Team:WHU-China/Experiments/Lab1
This subgroup took over the most of the molecular biology experiments including: interlab, parts production and experimental verification of the first plasmid. We have separately verified each part of the final pathway and obtained convincing results. Moreover, we have additionally built and validated the pathways for biosafety.
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I.Light control system
1.Light-controlled promoter—CcaS/R system+PcpcG
2.Light-controlled promoter+ Not Gate pathway
II.Functional protein to collect Phosphorus
1.PPK—phosphorus accumulation
2.PPX,PPN—phosphorus release
III.Pathway for bio-safety—kill switch
1.MazF/MazE toxic-antitoxic switch
2.ParD/ParE toxic-antitoxic switch
I.Light control system
We have done the experiments to examine the function of our light control system. Our light control system aims to switch between expression states to express different proteins according to different lights. There are two components: light-controlled promoter and a Not Gate. Firstly, we will show the light controlled promoter works and then add a Not Gate to prove that the whole pathway can work.
1.Light-controlled promoter —CcaS/R system+PcpcG
Since our light controlled promoter PcpcG is regulated by CcaR ,which can be phosphorylated by CcaS, and CcaS is the protein that can sense the light(activated by green light and ceased by red light)[1]. We must express this two proteins before use the PcpcG promoter. To test this system, we use GFP as indicator.
Firstly, we transformed four plasmids to E.coli and added IPTG to induce the expression of the CcaS and CcaR. Then, we examined the GFP expression.
1.lac operator + CcaR + CcaS (As negative control)
2.lac operator + CcaR + CcaS + Cpcg (light control promotor) + green fluorescence protein
3.lac operator + CcaR + CcaS + Cpcg (light control promotor) + TetR + TetO + green fluorescence protein
4.constitutive promotor + GFP (As positive control)
Figure 1 This picture shows the fluorescence intensity after induction for 5h.
Inevitably, the light control switch had a leaky expression, but the leakage was low in a certain period of time.
Then, we cultivated the induced E.coli under green light, red light and darkness for 2h, and examined GFP expression.
Figure 2 This picture shows the result after triggering by different light (the result is evaluated by fluorescence intensity).
The bacteria transformed with light control system and GFP had obvious increasing in fluorescence intensity after activated by green light and the negative control (only have the light control system) had the similar fluorescence intensity under different lights.
After that, we transformed the green light into red light, vice versa. We kept the bacteria in darkness dim and cultivated them for 1h to examine the GFP expression.
Figure 3 This picture shows the result after transforming light (the result is evaluated by fluorescence intensity).
After transforming the light, we can see the fluorescence intensity were changed dependent on the different lights. Positive control and Negative control had similar data in all kinds of light. But light control system play significant role in the expression of GFP under different light conditions. In conclusion, red light can cease the PcpcG, and green light can re-start the pathway ceased by red light. That suggests our promoter can work cyclically.
2.Light-controlled promoter + Not Gate pathway
We did this pathway experiments together with the light controlled promoter, and now we can focus on the blue one:
Figure 4 This picture shows the fluorescence intensity after induction for 5h.
Figure 5 This picture shows the result after triggering by different light (the result is evaluated by fluorescence intensity).
The bacteria transformed with light control system , not gate and GFP had a few increasing in fluorescence intensity in the red light and darkness. What’s more, The fluorescence intensity in green light had a relative lower fluorescence intensity compared with the samples that got before activated by light.
Figure 6 This picture shows the result after transforming light (the result is evaluated by fluorescence intensity).
In the transforming light experiment, the not gate play significant role in the expression of GFP to reverse the trend.
Conclusion: The verification of the entire pathway illustrates the coupling of the light-controlled promoter and the Not Gate, and preliminarily demonstrates that our system can work cyclically. Although in this experiment, the data is still not perfect. Factors such as expression time should be considered more in subsequent experiments.
II.Functional proteins to collect Phosphorus
Since we decided to take phosphorus as example, we must find the functional proteins to be controlled by the PcpcG and Not Gate. Two functional opposite protein can do this job, One to accumulate the Phosphorus from the environment and the other release them to the Ark. We found PPK to accumulate, and PPX to release. Later we add a part-PPN to strengthen the efficiency of PPX. In this section, we will test the function of these three proteins.
1.PPK—phosphorus accumulation
After a careful information retrieval, we decided to choose the Polyphosphokinase (PPK) from Team Manchester_2017 and we test the efficient of it again.
Firstly, we found that this PPK gene originally exists in our E.coli.
Figure 7 This picture shows colony PCR results, the wild type control originally possesses PPK.
This suggested that PPK protein is compatible in our chassis, and transformation of the PPK was actually an overexpression.
Then, we tested the function of PPK, We did this through measuring the change in phosphorus concentration in the medium. And we made a calibration curve before.
Figure 8 The calibration curve of phosphorus
Figure 9 This picture shows the effect of phosphorus accumulation of the engineered bacteria which we have transformed extra PPK gene.
This experiment verifies that BL21 transferred to the pET28a plasmid containing PPK has a certain phosphorus-concentrating effect, and it is not caused by bacterial growth and natural PPK.
2. PPX,PPN—phosphorus release
We refer to PPX’s data from the Team 15_York. Because our system requires faster and more complete release of phosphorus, we added PPN as a protein to assist in the release of phosphorus. Thus we built BBa_K2789032.
PPN is a protein that originally comes from Saccharomyces cerevisiae, before the function test, we must verify that PPN protein can be expressed in E. coli.
Figure 10 This picture shows the SDS-PAGE result of the E.coli that we have transformed PPN. Track 2 and track 3 show that PPN is expressed.
Figure 10 with color
We demonstrated that PPN can be expressed in E. coli by gel electrophoresis experiment.
Then, we use the same method to test the function of BBa_K2789032.
Figure 11 This picture shows phosphorus release of the experiment group.
From the picture 11, we can see obvious differences between the test group and WT. The more phosphorus in the sample, the bluer it is. Thus, the concentration P in the test group is higher than the control. However, as we measured the OD600 in the media, the result is largely due to the concentration of the bacteria instead of the ppx with ppn. The graph shows here is calculated by (C_1-C_2)/OD .C1 is the phosphorus concentration right before the induction .C2 is the phosphorus concentration right after the 16h induction. OD is the OD600 value which indicates the concentration of the bacteria. From the result, we think that the induction may be so strong that the protein produce is at a very high level which is not good for the bacteria growth. But if we compare the induced BL21 and the not induced group, we can still see a difference.
Figure 12 This picture shows the quantative result of phosphorus release.
Because of the growth of the bacteria, it is difficult to see the dramatic changes of phosphorus concentration of the medium. Thus we used the staining method - PolyP is metachromatic granules in the bacteria, which can be stained. It is possible to judge whether or not PolyP is present in the bacteria by staining to determine whether our protein is functional.
Figure 13 a. shows the staining result of wild type BL21 (blue spot in the picture shows polyP); b. shows the staining result of E.coli with PPX expressed (no polyP); c. shows the staining result of E.coli with PPN expressed (no polyP)
We use Albert stain for metachromatic granules, and PPX and PPN have the ability to release phosphorus from E. coli, respectively. It can be seen that BL21, which has not been transferred into the gene, shows a distinct blue color, indicating the presence of the heterochromatic granules; while the E. coli that was transferred to PPN and PPX, respectively, was not stained blue, demonstrating the release of phosphorus.
Conclusion: we proved that PPK,PPX,PPN both had the function and PPN can additionally enhance the ability to release the phosphorus.
III.Pathway for bio-safety—kill switch
To prevent bacterial leakage, we designed two toxin-antitoxin pathways to ensure that bacteria will commit suicide once they escape from the environment we provide. We use riboswitch to identify the different environment, antitoxic is continuously expressed but it can’t prevent the killing effect of toxins on cells, unless the corresponding counterpart of the riboswitch, theophylline, is added, and the antitoxin can inhibit the toxicity of the background expression level of the toxin[2].
Prefix + lac operator + parE+ riboswitch +parD + suffix
Prefix + lac operator + MazE + riboswitch + MazF + suffix
1.MazF/MazE toxic-antitoxic switch
Because theophylline itself may be harmful to cells, as a substance that turns off the riboswitch, we first made a gradient dilution and found the mildest range for the bacteria.
Figure 14 This picture shows the effect of theophylline to the E.coli.
Although we used a lower concentration of theophylline in this pathway, we finally found that this pathway did not work.
2. ParD/ParE toxic-antitoxic switch
We performed a second pathway validation with a lower theophylline concentration.
Figure 15 This picture shows the result of ParD/ParE pathway
We can drew the results that this pathway can work and the best effect of theophylline concentration is 0.2g/L.