Your group of researchers has been assigned the task of cloning a newly discover
ID: 278686 • Letter: Y
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.
Unfortunately, there are no convenient restriction sites on either side of the gene.
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 ?-gal gene. The enzyme ?-galactosidase normally breaks the disasaccharide 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 ?-galactosidase attacks it. Thus, if you grow E. coli cells on a plate containing X-gal, the cells expressing a functional version of ?-galactosidase will appear blue. Cells not expressing functional ?-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. Total RNA of dog cells will be first obtained using Trizol method of RNA extraction. The RNA needs to be further converted to cDNA using random primers or poly T based primers by reverse transcriptase enzyme. Once the cDNA is obtained, primers corresponding to bulD gene is used to generate full length bulD gene. The primers may or may not contain restriction sites. If it contains sites, then it will save the money and prevent us from ordering one more set of extra primers. Once the bulD gene is obtained, it needs to be cloned into an expression vector.
B. The gene obtained will have the restriction sites as I had included them during its amplification from the cDNA. If not, again design primers with restriction sites not there in the gene but present in vector for cloning purpose. Amplify them, PCR purify or gel purify them, cut both vector and amplified gene using specific restriction enzymes and using ligase enzyme ligate the insert into the vector (particular molar ratio of vector:insert used) and transform the mixture into a competent E.coli cells. Precautions that needs to be taken include: One needs to be careful while gel extracting the vector and the insert . The vector:insert molar ratio needs to be carefully determined for ligation. Restriction enzymes used should not have star activity, else use it for less time as mentioned in manufacturer's manual. One needs to be very careful while obtaining cDNA in terms of extraneous contamination.
C. One can insert bulD where gal gene is present. If the bulD is present in gal gene region, gal gene would not be functional and upon transformation followed by selection in X-gal containing media positive clones will show white color whereas negative or plasmids containing no inserts will show blue color of the cells.
D. To predict the orientation of bulD gene, one can use an internal restriction digestion, i.e usage of restriction enzymes present within the gene and outside. Based on the fragment size in an electrophoresis, one can understand the orientation of the gene and select only those clones that have forward orientation.
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