Demonstrate

This year we plan to verify the two systems. We did series of experiments to demonstrate that our system can work.
We plan to did these experiments to demonstrate:
1.verify the light control system
  1.verify the light-induced promoter-CcaS/R system and promoter cpcG
  2.verify the light promoter+ Not Gate
2.verify the phosphorus accumulation and release protein-PPK,PPX,PPN
  1.verify the PPK’s function
  2.verify the PPX+PPN’s function
3. Establish a symbiotic suspension system of algae and bacteria
  1. Determine the optimal ratio of bacteria to algae
  2. Determine the optimal conditions for symbiotic culture
4. Establish a means of forming symbiotic biofilm
5. Establish a means to quantitatively measure the stability of biofilm
6. Enhance biofilm stability with three synthetic biological pathways

1.verify the light control system

1.verify the light-induced promoter-CcaS/R system and promoter cpcG+

2.verify the light promoter+ Not Gate

We could draw the results that GFP have some leaking risks though we haven’t activated the bacteria with light.

The bacteria transformed with light control system and GFP have obvious increasing in fluorescence intensity after activated by green light and the negative control (only have the light control system) have the similar fluorescence intensity in the different lights.

The bacteria transformed with light control system , not gate and GFP have a few increasing in fluorescence intensity in the red light and darkness. What’s more, The fluorescence intensity in green light have a relative lower fluorescence intensity compared with the samples that got before activated by light.

After transforming the light, we could see the fluorescence intensity are mostly dependent on the different lights. Positive control and Negative control have similar data in all kinds of light. But the not gate and light control system play significant role in the expression of GFP under different light conditions.

2.verify the phosphorus accumulation and release protein-PPK,PPX,PPN

1.verify the PPK’s function

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.verify the PPX+PPN’s function

The picture shows the WT stains darker than the ppx and ppn which means the ppn and ppx strain have lower phosphorus concentration. The photo is taken with same parameter at 1000×.Each group has 3 sample，they show the similar results with the picture here. The ppx and ppn strain are constructed on the pET28a(+) vector, also using IPTG to induce the expression. The result is acquired after 72 h induction.
PPX and PPN works well due to these experiments

3.verify the PPN’s function and demonstrate that the PPX+PPN can do better than PPX alone

It is a pity that we have not done the experiment in phosphorus absorption in PPX alone, and also the staining experiments can’t quantitatively illustrate how PPN can accelerate the reaction. To make up for this vacancy, we used modeling to simulate this process.

3. Establish a symbiotic suspension system of algae and bacteria

1. Determine the optimal conditions for symbiotic culture

We did a series of gradient experiments and finally determined that the culture conditions of the mixed system were LB: BG11=7:1

2. Determine the optimal ratio of bacteria to algae

We did a series of gradient experiments and finally determined that the ratio of bacteria to algae should be 1:1-1:10

4.Establish a method to form symbiotic biofilm

We use suction filtration to obtain biofilm

5.Establish a means to quantitatively measure the stability of biofilm.

After so many experiments, we used blender device and shear force as interference(250r/s,3min). And we used chlorophyll method and hemocytometer counting method to test the shedding rate. This together can build a quantitative measurement to test the stability of biofilm.

6. Enhance biofilm stability with three synthetic biological pathways

This picture indicated that our binding part worked well and showed it amazing potential in binging field. We felt quite pity that due to time limit, we hadn’t explored the best experiment condition, and we will optimize this in the future.

Although we verified each part to work successfully,we still planned to combine the PPX,PPN and PPK into the light control pathway to further illustrate that our bacteria can absorb P in green light and release it in red light. However, DDl was around the corner. We designed the primers in October but didn't catch up to assemble the entire pathway. Although we have verified all the components and they worked well, we will still continue to complete the verification of this huge pathway ,maybe in the next iGEM season.

Team:WHU-China/Demonstrate