BLAST Topic - (Nov/16/2010 )

• Task 1. The protein sequence provided below is from a protein that has just been characterised. Use the protein sequence to identify the likely function of the protein from which the sequence is derived.
• Explain your approach and present your results. You will probably find the following web site useful but be careful to select
the appropriate searches http://blast.ncbi.nlm.nih.gov/Blast.cgi ... =BlastHome

TCPFADPAALYSRQDTTSGQSPLAAYEVDDSTGYLTSDVGGPIQDQTSLKAGIRGPTLLEDFMFRQKIQHFDHERVPERAV

• Task 2. The coding region for this protein, which is over 600 amino acids in length, has not yet been cloned.
• Explain how you would use the protein sequence in Task 1 to clone the coding region for this protein.
Tasks 3 and 4 require you to do some reading of the scientific literature and to understand Quikchange mutagenesis, how it
works, and how you can use it to perform mutagenesis experiments. This aspect is related to BIOL5206.

• Task 3. While you are performing Tasks 1 and 2, you also start work on a second project involving a different protein.
Identify this new protein for which you are given a region of DNA sequence below.

• Describe details of the protein that you have identified including any unusual features. This will require you to look up some
literature. Remember to cite your sources of information and references in a scientifically correct manner.
Gacggaagcacgtttattacaggaggccaacgacgtggaattccgttcgaggattcaaccccggtatttacacctgagatctacgtccctgaacaagacac
Tttctacaagcagaaccccaactccattgttcgcgtctaccatagcatttcccttttgttacctgatggcagggtatttaacggtggtggtggtctttgt
Task 4. In order to investigate this protein in more detail you undertake some mutagenesis experiments; you decide to
mutate cysteine 228.

• From your answer to Task 3, can you say why residue 228 has been selected for mutagenesis and what effect you predict
the mutation would have on the structure and function of the protein?

• Task 5. Design primers for Quikchange mutagenesis to change Cysteine228 to Alanine. What rules did you follow during the
design process? What is the melting temperature of each primer? The web site to use for this task is
http://depts.washington.edu/bakerpg/pri ... rtemp.html

Task 6 requires you to do some reading about the features of bacterial expression vectors and to consider the important features
of expression vector design. Again this is related to BIOL5206.
• Task 6. You now need to clone the coding sequence for the mature protein you identified in Task 3 into the expression
vector pET28c GFP. The coding region for your protein must replace the existing coding region of GFP. A restriction map of
the pET28c GFP plasmid is shown in Figure 1. The DNA sequence regions of the 5’ and 3’ cloning junctions of pET28c with
the GFP coding region are shown in Figures 2 and 3 respectively .
• Why is a version of pET28c that already contains an insert being used as the cloning vector?
• Describe how you would perform the cloning experiment. Use a flow diagram to explain the steps and include experimental
details such as primer sequences.

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Regards,
p