Team:BUCT-China/Demonstrate
Demonstrate
Major achievements:
（1） Complete assembly of basic parts (operator gene, fluorescent protein gene, vector plasmid gene, etc.) and successfully construct composite parts: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon)；
（2） Compound parts in trans10 strain: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon) functional verification；
（3） Preliminary quantitative tests for composite parts: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon)；
（4） Modeling of composite parts: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon)；
Results：
（1） Electrophoresis verification for constructed composite parts: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon)：
（2） At the same time, the constructed plasmid is verified by enzyme digestion and electrophoresis to ensure that the plasmid is safe.:
（3） Design comparison test to verify the function of composite parts: BBa_K2782011 (glyoxylic acid operon), BBa_K2782012 (fatty acid operon)：（Experimental condition）
a.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.
Figure 2: Experimental results of fatty acid ec operon
Analyse：
Combined with the experimental conditions of the fatty acid ec operon validation, the results are inconsistent with the expected, indicating that the fatty acid ec operon is ineffective；
b.Glyoxylic acid operon：
No. 1 tube and No. 4 tube have no fluorescence, and No. 2 tube and No. 3 tube have fluorescent expression.
Figure 3 shows the results of the glyoxylic acid operon experiment
Analyse：
Combined with experimental predictions, the results are consistent with the expected, indicating that the glyoxylate operon experiment was successful.
c.Fatty acid bh operon：
No fluorescent expression was observed in No. 1 tube, No. 2 tube, No. 3 tube, and No. 4 tube.
Figure 3 shows the results of the glyoxylic acid operon experiment
Analyse：
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.
（4） Supplementary experiments to change the substrate (the experiment of fatty acid ec, fatty acid bh operon, respectively)：
a.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..
Figure 5: Experimental diagram of fatty acid ec operon with substrate replaced with hydroxy fatty acid
Analyse：
The experimental results are consistent with the predictions that the hydroxy fatty acid is a suitable substrate for the fatty acid ec operon and successfully verified our fatty acid ec operon.
b.Fatty acid bh operon：
None of the 6 tubes showed fluorescence.
Figure 6 : Experimental diagram of the fatty acid bh operon after the substrate is replaced with hydroxy fatty acid
Analyse：
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 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).
Model establishment：
Fluorescence Model
In the case of adding arabinose to all samples, we set up eight fatty acids with different concentration gradients, which were added to the test tubes, and a set of fluorescence was measured every 10 minutes. The x-axis was the time after the addition of fatty acids, and the Y-axis was added. The concentration of fatty acids, the Z axis is the measured fluorescence value.
Through the model, we can see that the initial measured fluorescence value is relatively high, then it decreases sharply and then gradually increases. We speculate that this is caused by the Fade gene in E. coli, which will affect the added fatty acid and thus affect Expression of fluorescent proteins.
Biological Model：
The picture on the right shows the biological model of Fadr. In order to eliminate the influence of fadE gene on fatty acids, we used RED recombination system to knock out the fadE gene, then transferred it to our constructed plasmid, re-measured the fluorescence value, and verified the fatty acid ec operon.
Circuit model：
We all know that operons are the switch of gene expression, and synthetic biology means that people connect "genes" into a network, allowing cells to accomplish the tasks that the designer envisions. So we modeled the plasmid we constructed into a circuit network as a model. Of course, this is an ideal state model, and the factors in the cell will be more and more complicated.
A: add arabinose A=1
No arabinose A=0
B: fatty acid/glyoxylic acid B=1
No fatty acid / glyoxylic acid B = 0
S: The switch is turned on, representing the inhibition of gene transcription by the synthesis of a repressor protein representing the arabinose operon
OR1 gate: arabinose operon
Y=1 turns on transcription, followed by fatty acid operon repressor gene expression to produce repressor
Y=0 arabinose operon binds to repressor and blocks gene transcription
NOT gate: a repressor of the trans-acting factor fatty acid/glyoxylate operon, which hinders gene transcription after synthesis, cannot be synthesized, and does not hinder gene transcription.
OR2 gate: Switching effect of fatty acid/glyoxylic acid operon
X=1 fatty acid/glyoxylate operon does not bind to repressor protein, no hindrance
X=0 fatty acid/glyoxylate operon binds to repressor and blocks gene transcription
AND gate: continuity of prokaryotic genes, directionality of transcription.
Z=1 open
Z=0 transcription is blocked, fluorescent protein gene cannot be expressed, and fluorescent protein cannot be produced.