Title: Bioengineering of synthetic internal ribosomal entry site (IRES) for efficient bicistronic expression for application in genome editing and gene therapy
In contrast to polycistronic transcripts in prokaryotes, eukaryotic mRNA generally only express a single gene. Several strategies, such as ribosomal skipping peptide sequences or internal ribosomal entry sites (IRES), have been used to create bicistronic transcripts. However, their efficiency has been underwhelming. Inspired by naturally existing sequences from viruses and human genes, we will screen and determine small nucleic acid sequences for efficient use in viral production, genome editing constructs, and gene therapy.
The projected difficulty is suitable for undergraduates while providing a sufficient challenge that facilitates the student’s learning and growth. The student will be exposed to a research laboratory environment and learn vital molecular biology methods (PCR, cell culture, molecular cloning, bacterial culture, DNA and RNA preps, western blot, flow cytometry, transfection, viral transduction, etc.). The experience and mentorship from this project will prepare the student for a successful FYP and eventual employment or graduate degree pursuit.
We are looking for students who:
1) is highly motivated and passionate about learning;
2) takes ownership of their work and progress;
3) is adaptable in a fast pace environment; and
4) being a contributive team member.
1) Molecular cloning of human 5’ UTR and viral sequences in plasmid constructs
2) Screen these sequences for IRES activity and the effect on upstream and downstream genes
3) Create a truncation construct to screen for a minimal functional portion of the IRES
4) Application of these characterized IRES in lentiviral and AAVs constructs for efficient reporting of gene expression and transduction
Field of Research:
Molecular Biology, Bioengineering, Biochemistry, Cell biology, Synthetic biology, Genome editing
- Polymerase chain reaction (PCR)
- Molecular cloning via gene fragments assembly
- Bacterial transformation
- Bacteria culture
- DNA, RNA, Protein preps
- Cell Culture
- Flow cytometry
- Western blot
- Quantitative PCR
Successful engineering of short and efficient IRES sequences would serve as excellent tools to report gene delivery. Furthermore, multicistronic transcript would help alleviate the size limitation of common gene therapy vectors such as AAVS.