Difference between revisions of "Team:NUS Singapore-Sci/Safety"
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In our project, we created a fusion protein (RESCUE editor) made up of dCas13b and rAPOBEC1, and a RESCUE reporter to indicate editing efficacy. Our fusion protein is designed to edit a specific RNA target from C-to-U. As the RESCUE editor targets only RNA, editing would be transient and reversible. To add on, the RESCUE editor consists of a ‘dead’ Cas13b, thus preventing cleavage of RNA strands. Our fusion protein gene was assembled together and cloned into a mammalian expression plasmid vector using competent <i>E. coli</i> cells. Subsequently, we transfected the fusion protein and a gRNA targeting a specific base on mRNA into the HEK293T human cell line to test the RNA editing efficacy <i>in vivo</i>. <br><br> | In our project, we created a fusion protein (RESCUE editor) made up of dCas13b and rAPOBEC1, and a RESCUE reporter to indicate editing efficacy. Our fusion protein is designed to edit a specific RNA target from C-to-U. As the RESCUE editor targets only RNA, editing would be transient and reversible. To add on, the RESCUE editor consists of a ‘dead’ Cas13b, thus preventing cleavage of RNA strands. Our fusion protein gene was assembled together and cloned into a mammalian expression plasmid vector using competent <i>E. coli</i> cells. Subsequently, we transfected the fusion protein and a gRNA targeting a specific base on mRNA into the HEK293T human cell line to test the RNA editing efficacy <i>in vivo</i>. <br><br> | ||
| − | Even without a gRNA, the RESCUE editor may also make non-specific edits. However, Cas13b targets RNA strands and not DNA. Therefore, any non-specific edits would not be passed on to the daughter cells. In addition, mRNAs have relatively short half-lives. Thus any lethal changes made would also be degraded quickly and will not be passed down to the next generation. | + | Even without a gRNA, the RESCUE editor may also make non-specific edits. However, Cas13b targets RNA strands and not DNA. Therefore, any non-specific edits would not be passed on to the daughter cells. In addition, mRNAs have relatively short half-lives. Thus any lethal changes made would also be degraded quickly and will not be passed down to the next generation. <br><br> |
| − | + | ||
| − | The RESCUE reporter system was cloned into a mammalian expression plasmid vector using competent <i>E. coli</i> cells. It was subsequently transfected and tested <i>in vivo</i> using a fusion protein made up of Cas9 and rAPOBEC1 to test for the functionality of the reporter system. | + | In such a case, our fusion protein may be used for targeted RNA editing. We can generate specific gRNAs to target RNA sequences and modify the RNA sequences in the body. This is also to minimise off-target editing. <br><br> |
| + | |||
| + | The RESCUE reporter system was cloned into a mammalian expression plasmid vector using competent <i>E. coli</i> cells. It was subsequently transfected and tested <i>in vivo</i> using a fusion protein made up of Cas9 and rAPOBEC1 to test for the functionality of the reporter system. <br><br> | ||
All bacterial work, transfections and cell cultures were done in certified BSL2 biosafety cabinets. | All bacterial work, transfections and cell cultures were done in certified BSL2 biosafety cabinets. | ||
</div> | </div> | ||
Revision as of 18:40, 15 October 2018
Safety
Safety is imperative for the smooth functioning and progress of our project, especially since it involves the use of a variety of biological agents. Therefore, the team views safety very seriously and has put in much effort to ensure all protocols are up to standard.
Biosafety in our project involves minimising the risks to the researchers working in the laboratory, as well as the general public in future medical applications based on our research.
In line with our emphasis on safety in the laboratory, we are proud to announce that we have obtained an ‘A’ grade for the annual Faculty of Science Housekeeping Inspection 2018 for lab safety over the summer of 2018, an achievement for four consecutive years. We endeavour to continue maintaining a safe working environment for all researchers.
Biosafety in our project involves minimising the risks to the researchers working in the laboratory, as well as the general public in future medical applications based on our research.
In line with our emphasis on safety in the laboratory, we are proud to announce that we have obtained an ‘A’ grade for the annual Faculty of Science Housekeeping Inspection 2018 for lab safety over the summer of 2018, an achievement for four consecutive years. We endeavour to continue maintaining a safe working environment for all researchers.
Safety when handling biological organisms
Non-pathogenic strains of E. coli such as BL21 and DH5α from Life Technologies and NEB Stable Competent E. coli were used for cloning of plasmids and expression of proteins of interest. Such strains have been modified such that the pathogenic gene islands has been removed and the strain used is considered non-pathogenic. These strains are Risk Group 1 and were handled in a BSL2 Biosafety cabinet. The mammalian human cell line, HEK293T is classified under Risk Group 2 and was also handled in a BSL2 Biosafety cabinet. Human embryonic kidney cells that have been transformed and immortalised with SV40 large antigen. Training for handling of mammalian cells or any new experimental protocol was provided by the principal investigator (PI) to all team members prior to the experiments.
Safety in project design
In our project, we created a fusion protein (RESCUE editor) made up of dCas13b and rAPOBEC1, and a RESCUE reporter to indicate editing efficacy. Our fusion protein is designed to edit a specific RNA target from C-to-U. As the RESCUE editor targets only RNA, editing would be transient and reversible. To add on, the RESCUE editor consists of a ‘dead’ Cas13b, thus preventing cleavage of RNA strands. Our fusion protein gene was assembled together and cloned into a mammalian expression plasmid vector using competent E. coli cells. Subsequently, we transfected the fusion protein and a gRNA targeting a specific base on mRNA into the HEK293T human cell line to test the RNA editing efficacy in vivo.
Even without a gRNA, the RESCUE editor may also make non-specific edits. However, Cas13b targets RNA strands and not DNA. Therefore, any non-specific edits would not be passed on to the daughter cells. In addition, mRNAs have relatively short half-lives. Thus any lethal changes made would also be degraded quickly and will not be passed down to the next generation.
In such a case, our fusion protein may be used for targeted RNA editing. We can generate specific gRNAs to target RNA sequences and modify the RNA sequences in the body. This is also to minimise off-target editing.
The RESCUE reporter system was cloned into a mammalian expression plasmid vector using competent E. coli cells. It was subsequently transfected and tested in vivo using a fusion protein made up of Cas9 and rAPOBEC1 to test for the functionality of the reporter system.
All bacterial work, transfections and cell cultures were done in certified BSL2 biosafety cabinets.
Even without a gRNA, the RESCUE editor may also make non-specific edits. However, Cas13b targets RNA strands and not DNA. Therefore, any non-specific edits would not be passed on to the daughter cells. In addition, mRNAs have relatively short half-lives. Thus any lethal changes made would also be degraded quickly and will not be passed down to the next generation.
In such a case, our fusion protein may be used for targeted RNA editing. We can generate specific gRNAs to target RNA sequences and modify the RNA sequences in the body. This is also to minimise off-target editing.
The RESCUE reporter system was cloned into a mammalian expression plasmid vector using competent E. coli cells. It was subsequently transfected and tested in vivo using a fusion protein made up of Cas9 and rAPOBEC1 to test for the functionality of the reporter system.
All bacterial work, transfections and cell cultures were done in certified BSL2 biosafety cabinets.