Team:WHU-China/Design
In this section, we will show our design—how does our pathway work. Since P is one of the most classical CC in environment and causes local pollution so We choose phosphorus as example to test this pathway this year.
In our project, we transform two plasmids into bacteria and they render two amazing function to our bacteria.
1.the first plasmid—the element collection pathway
As we have mentioned before, our Ark need to function as a vacuum cleaner— collect and reserve CC in polluted water body. This requirement demands our engineered bacteria to have this character: work periodically based on given signal.
This picture shows how our bio-conveyor work. The functional part of the bio-conveyor is the biological part on it—biofilm which contains engineered E.coli.
To collect phosphorus, bacteria must express different protein in different time and place. Thus, bacteria can absorb phosphorus in environment and release them in Ark’s interior to accomplish its collection mission.
To control that behavior, we design a control pathway. Once we give a signal, bacteria can express a corresponding protein and if we give another signal, it can express another protein. That means it can express different protein periodically by our control. The pathway looks like a “Tai Chi diagram” and this running loop expresses ancient Chinese philosophy towards the harmonious cycle of the universe.
We use light as a control signal
Since we want it to work periodically in order to control its expression, we choose a light-induced system to control it instead of chemicals. Because the chemical signal may accumulate in the bacteria which will cause the system gradually collapse in the process of recycling, but light won’t accumulate(We simulated the process through modelling, see our modelling.).
We found the green light-induced and red light-cutout promoter — PcpcG[1][2]. After transformed a two-protein(CcaS+CcaR) pathway, the coding sequence downstream the PcpcG can be expressed by green light, and ceased by red light.
How does the PcpcG+CcaR/S system work?
CcaS can sense the green light, and then dimerize and activate the CcaR, which is a transcription factor of PcpcG. Then the pathway on. This process can be ceased by red light since it can stop the dimerization of CcaS.
How to build the pathway?
To make it work better, we must simplify the pathway as much as possible. Finally we find a light-induced promoter plus a NOT GATE is enough to control the periodically expression.
To collect different CC, we must find the protein correspondingly, one to absorb and one to release that CC. This is a standard procedure. This year we chose the phosphorus as an example(click HP to see why), and we found the corresponding protein—PPK to absorb the phosphorus and PPX,PPN to release phosphorus.
What is PPK?
PPK , short form of polyphosphokinase, can catalyze the synthesis of PolyP. it is just like a glue to string phosphorus and store them as PolyP.
What is PPX,PPN?
PPX,PPN(exopolyphosphatase, endopolyphosphatase), are two different proteins that can catalyze the degradation of PolyP[3], thus they together just like scissors and can reverse the reaction of what PPK catalyze and release the phosphorus.
Here shows the whole pathway we build this year:
More specifically, 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.
More specifically, 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.
Through this pathway, we can control the expression to collect phosphorus. This design can also apply to collect other CC as long as we find other two proteins can work opposite and cooperately to collection the CC we want!
2. the second plasmid—the construction of biofilm
Why we use symbiotic biofilm?
As mentioned earlier , algae is a perfect match with bacteria especially in swage treatment—algae can give bacteria resistance to the environment and energy they needs.
There are two kinds of symbiotic system of algae and bacteria—suspended system and biofilm system. In order to fix the organisms into the track to form a conveyor, We choose the biofilm system.
Why we use symbiotic biofilm?
Biofilm is a syntrophic consortium of microalgae and bacteria. These adherent cells are embedded in a slimy extracellular matrix which is composed of extracellular polymeric substances(EPS). EPS, secreted by microalgae , fill the entire biofilm, adhere to the surface of organisms in biofilm and connect them together. It is EPS that mainly conduct the mission of protecting the bacteria. Biofilm provides a micro-environment for bacteria to grow well and enable them to perform many information communication.
The bacterial algae biofilm has already been an appropriate system for advanced sewage treatment. We learned this technology from institute of .hydrobiology, Chinese academy of science.
Why we build pathway?
Biofilm is a very suitable candidate for our system. We were very excited and set about the experiment related to biofilm immediately. Later we found a tough problem and looked up the papers. The same problem occurred in biofilm research and application areas nowadays—the biofilm of symbiotic system are very loose and easily detached from the surface, which make the system unstable.
Thus here we plan to use synthetic biology method to solve this problem. The detach happens when the connection between the algae and bacteria breaks. Natural connection between them is EPS, those poly substances can not only protect the bacteria but also play a important role in building the biofilm. Our method is to build an extra and direct connection between two kind of organisms. We design three parallel pathways to do that job.
EPS has a high density of negative charges. We found a protein called lectin, which can greatly bind the EPS through its high density of opposite charges.
We use surface display technology to express lectins on the cell membrane to create an extra connection between the EPS of algae and bacteria
Similar to the first pathway ,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
Similar to the first pathway , we use surface display technology to express dCBD in the cell membrane. dCBD(double cellulose binding domain) can directly bind the cell wall of algae since the cell wall is made of cellulose. This is a directly way to connect two kinds of organisms.
Three pathways are parallel relationships. We don’t know the best way to solve the problem before the experiment. For the sake of insurance, we make three pathways to compare the effect each other and finally choose the best one.