As you are getting ready to write up the role of each labeled segment of the pla

Question

As you are getting ready to write up the role of each labeled segment of the plasmid, the phone rings. It's Mr. Rawley again. He just came across an article about a frog protein that kills corn borer. (Where does he get this stuff?). He wants to discuss how one could clone a frog gene and express it at high levels in Escherichia coli. Oh Wow. A whole new mess to describe! Time to get ready for the meeting!. The frog protein Mr. Rawley read about is produced only in small amounts by a rare tree frog. Mr. Rawley would like to use this protein to protect crops, but he certainly doesn't want to kill any of these frogs. His idea is to use E. coli to make large amounts of the frog insecticidal protein (FIP) by: (i) cloning the frog fip gene into a plasmid that replicates in E. coli, (ii) purifying FIP from large cultures of these recombinant bacteria. You consider... Would coli be able to express a frog gene?? (Frogs are eukaryotes and a frog gene most likely will contain introns.) How would one alter the cloning protocol to ensure expression of the fip gene in E. coli? 3b. What special problems would be involved in cloning and expressing the frog gene in E. coli? How would you alter the cloning protocol to allow for expression of the frog gene in E. Coli? 3c. Describe steps to transfer the frog gene from E. coli to a plant cell.

Explanation / Answer

Problems with cloning and expression of frog gene in E. coli:

1.  no splicing of introns/exons

The solution may be:

             ----The solution has been to make artificial genes that lack introns.

            -----Scientists isolate fully processed mRNA from eukaryotic cells (i.e., mRNA which has had the introns removed).Reverse transcriptase (obtained from retroviruses) is added to make DNA transcripts from this mRNA.

           ------This is called complementary DNA (cDNA) and is distinguished by the fact that it has genes with no introns

          ------The cDNA can be added to a plasmid and then be expressed.

2.  foreign promoters do not work in E. coli

(unless from closely related Gram negative bacteria)

– cDNAs do not have promoters

– need promoter from E. coli gene (–35, –10 sequences)

3.  proteases may degrade foreign proteins

4.  proteins may form inclusion bodies

------fusion proteins – may solve problems 2, 3, &/or 4 at once

– from vectors with 5' ends of E. coli genes

– multiple cloning site in gene

– allows in-frame cloning of cDNA

– E. coli promoter & ribosome binding site provided

– E. coli protein N-terminal domains

5.  codon usage

6.  post-translational modifications

The steps of gene transfer from E.coli to plant cell are:

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