Difference between revisions of "Team:Fudan-TSI/Safety"

• MMLV-RT’s enhanced version is commonly used in in vitro reverse transcription, which guarantees the safety and well characterization of this part.
• Unlike the reverse transcriptase of HIV, MMLV-RT acts as a monomer, this brings less trouble to its production in an heterogenous host.
• MMLV is a eukaryotic virus, which means it’s orthogonal to prokaryotic species. This orthogonality makes up of the host adaptability of our system.
• MMLV-RT has a higher processivity in relation to other viruses’ RT such as HIV and AMV. This feature gives our system a greater range of mutagenesis length.
• MMLV-RT has low primer specificity, which means that if we change the sequence of its primer and its corresponding primer binding site (PBS), unlike HIV, the change will not be reverted in the following reverse transcription process. This makes it possible for us to customize the sequence of the tRNA primer used in accord with the target sequence in our software.

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.

We used both original and mutated version of reverse transcriptase (RT) from Moloney murine leukemia virus. The mutation is made to enhance the error-prone ability of the reverse transcriptase and poses no risk to the experimenters or the environment. When expressing the gag-pol polyprotein with the reverse transcriptase in it, we made sure that the Integrase fraction was removed, so that the construct does not affect host cells’ genome. The capsid protein and reverse transcriptase are both not infectious on its own. In our project, the RT will strictly reverse transcribe the sequence we assigned as it is acting in a non-native system and other mRNAs do not contain its initiation sequence. To know more about it, please check our check-in forms.

Ampicillin resistance and kanamycin resistance have been introduced to E. coli strains used in our project as selectable markers to examine whether the plasmids carrying the resistance gene are transformed into the bacteria successfully. We also use chloramphenicol resistance to prove and evaluate the efficiency of our system. As all the microorganisms used in the project need to be strictly kept in the laboratory, we made certain that the antibiotics resistance poses no threat to the ecological environment.

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.