PART 1

You have an idea for a vaccine to prevent Group A Strep (GAS) infections. You know that Streptococcus bacteria are fastidious organisms, and you don't expect to be able to produce your protein of interest in large quantities in S. pyogenes. Based on your knowledge of GAS, design a recombinant vaccine candidate to protect against GAS infection using E. coli as your recombinant protein production organism. Your choice of GAS antigen should be something produced by GAS that is not produced by humans. Your GAS protein(s) of choice will be expressed in E. coli and then purified for use in vaccine production. (Yes, I am expecting you to find a GAS antigen on your own.) To express your protein in E. coli, you need to clone the gene(s) of interest into a plasmid under the control of the Plac promoter. Create and upload a diagram that shows each step of your design strategy and cloning process. Start with getting your gene of interest out of S. pyogenes and end with your selection and screening process in E. coli. Be sure to include all of the following:

– What GAS protein(s) will be expressed by your recombinant E. coli?

– Show all components that need to be present on your plasmid for replication, selection, screening, and for regulation by the Plac promoter.

– How do all of the parts come together?

– A selection mechanism to ensure that only recombinant E. coli expressing your plasmid can grow. Any selection mechanism is ok. Indicate what media needs to be used and what you expect to see.

– A screening mechanism to show that your gene(s) of interest is being expressed. Indicate what media needs to be used and what you expect to see.

PART 2
1. What S. pyogenes protein(s) are you expressing in E. coli for your recombinant vaccine candidate?

2. What function does your protein provide in S. pyogenes? Why do you think it will make a good GAS vaccine candidate?

3. Are there any concerns about negative side effects or potential toxicity based on the GAS protein you have chosen? Would you expect your protein to cause an infection or disease symptoms in the host or have some other negative impact? Justify your answer.

4. If your protein of interest proves to be only mildly immunogenic when tested, what could you do to improve its immunogenicity? (1-2 sentences is fine)

5. Do you expect to see a cellular or humoral immune response to your vaccine? What part of the immune response could you measure to determine whether or not the vaccine is effective?

6. What advantage(s) does your vaccine have compared to a live GAS vaccine? Provide at least one advantage.

7. What disadvantage(s) does your vaccine have compared to a live GAS vaccine? Provide at least one disadvantage.

8. What media component(s) are necessary to ensure that your gene of interest is expressed in the recombinant E. coli cells?

9. What media component(s) would you need to add to shut down the expression of your gene in the recombinant E. coli cells?(I've attached my previous diagram of recombinant DNA)

Requirements: One diagram with explainations to the questions