a. You are interested in the results you have obtained and think that they may be important in explaining the differences in the responses of each family member to the infection. To find this out, undertake a literature search to identify any links between the gene you have identified, the mutations and the infection. Is there any scientific literature, which links the gene/protein and associated mutations with susceptibility to the disease? Investigate the evidence and illustrate concepts with figures where necessary.

b. Find the accession page for sequence NM_000492.3, which encodes the full mRNA for your gene of interest.

Using this sequence, design primers for amplification of the coding sequence into the pPAL7 vector for expression as an N terminal fusion protein in E.coli .

Details of pPAL 7 vector

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5867.pdf

Your strategy should cover all aspects of the cloning process and should be justified with scientific explanations throughout. The following aspects will need to be covered and you should ensure to include appropriate screen shots or other information and diagrams to evidence your strategy. Ensure you show full evidence of your analysis and design, giving annotated screenshots and web addresses where appropriate.

1.Outline of cloning strategy to produce the recombinant plasmid construct with appropriate justification at all points.

2. Design appropriate primers to allow the DNA to be cloned into the vector to enable the expression of the protein as an N terminal fusion protein with the Profinity exact tag which will help you to be able to purify the expressed protein. Provide justification for your choice of primers and highlight all the important aspects of the primers that you have designed and give the PCR cycling conditions that would be appropriate for amplification.

3. Details of controls to be included at various points throughout this experimental process to allow you to determine the causes of any problems etc.