Your group of researchers has been assigned the task of cloning a newly discover

Question

Your group of researchers has been assigned the task of cloning a newly discovered fluorescent protein and expressing it in E. coli so that you can study it further. When stimulated by UV light, this protein happens to emit purple light. The gene for this protein is named bulD and is naturally found in the genome of the seldom-seen and notoriously temperamental Wild Golden Dog (Canis aurelia).   Fortunately, you already have a frozen sample of the dog’s cells. The section of the chromosome containing bulD looks like this.

Unfortunately, there are no convenient restriction sites on either side of the gene.

The vector into which you need to put the bulD gene is shown below. It only has two restriction sites, one for the restriction enzyme EcoRI and one for NotI.

Please come up with a detailed plan explaining how you will get the bulD gene into pCM981, how you will get that vector into E. coli, and how you will select E. coli colonies that have the gene. As you prepare your plan, be sure to address the following issues.

A. The Wild Golden Dog is obviously a eukaryotic organism, so its genes have introns. These are removed during normal eukaryotic mRNA processing, but prokaryotes don’t know how to handle introns. Furthermore, you don’t know exactly where the exons and introns are in this gene. How will you create a version of the bulD gene without introns so that the mRNA will be translated correctly by E. coli?

B. There are no obvious restriction sites surrounding the bulD gene, yet you still need to insert this gene into pCM981. How will you do this? What precautions do you need to take?

C. The pCM981 plasmid normally expresses the b-gal gene. The enzyme b-galactosidase normally breaks the disaccharide lactose into the monosaccharides galactose and glucose. If the artificial substrate X-gal is provided instead of lactose, a blue product will be formed when b-galactosidase attacks it. Thus, if you grow E. coli cells on a plate containing X-gal, the cells expressing a functional version of b-galactosidase will appear blue. Cells not expressing functional b-galactosidase will appear white. This is called “blue-white screening.”

With all of that as a preamble, how can you use blue-white screening to help you determine which E. coli cells contain copies of pCM981 with bulD?

D. If bulD gets incorporated into pCM981, it might be inserted in the “forward” orientation or the “reverse” orientation. Is one orientation preferable to the other? If so, how can you tell which plasmids have bulD in the correct orientation? Design the experiment and predict the results (Draw a diagram indicating the results you would expect if the insert is in the forward or reverse orientation).

Explanation / Answer

A) You isolate mRNA and prepare cDNA libraries. Then sequence these to identify exons.

B) when you have no restriction sites in the desired gene to be inserted. You design the restriction enzyme sequence into the 5'end of the primers that you use to amplify the target. This will insert the restriction site sequences into the desired gene amplicon which can be then digested by that specific restriction enzymes. The digested insert can be then inserted into the digested vector (vector digested by same enzymes as digested by an insert). In this question, Not 1 and EcoR1 sequences are designed into primers. Before the restriction enzyme sequence you need to place 3-4 nucleotides overhang. So the primer will have an overhang, followed by restriction sequence and oligo primer.

C) To perform blue white screening, the gene of interest need to be inserted into Beta-galactosidase site. If the gene is inserted into Beta-galactosidase gene the gene becomes inactive. Hence in the presence of X gal, the colonies remain white. When the gene is not inserted into the beta-galactosidase site, the beta-galactosidase is active and in the presence of X gal, it cleaves X gal to form a blue product. So the colonies are blue when no insert is placed in beta-galactosidase site.

D) The orientation of insert can be checked by performing nucleic acid sequencing of the recombinant plasmid using vector primers which are close to insert.

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