Advanced Cloning Exercise PDF 2019

BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington ADVANCED CLONING EXERCISE Your group of researchers has been assigned the task of clo...

BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington ADVANCED CLONING EXERCISE 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 husK and is naturally found in the genome of the seldom-seen and notoriously temperamental Wild Golden Dog (Canis insanus). Fortunately, you already have a frozen sample of the dog’s cells. The section of the chromosome containing husK looks like this. BamHI 2000 bp Unfortunately, there are no convenient restriction sites on either side of the gene. On the gene, the distance between the ‘left’ edge and the BamHI restriction site is 150 bp. The vector into which you need to put the husK gene is shown below. It only has two restriction sites, one for the restriction enzyme EcoRI and one for NotI, which are 300 bp apart. NotI promoter EcoRI EcoRI restriction site: lacZ gene 5’..N N N G A A T T C N N N..3’ 3’..N N N C T T A A G N N N..5’ pCM999 NotI restriction site: (4000 base pairs) 5’..N N G C G G C C G C N N..3’ 3’..N N C G C C G G C G N N..5’ Kanamycin resistance gene (KanR) Please come up with a detailed plan explaining how you will get the husK gene into pCM999, 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 questions: 1  BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington 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 husK gene without introns so that the mRNA will be translated correctly by E. coli? (Hint: the enzyme reverse transcriptase, found in retroviruses and commercially available, makes DNA copies from RNA strands.) B. There are no obvious restriction sites surrounding the husK gene, yet you still need to insert this gene into pCM999. How will you do this? (Hint: The 5’ end of a PCR primer does not need to be complementary to anything as long as there is a long stretch of complementary bases at the 3’ end. Thus, when you design a primer, the 5’ end can include any sequence of nucleotides that you want.) C. The pCM999 plasmid shown on the previous page normally expresses the beta-galactosidase enzyme (from the lacZ gene). How can you use blue-white screening to help you determine which E. coli cells contain copies of pCM99 with husK? D. If husK gets incorporated into pCM999, it might be inserted in the “forward” orientation or the “reverse” orientation. Is one orientation preferable to the other? If so, how can you use a restriction digest to tell which white colonies have plasmids with husK in the correct orientation? Draw a constructs (plasmids including the gene of interest) showing the gene inserted in the forward orientation and one showing the gene in a reverse orientation. Be sure to include the location of all restriction sites. Complete the table shown on the next page. 2  BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington Draw your two constructs (vector plus insert) here: Complete the following table: How many fragments would you get after restriction with the following enzyme(s)? What is the approximate size of each fragment? NotI EcoRI BamHI NotI + EcoRI husK in husK in husK in husK in husK in husK in husK in husK in forward reverse forward reverse forward reverse forward reverse orientation orientation orientation orientation orientation orientation orientation orientation Number of expected fragments after digestion Size of expected fragments (bp) after digestion How many fragments would you get after restriction with the following enzyme(s)? What is the approximate size of each fragment? EcoRI + BamHI NotI + BamHI NotI + EcoRI + BamHI husK in husK in husK in husK in husK in husK in husK in husK in forward reverse forward reverse forward reverse forward reverse orientation orientation orientation orientation orientation orientation orientation orientation Number of expected fragments after digestion Size of expected fragments (bp) after digestion 3  BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington E. Which enzyme(s) will you use to detect the orientation of your gene of interest? Briefly explain why. F. Regarding the expression of a eukaryotic gene in a prokaryotic cell, are there any concerns? Will you get the product you expect? Will it be functional? G. Imagine that, instead of incorporating husK into a prokaryotic vector, you would like to incorporate it into a eukaryotic cell. One way to do this is to microinject the DNA straight into the zygote or early embryo. Let’s say you want your gene to be expressed in the pancreas. What would you do differently? What other factors should you consider? H. To wrap up this exercise, draw a flow chart that describes the full process of molecular cloning. Be sure to include all steps and use the appropriate technical vocabulary. 4