Our project observes all the safety rules and policies required by iGEM organizers. Safety has been carefully and thoroughly considered throughout our project design and experimental operation. We made sure to keep all the microorganisms used in our project in control and avoid the unintentional exposure to pathogens and toxins, or their accidental release. Our project remains at an experimental stage and any application outside the lab is strictly forbidden.

Design

E. coli

To accomplish molecular cloning and obtain the designed plasmids, E. coli strain T-FAST and DH5α have been used in our project. Both of them are non-pathogenic and can only live in provided environment. E. coli strain BL21 has been used for construct test. All the strains of E. coli used in our project are in Group 1 and have been handled properly in Safety Level 1 and 2 laboratories.

Reverse transcriptase

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 form.

Anti-microbial resistance (AMR)

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.

Working safely

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 experimental 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.