operon, we can conclude that hydroxy fatty acids are not a suitable substrate for the induction of the fatty acid bh operon and, therefore, the fatty acid bh operon experiment failed.
Qualitative analysis total results:
The effectiveness of the fatty acid ec operon and the glyoxylate operon was successfully verified, and the induced substrates were hydroxy fatty acid and glyoxylic acid, respectively. Therefore, based on the success of the experimental results, we have uploaded our fatty acid ec operon and glyoxylate operon (as composite part numbers: BBa_K2782012 and BBa_K2782011).
Fatty acid ec operon(to upload as a composite part number: BBa_K2782012)
Glyoxylic acid operon (to upload as a composite part number: BBa_K2782011)
View the corresponding composite parts
Quantitatively verify the results of the operon:
Fluorescence value is proportional to the relationship between time.
Experimental protocol: For the fatty acid operon, we designed four control groups to measure the fluorescence value. The first group only added arabinose; the second group added different concentrations of fatty acid after adding arabinose for one hour; The group only added gradient fatty acids; the fourth group was a blank control group.
Stage results: The data on the right is the fluorescence value measured for the first three hours after eight hours of bacterial culture. From this set of data, it can be seen that the fluorescence value of the second group is higher than that of the first group and the blank control group, indicating that the fatty acid operon plays a role.
Follow-up experiment: We want to knock out the Fade gene in E. coli in subsequent experiments to eliminate the effect of this gene on the detection of fluorescence. This experiment is in progress.
Experimental protocol: For the glyoxylic acid operon, we designed four sets of control experiments to measure the fluorescence value, the first group only added arabinose; the second group added glyoxylic acid after one hour of arabinose reaction; the third group B Aldehydic acid; the fourth group was a blank control group.
Stage results: The data on the right is the fluorescence value measured for the first three hours after eight hours of bacterial culture. From this set of data, it can be seen that the fluorescence value of the second group is higher than that of the first group and the blank control group, indicating that the glyoxylic acid operon plays a role.
