Molecular Cloning Exercise - BIOL 3321 - Spring 2022 PDF

BIOL 3321 Genetics / Dr Simmons / Spring 2022 Modified from the University of Washington OVERVIEW: MOLECULAR CLONING Molecular Cloning is a laboratory technique that aims to eit...

BIOL 3321 Genetics / Dr Simmons / Spring 2022 Modified from the University of Washington OVERVIEW: MOLECULAR CLONING Molecular Cloning is a laboratory technique that aims to either amplify large DNA segments (that could not be amplified via PCR), or to study the protein encoded by a specific gene. Please check out Chapter 14 of your textbook (Genetics Essentials, Pierce, 4th Ed) pages 395-398, to learn more about Cloning. You may also need to review restriction enzymes and their function (see pages 389-390). To clone, one uses a cloning vector, which is a circular, dsDNA molecule (like a plasmid) into which a foreign DNA fragment can be inserted. To be effective, the cloning vector requires an origin of replication, selectable markers, and one or more unique restriction sites (also called multiple cloning site -MCS- or polylinker region). The general steps of molecular cloning include: vector and insert preparation, ligation, transformation and colony screening. These steps are described below: The segment of gene to be cloned must be isolated and prepared to be inserted into the vector (insert preparation). This usually includes cutting with one or two restriction enzymes, but may require additional steps. The vector also must be cut using a restriction enzyme (vector preparation). For these experiments, restriction enzymes that produce staggered cuts are preferred as they leave complementary sticky ends. It is also preferable to use just one restriction enzyme to cut the insert and the vector, if possible, as it limits the number of fragments that could potentially recombine with each other and the vector. The cut foreign DNA and open vectors are mixed together and allowed to incubate with DNA ligase (ligation). Due to complementarity, cut end of the fragments will pair with cut ends of the vectors. DNA ligase will seal the sugar-phosphate backbone to create the recombinant plasmid, also known as a construct. Bacterial transformation requires inserting the construct into live bacterial cells, for example, E. coli DlacZ, which lack a wild-type copy of the lacZ gene. These are also called competent cells. The transformed E. coli culture is then plated on plates with media containing a specific antibiotic and Xgal, a lactose analog. Colony screening is the process by which one identifies the colonies bearing the constructs of interest. The functional or disrrupted copy of the lacZ gene on the plasmid, together with Xgal, permits blue-white screening. Other colony screening protocols include studying the DNA of the construct from select colonies to perform restriction analysis, PCR or sequencing to confirm the presence of the fragment of interest and the correct orientation, before continuing with other downstream applications. ACTIVITY: SIMPLE CLONING EXERCISE In the genome sequence of Propionibacterium acnes (bacterium that causes acne), you have discovered a gene predicted to encode a lipase (see next page). This is an enzyme that breaks down lipids in the skin, and if an inhibitor could be developed, it might lead to a therapy for acne. You want to find out more about this enzyme. To be able to produce large quantities of lipase you plan to clone the gene into a suitable vector, insert it into E. coli, allow for expression of your gene of interest, collect the protein, and study its properties. How would you clone this gene into competent E. coli cells? The key here is how to figure out how you would get the lipase gene, and only that gene, into a cloning vector, and then insert the construct (cloning vector + gene of interest) into E. coli. The location and orientation of lipase (your gene of interest) is depicted in the diagram below (next page). The tags correspond to the location of various restriction sites. 1  BIOL 3321 Genetics / Dr Simmons / Fall 2019 Modified from the University of Washington BIOL 3321 Genetics / Dr Simmons / Spring 2022 Modified from the University of Washington Lipase gene (743 bp) as positioned ingene Lipase the P. acnes (743 chromosome bp) as positioned in the P. acnes chromosome ScaI BamHI SspI ScaI ScaI BamHI SspI ScaI SspI PstI PstI PvuI PstI BamHI SspI PstI PstI PvuI PstI BamHI Assume you have a suitable cloning vector, a modified plasmid used for cloning, such as the one shown below Assume you have a suitable cloning vector, a modified plasmid used for cloning, such as the one shown below (pGOblue): (pGOblue): The sequence of multiple restriction sites on the vector are identified with standard name abbreviations The sequence of multiple restriction sites on the vector are identified with standard name abbreviations (PvuII, ScaI, SspI…) (PvuII, ScaI, SspI…) The ampRR gene is highlighted in red. This gene allows any bacterium that contains the plasmid to be resistant The amp gene is highlighted in red. This gene allows any bacterium that contains the plasmid to be resistant to a particular antibiotic (in this case, ampicillin). The ampRR gene makes a protein that breaks down to a particular antibiotic (in this case, ampicillin). The amp gene makes a protein that breaks down chemicals with a beta-lactam ring, structure that is present in ampicillin. This way the antibiotic around the chemicals with a beta-lactam ring, structure that is present in ampicillin. This way the antibiotic around the bacteria will get degraded and the bacteria will be able to survive. bacteria will get degraded and the bacteria will be able to survive. The lacZ gene is highlighted in blue. When the plasmid is inserted into a bacterium, this gene produces the The lacZ gene is highlighted in blue. When the plasmid is inserted into a bacterium, this gene produces the enzyme ß-galactosidase, which normally breaks the disaccharide lactose into the monosaccharaides enzyme ß-galactosidase, which normally breaks the disaccharide lactose into the monosaccharaides galactose and glucose. If the artificial substrate X-gal is provided in the media (instead of lactose), a blue galactose and glucose. If the artificial substrate X-gal is provided in the media (instead of lactose), a blue product will be formed when ß-galactosidase cleaves it. Thus, if you grow E. coli cells that contain the product will be formed when ß-galactosidase cleaves it. Thus, if you grow E. coli cells that contain the plasmid on a plate containing X-gal, the cells expressing a functional ß-galactosidase gene will appear blue. plasmid on a plate containing X-gal, the cells expressing a functional ß-galactosidase gene will appear blue. Bacterial cells that do not contain or express beta-galactosidase will appear white. This is called “blue-white Bacterial cells that do not contain or express beta-galactosidase will appear white. This is called “blue-white screening”. screening”. PvuII PvuI HindIII PvuII PstI ScaI BamHI KpnI SspI EcoRI PvuI pGOblue Plasmid (Cloning Vector). Red: ampR gene. Blue: lacZ gene. Gray: non-coding sequence. pGOblue Plasmid (Cloning Vector). Red: ampR gene. Blue: lacZ gene. Gray: non-coding sequence. 2 2  BIOL 3321 Genetics / Dr Simmons / Spring 2022 Modified from the University of Washington Here is what you need to do: 1. Complete the following table. Keep in mind that in cloning experiments, the transformed bacteria (bacteria that are expected to have acquired the construct) are plated on media that contains basic nutrients, an antibiotic (in this example, ampicillin) and Xgal. Be sure to explain your answers. Completing the table should help you learn the outcomes (advantage or disadvantage) of cloning your gene of interest into different sites on the plasmid (ampR gene, lacZ gene, or a non-coding sequence). Condition Would the bacterial colonies If colonies grow, what color should survive on the amp + Xgal plates? they be? Neither construct nor empty vector are inserted into the bacterium Empty vector is inserted into bacterium Construct is inserted into bacterium. The gene of interest is inserted in the ampR gene. Construct is inserted into bacterium. The gene of interest is inserted in a non-coding region. Construct is inserted into bacterium. The gene of interest is inserted in the lacZ gene 2. Keep in mind that the foreign DNA is not just ‘randomly’ inserted into the vector. The last three scenarios on the table (on top) are meant for your to understand where the foreign DNA should be inserted and why. One of the main goals of using blue-white screening is to be able to distinguish these two scenarios: the bacterium has acquired an empty vector vs. the bacterium has your desired construct. Based on this, where should the gene of interest be inserted? Why? 3. Based on your answer for question 2, observe the restriction sites present in the vector as well as in and around the lipase gene of Propionibacterium acnes. Choose the restriction enzyme you would use to ‘cut out’ the gene. Ideally, the same enzyme you use to cut out your gene of interest should be used to clone the gene of interest into the vector and create the construct. For your cloning experiment to be successful you must be able to prove that the following has occurred: (1) your gene of interest -lipase- is in the plasmid (pGOblue) and (2) your construct (gene of interest + vector) is in the bacterial cell (E. coli). 3  BIOL 3321 Genetics / Dr Simmons / Spring 2022 Modified from the University of Washington 4. Based on your observations, describe how can you use blue-white screening to help you determine which E. coli cells contain copies of pGOblue with the lipase gene? 4