You have been allocated a cDNA accession number from the NCB' 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 using the Gibson assembly cloning method 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: Submissions are made on Turnitin in sections. The following sections to be submitted separately.
First submission : Section 1
Second submission: Sections 2 - 7
Third submission: 8 - 9
Final submission: 10 -11.
Please note: All submissions to be made before the due date of 19/10/2018
1. A completed assignment cover page (provided)
2. A 150 word description of the function of your allocated protein
3. A 150 word explanation of your cloning strategy
4. The gene sequence of your allocated sequence with introns and exons clearly highlighted
5. Plasmid maps for both plasmids
6. You must use the following plasmids (details available online) for cloning:
i). pEGFP-N1
ii). pH6HTN His6HaloTagT7
7. The translated sequence of your full length fusion proteins (using ORF Finder software) aligned with the coding sequence
8. 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.
9. 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.
10. A theoretical tryptic digest of your tagged/fused protein (no missed cleavage sites)
11. Results from MASCOT to demonstrate that the tryptic peptides obtained from your untagged protein actually correspond to your allocated cDNA sequence (you need to show results from both fusion proteins)
Attachment:- assignment.rar