Task 1
Design a hypothetical peptide for recombinant expression in the designated host organism (mammalian/plant/yeast/insect/worm) using your name as the amino acid sequence.
For example, the amino acid sequence for John Smith, using the one-letter code would be: JOHNSMITH.
If your name contains alphabets that are not part of the 20 common amino acids, use the following table for codon assignment:
1.1 Produce the back-translated DNA sequence for your hypothetical peptide in the form that is ready to be expressed. The sequence should be provided in FASTA (plain text) format instead of in graphical form to allow for sequence verification by the tutors.
HINT: consult the codon optimisation tutorial and think of special codons that are relevant during translation.
1.2 Provide screenshot of program output and URL of programme(s) used for codon optimisation. Be sure to include parameters used, starting with specifying the codon usage profile.
Task 2
Design a vector construct for amplification of your recombinant DNA in E. coli.
2.1 Select a plasmid backbone best suited for amplification of the recombinant DNA. Use the Addgene plasmid repository (https://www.addgene.org/) for this task and provide the plasmid name and catalogue number.
2.2 Pick a suitable E. coli strain for the cloning of the DNA sequence made, explain your choice.
2.3 Justify your plasmid selection for this task.
2.4 How are you going to select for positive E. coli transformants? Explain your strategy.
Task 3
Design a vector construct for the expression of your recombinant DNA, generated in largf quantities in Task 2, for expression in your designated host.
HINT: You still have to amplify the vectors first... refer to Helena's recent lectures for information.
3.1 Select a plasmid backbone best suited for the recombinant expression of the DNA encoding your namesake peptide in your host organism. Use the Addgene plasmid repository (https://www.addgene.org/). Justify your choice.
3.2 You have isolated a few transformants after a successful transformation. Describe a strategy to verify integration of the recombinant DNA into the host gDNA and design a set of PCR primers for this task. Discuss salient features starting with the orientation and complementarity of the primers.
3.3 In order to facilitate extraction and purification of the recombinant peptide, you can incorporate a purification tag (as indicated by the purification method assigned to you) into your DNA construct. Describe how this can be done by PCR and design a set of primers for this task.