Team:WHU-China/Demonstrate
Introduction
For many precious resources we are using now, on the one hand, they act as vital materials and show a great side; on the other hand, they become dangerous evil to the environment once the improper emission or leakage happens. We call these kind of resources coin chemicals (CC).
In this project, we use algae-bacteria symbiotic system to assist engineered bacteria in order to recycle phosphorus (a typical CC) from polluted water.
The bacterial algae biofilm has already been an appropriate system for advanced sewage treatment. It can provide a micro-environment for bacteria to grow well and enable them to perform many information communication.
Design
We designed to pathway this year.
1. the first plasmid—the element collection pathway
When green light is given, PcpcG is activated, PPK is expressed,and NOT GATE make the downstream PPX,PPN silent, so only PPK is expressed. When red light is given, PcpcG is stoped, PPK silent,and NOT GATE make the downstream PPX,PPN expressed. PPX,PPN expressed only.
PcpcG: the green light-induced and red light-cutout promoter PPK: polyphosphokinase, can catalyze the synthesis of PolyP. it is just like a glue to string phosphorus and store them as PolyP. PPX, PPN: exopolyphosphatase and endopolyphosphatase, are two different proteins that can catalyze the degradation of PolyP.
2. the second plasmid—the construction of biofilm
We use surface display technology to express lectins on the cell membrane to create an extra connection between the EPS of algae and bacteria
we use the “flagellum display ” to express lectin in the surface of cell to create an extra connection between the EPS of algae and bacteria
we use surface display technology to express dCBD in the cell membrane.
Experiment:
Light-controlled promoter —CcaS/R system+PcpcG
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)
Then, we cultivated the induced E.coli under green light, red light and darkness for 2h, and examined GFP expression.
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.
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.
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.
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.
Functional proteins to collect Phosphorus
1.Function of ppk
we tested the function of PPK, We did this through measuring the change in phosphorus concentration in the medium.
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 PPX+PPN pathway.
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.
We demonstrated that PPN can be expressed in E. coli by gel electrophoresis experiment.
By induction 2, its average phosphorus uptake capacity is significantly reduced, indicating that phosphorus is released.
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. 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.