Team:THIS-China/Future work

Future Goals

During the experimental process, we discovered that it was nearly impossible to grow Bacillus subtilis under normal circumstances, and the corresponding competent cells were extremely unstable. However, the membrane proteins comD and comE can be originated in bacillus subtilis. Locating the comD protein on E. coli’s membrane has become a great challenge for us. In order to solve this problem, we performed some extra research and present some possible solutions for future teams.

Changing the Host - S. mutans

During the research, we learned about the comCDE system inside of S.mutans. The comC proteins are required to send out the CSP signal in order to be sensed by other S.mutans bacteria. The comDE system senses the CSP signal and activates the toxin to kill all of the other bacteria and create a plaque full of S. mutans. Therefore, this gives us the idea of directly using S. mutans as the host. We can knock out the comC gene in the S.mutans so the S.mutans does not properly produce CSP. Then, we can add in the reporter gene cotA under the promoter nlmc to make sure it will be activated by the comDE system and show color degradation. This method allows us to work around the problem of comD not getting onto the membrane.

Figure 1: The S. mutans signaling circuit.

Nanodisk Technology – Creating a Cell-Free System

Due to failures in determining the position of membrane proteins, the membrane protein comD was unable to trigger comE to phosphate. In order to solve this existing problem and establish a cell-free system, we have come up with some troubleshooting and goals of future research. Thermofisher’s scientific Membrane max protein expression kit expresses membrane proteins without the existence of bacteria. After expressing our target membrane protein, we stabilize these proteins using Nanodisk technology—a disk-shaped protein with a similar biological structure as a lipid bilayer, surrounded by proteins, peptides, and synthetic polymers. The comE and cotA proteins can be expressed using Thermofisher’s scientific Expressway protein expression kit. By mixing the stabilized comD protein with the comE and cotA proteins, these proteins will connect to their corresponding binding sites and form a long chain of proteins that will transmit specific signals to trigger biological changes.

This procedure prevents all possible biohazards and reduces the cost of manufacturing products. In addition, we would solve the problem of being unable to preserve membrane proteins in harsh environments by stabilizing them using nanodisks. However, successfully fusing the nanodisk and our target comD membrane protein has not yet been demonstrated to be feasible, and it has been said that this process is so challenging that only a few labs have achieved the expression of proteins without the involvement of bacteria.

The Thermofisher MembraneMax and Expressway protein expression kits can be replaced by normal protein expression in E. Coli, and synthesized into larger chains of linked proteins after target proteins are extracted from E. Coli by professionals.

Protein Fusion Method – Expression Within Cell

In addition to the nanodisk technology method mentioned above, we conclude that protein fusion may also be a great choice for this experiment. By regulating protein translation, we can code the genetic information of our comD membrane proteins connected to the comE and cotA proteins to form one large chain of proteins connected to each other. This method decreases the hassles involved in the previous method and increases stability by decreasing the needed protein connection. Unfortunately, this experiment has not yet been justified and will serve as a future research goal.