NA sequence analysis, primer design, protein expression and mutagenesis assessment

For Task 1 and 2 I identifed the protein with sequence which is provide with the attachmment.

Tasks 1 and 2 provide some experience of data analysis and using software for
analysing sequence information
” Task 1. Read the DNA sequence from the autoradiogram of a sequencing gel in
Figure 1. Indicate how you went about reading the sequence and note any
difficulties you encounter.
” Task 2. Use the DNA sequence data you have read in Task 1 to identify the gene
from which it is derived.
 To do this use software available on the internet to
(a) Translate the DNA sequence into protein coding sequence to identify
the coding region Eg. http//us.expasy.org/tools/dna.html
(b) use this coding region to perform a database search to identify the
full length coding sequence. Eg. http//nih.gov/B:AST
 From these database entries you should be able to get both the protein
sequence and the DNA sequence of the gene. Remember that the start of
the protein will probably not be at the start of the cDNA sequence as cDNA
usually has 5 and 3 untranslated regions.
 Be careful that your answer is based on sequence alignments with the
coding strand!
 Could you have identified the gene more directly?
Tasks 3 and 4 require you to do some reading of the scientific literature
” Task 3. 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.
” Task 4. In order to investigate the protein in more detail you undertake some
mutagenesis experiments; you decide to mutate lysine 83 to alanine. From your
answer to Task 3, can you say why residue 83 has been selected for mutagenesis,
why an alanine substitution has been chosen and what effect this mutation might
have on the protein?
Task 5 requires you to understand what Quikchange mutagenesis is, how it works, and
how you can use it to perform mutagenesis. This aspect is related to BIOL5204.
” Task 5. Based on the full length coding sequence that you identified in Task 2
design primers for Quikchange mutagenesis to change lysine 83 to alanine 83.
What rules did you follow during the design process? What is the melting
temperature of each primer? A web site that is useful for checking the primers is
primertemp.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 BIOL5204.
” Task 6. How would you clone the wild-type and the mutant coding regions into a
bacterial expression vector in order to produce sufficient protein for structural
studies. What type of bacterial expression vector would you choose to express
these proteins and what features would the vector possess? What strategy would
you choose for the detection and future purification of the recombinant proteins?