An overproducer E. coli strain is an E. coli strain, which carries a recombinant plasmid with an ORF of a protein of interest under the control of a very strong promoter, therefore it produce large quantities of the protein of interest. For example, recombinant human insulin is produced using an overproducer E. coli strain, which carries a plasmid with the ORF of human insulin.

(a) You want to make an overproducer E. coli strain, which will make a protein from bacterium Salmonella, YFPS (your favorite protein from Salmonella). Below is a restriction map of a fragment of Salmonella genomic DNA, which contains ORF of YFPS and a restriction map of a plasmid, which you are using to make the overproducer. Which pair of restriction enzymes would you use?

b.) You want to use the same strategy to produce a human protein in E. coli cells, YFPH (your favorite protein from humans). You have two potential sources to make the insert: a fragment of human genomic DNA, which contains YFPH gene, and a fragment of human cDNA, which contains YFPH ORF. Which one would you use?

c.) What enzyme synthesizes DNA using mRNA as a template during cDNA construction?

a) Restriction enzymes are defined as those enzymes that cut DNA. These enzymes recognize a target sequence and cut DNA near those sequences. Choosing these two restriction enzymes allows obtaining a full length protein.

b) An intron is defined as a part of the gene that does not code for any amino acid. The regions in a sequence that are expressed in proteins are called exons because they are expressed, and human genomic DNA has introns, which would not be part of the desired protein.

c) Reverse transcriptase is a DNA polymerase type enzyme, its function is to synthesize double-stranded DNA using single-stranded RNA as a template. This enzyme aids in the formation of the double-stranded DNA when RNA has undergone reverse transcription in the cDNA strand. Reverse transcription means the synthesis of DNA from RNA.