Moloney murine leukemia virus reverse transcriptase
Even though RT does the function of reverse transcription, what is expressed in the cell is its polyprotein version. The gag-pol polyprotein has four parts—capsid protein, protease (stop codon separated), reverse transcriptase, and integrase. The integrase is deleted from the polyprotein to eliminate the possibility of genome interference. The protease contains a UAG stop codon at its 5th amino acid site1, which is readthrough as glutamine at a 5% efficiency in its native host cells, to enable the 20:1 ratio between capsid and reverse transcriptase protein. As its readthrough efficiency is much lower in E. coli cells, and studies have shown that lower efficiency greatly damages the activity of reverse transcriptase2, we mutated the UAG codon into CAG, making a complete readthrough of the polyprotein. This will slightly decrease the activity of RT but within an acceptable range. The capsid protein is necessary as it has been found to promote the annealing of tRNA primer to the primer binding site (PBS) in MMLV, and plays an important role in the following two strand transfer steps3,4. To be certain of this design, we have consulted Prof. Alper through mail and received his confirmation on the necessity of the capsid protein.
To increase the mutation of our RT, we built a mutated version (Y1245F). This mutation has been shown to increase the mutation level by 5 times5.
The gag-pol polyprotein is placed under Lac operon, whose expression controlled by IPTG.
Anti-microbial resistance (AMR)
Our experiments are all performed in a standard laboratory where all the equipment and reagents needed are prepared and placed appropriately. We have the dedicated refrigerators to store our experimental materials such as plasmids and primers. There is also a cabinet to store the hazardous reagents which is usually locked and only the PI has the key. To make sure all the processes are operated correctly and canonically, everyone in our team has been trained in experiment skills by experienced colleagues before we started our project. There is also a laboratory safety knowledge test organized by our university to ensure all the members joining our project are qualified for performing experiments. Everyone performing the experiment must wear the lab coat and nitrile gloves all the time in the laboratory.
Simultaneously, a lot of efforts have been made to prevent contamination.
All the experiments that might cause direct contact between the bacteria and the environment are performed in the clean bench, which is sterilized with 75% ethanol and ultraviolet light before and after each operation. We also seal our culture medium and plates with parafilm before putting them into refrigerators. All the vessels that has been in contact with microorganisms are sterilized after use to keep them away from the environment.
At the same time, as the ethidium bromide used to visualize double stranded DNA can be toxic at high concentrations and poses potential risks to the experimenters’ health, there is a special area divided from the console to operate the experiments related to ethidium bromide. In that area, experimenters must wear an additional pair of plastic gloves when contacting with the materials containing ethidium bromide.
Furthermore, we have the laboratory waste bins which will be emptied regularly to collect the normal waste. There are also special containers for contaminative and toxic materials such as agarose gel containing ethidium bromide. All the waste will be treated uniformly by specialists at regular intervals.