Integrated Assignment
You have been allocated a cDNA accession number from the NCBI database (see table below on page 2). Your task is to design experimental strategies that will allow you to: clone the cDNA in different expression vectors and show the protein products; knock out the function of your target using CRISPR; simulate a mass spectrometry analysis of your protein using a tryptic digestion and MASCOT analysis.
This assignment is designed to be completed throughout the semester, alongside the various lecture blocks. You should be able to perform each task immediately after you have learnt the appropriate techniques in lectures and practiced the analysis in workshops. If you leave it all until the final week of semester, you may find this assignment very, very difficult.
You must include the following in your report which must be submitted via LMS (word document) before 5pm Thursday October 26th.
Please submit the following:
- A completed assignment cover page (provided)
- A 150 word description of the function of your allocated protein
- A 150 word explanation of your cloning strategy
- The gene sequence of your allocated sequence with introns and exons clearly highlighted
- A restriction map of your coding region (include a list of non-cutting enzymes)
- Plasmid maps for both plasmids
- You must use the following plasmids (details available online) for cloning: i). pEGFP-N1 andii). pH6HTNHis6HaloTagT7
- The translated sequence of your full length fusion proteins (using ORF Finder software) aligned with the coding sequence
- The results of a BLAT IT analysis of your gene:On your gene sequence, highlight a region suitable to knock out using CRISPR and explain your choice.
- Primers suitable to knock out the region you have chosen using CRISPR. Give forward primer sequence, reverse primer sequence and clearly indicate where they will bind on your gene sequence.
- A theoretical tryptic digest of your tagged/fused proteins (no missed cleavage sites)(you need to show results from both fusion proteins)
- Results from MASCOT to demonstrate that the tryptic peptides obtained from your untagged protein actually correspond to your allocated cDNA sequence (HINT: Use the results from the digestions above to work out which peptide masses to analyse).
Attachment:- Assignment Files.rar