AraC and pBAD promoter expression part
Fusion expression of AraC and proinsulin
In Pathway 1 + Pathway 3 system, we used a final concentration of 0.5 mmol/mL IPTG to induce expression of the AraC protein in Pathway 1. An increase in the expression of the AraC protein inhibits the pBAD promoter in Pathway 3, resulting in a decrease in the expression of the downstream GFP protein, manifested as a decline in GFP/OD600. Our expectation was that GFP/OD600 for three kinds of E.coli (Normal, Mutation I (which replaces 12 bases of the human proinsulin and AraC fusion protein sequence) and Mutation II (which deletes 8 bases of the human proinsulin and AraC fusion protein sequence )) should all decrease as the induction time increasing. Due to the structural abnormalities of AraC protein caused by the mutations, the AraC inhibition effects of Mutation I and Mutation II should be lower than the Normal one, and the GFP/OD600 should be higher.
In our results, the blank control never showed fluorescence as the induction time increasing, successfully eliminating the interference of unrelated factors. In Normal, Mutation I, and Mutation II, the GFP/OD600 decreased with the prolongation of IPTG induction, which were consistent with our expected results. The GFP/OD600 at different induction times showed Mutation II > Mutation I > Normal, which was also in accordance with our expectation.
mRNA switch part
Bacteria internal experiments
As we expected, the Normal E.coli should not glow fluorescent because the certain 12bp sites in insulin proteinogen combined with complementary sequences in Pathway 2.However, in the case for MutationⅠand MutationⅡ E.coli, it should glow flurescent as for sequences mutation.
But according to our results of bacteria internal experiments, the Normal, MutationⅠand MutationⅡ E.coli all could glow bright fluorescent within 1-12 hours, which means unfortunately mRNA switch had not work.
We proposed two possible reasons for the outcome after discussing with professors and analyzing the results. (1)12bp mRNA bounding site is not enough for mRNAs’ interaction. (2)The efficacy of this switch is poorly guaranteed. We continued to design a series of experiments to test it.
Endocytosis experiments
The results showed that if 12bp mRNA were added to the medium at the concentrations of 0.005μM/μL, 0.01μM/μL and 0.03μM/μL, the fluorescence intensity of bacteria containing the Pathway 1 + Pathway 2 system would become increasingly lower with the incubation time, indicating that bacteria could absorb short mRNA under normal conditions and the mRNA interaction could inhibit the expression level of GFP. We also found that each generation of bacteria could continuously absorb it to maintain an overall continuous decline in GFP/OD600.
mRNA transformation experiments
We used mRNA transformation assay to artificially transform mRNA into cells and detect fluorescence intensity during the recovery period and the breeding period. As expected, bacteria can emit fluorescence, and its intensity decreases with time. The results showed that the fluorescence intensity per unit volume would decrease in 0-150 minute of the 12bp and 24bp mRNA transformation experiments. This proved that the simple 12bp and 24bp mRNA sequences could bind to certain sites in the mRNA transcribed from Pathway II and serve as switches. It meant that our vision of mRNA switching was possible to some extent.
However, we also found that the efficacy of the mRNA switch did not improve with the increase of mRNA concentration. The possible reasons may lay in the low transformation efficiency or the length of the mRNA binding sites. We will design further experiments to test the optimal length of mRNA binding site in the case of strain self-expression to truly reflect the efficiency of our switch.
Guard Module
Verification of Plasmid Construction
We inserted the gene fragment into the plasmid ptr99a in two steps by restriction endonuclease cleavage and ligation. Then we verified the correctness of plasmid construction and product length by colony polymerase chain reaction and gel electrophoresis. The gel electrophoresis pattern is as follows:
OmpT Function Verification
We inserted the OmpT signal peptide sequence at the upstream of mCherry, the plasmid was verified by colony PCR. The result is as follows:
We transformed E.coli DH5α with our validated plasmid and carried out the function verification experiments as we described in the Experiment section. At first, we attempted to take pictures of secreted mCherry with fluorescent microscope. However, the fluorescent images didn’t show much difference between test group and control group. The images are shown below:
Therefore, we did further exploration and redesigned our verification plan. We carried out more accurate testing experiments with microplate reader and carefully analyzed the mCherry fluorescent intensity data of the test group and the control group.
Here we used the method of paired sample t-test to compare the differences between the two groups of data. And the p-value we got is 1.665e-07 which was far less than 0.01. Therefore, we concluded that there were significant differences between the two groups of data, which successfully confirmed the OmpT guidance on mCherry extracellular secretion. The data of fluorescent intensity measured by microplate reader is as follows:
Promoter Ptrc Examination
We obtained two plasmids in which the green fluorescent protein was located at downstream of the promoter Ptrc. The difference between them is whether they contains lacI. To verify the construction of the two plasmids, we adopted colony PCR,and the gel electrophoresis profiles of the plasmid with or without lacI are as follows:
We then transformed E.coli DH5α with the two kinds of plasmids respectively and measured the intensity of green fluorescence with fluorescence microscope and microplate reader. E.coli with lacI expression shows significantly less green fluorescent density, which proved that lacI can inhibit the downstream transcription of Ptrc. Below is the images observed under fluorescence microscope with or without lacI.
Toggle Switch Verification
When a certain amount of synthetic AIP was applied to the bacterial solution, red fluorescence was observed under the fluorescence microscope(Figure12.A). After a period of time, red fluorescence disappeared, and green fluorescence was observed under the fluorescence microscope(Figure 12.B).
We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.
The corresponding curve is shown below:
Verification of killing pathways and parts
When guards perceive AIP signals, they would secrete mCherry outside of the cell, which could trigger the cheater killing pathway. In this part of the experiment, we were intended to verify the mCherry's killing effect on cheaters.
1.Verify the correctness of the plasmids:
The PCR products of bacterial colonies were verified by gel electrophoresis to determine whether the strip length was correct. Plasmids were extracted and verified by enzyme digestion. The fragment are then sequenced.
The gel electrophoresis bands of the two plasmids were as follows:
Through the above two gel electrophoresis stripes, we can see that the stripes are correct, which means that our plasmids are correct as well.
2.Conduct confirmatory experiments:
We expanded the culture of bacteria containing plasmid-G, and divided these bacteria into several groups. Then we added mCherry protein solution to one of them, made mountings and observed the fluorescence using the fluorescence microscope. Subsequently, we set the concentration gradient of mCherry protein solution, and measured the fluorescence intensity of GFP under different mCherry protein concentration using microplate reader.
Figures under fluorescence microscope before and after adding mCherry protein solution are as follows:
The GFP production represented the switching on of the cheaters’ killing pathways. mCherry conglobation leads to a positive feedback regulation. Once cheaters bind to one mCherry protein molecule, they tend to bind more . And the result is consistent with the prediction of our modeling.
The fluorescence intensity of GFP under different concentrations of mCherry protein solution is shown below:
The fitting curve showed that the expression of GFP may improve with the increase of mCherry. This proves that the intensity of the killing pathways may be enhanced when the concentration of mCherry protein grew higher.
We expanded the culture of bacteria containing plasmid-B, and divided these bacteria into several groups. According to the data measured in the previous experiment, mCherry protein solution of appropriate concentration was added to several groups of bacteria and incubated for a period of time. The remain was added with buffer as control. Afterwards, we applied the culture medium to the solid medium to form single colony. Then we counted the number of growing colonies and calculate the mortality, so as to obtain the killing efficiency.
The colony growth after adding mCherry or buffer solution are as follows:
Through spreading plate verification, we found that most cheaters died in our system, which indicated that our platform possesses high killing efficiency. Additionally,, we find that our killing efficiency has reached 90% through further calculation.
Verify interaction between the cheater and the guard
We cultured a certain amount of cheaters for a period of time, and then put a certain amount of cheaters’ culture medium into the guard's culture medium. Finally, we measured the concentration of mCherry protein produced by the guard with a microplate reader.
The concentration of mCherry protein produced by guards is shown below:
According to the above figure, we can see that cheaters can produce AIP, which will stimulate guards to produce mCherry protein subsequently.
Measure the environmental capacity and glucose consumption of three roles (worker, guard and cheater)
1.Measure the environmental capacity：
The same amount of three bacteria was inoculated in a certain amount of LB medium. Then we used microplate reader to measure the value of OD600 every 30 minutes. We continued the detection until stability has been reached. Through modeling, we obtained the value of environmental capacity.
The fitting curves are as follows:
2.Measure glucose consumption：
The environmental capacity of the three bacteria was inoculated into SOC culture medium, and samples were taken once every 5 minutes. After the D-glucose content determination kit was used, the OD505 reaction value was performed with a microplate reader, and the absorbance was measured to obtain the glucose concentration in the sample.
The sugar consumption table is shown below:
