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BISC219/F12: RNAi Lab 7: Difference between revisions
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→Picking a Bacterial Colony
Tucker Crum (talk | contribs) |
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How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the ''C. elegans lys-2'' gene.<BR><BR> | How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the ''C. elegans lys-2'' gene.<BR><BR> | ||
This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, it is likely that the hundreds of thousands of bacteria making up that colony are genetically identical daughters of a single cell. This allows us to take bacteria from a colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your agar with ampicillin all have the plasmid responsible for amp resistance, that does not mean that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that may have a plasmid without the gene of interest. That can happen when a vector plasmid is "empty". Our plasmid, pPD129.36 ''lsy-2'', was genetically modified from a purchased base plasmid that was genetically synthesized by a pharmaceutical company. Remember that researchers need to be able to study any gene of interest; therefore, base plasmids are created that allow insertion of any gene of interest in proper alignment with synthetic gene promoters so that the gene of interest can be expressed as desired in a bacterial or a yeast model system. The first step in this process after acquiring a base plasmid such as pPD129.36 is to isolate small segments of chromosomal DNA that contains your gene. Then you must make lots of copies of it by PCR after designing primers that will copy just your gene, and then ligate the gene from the PCR product into the vector plasmid so that it is in proper alignment with an effective synthetic promoter. If all goes well, after transformation (uptake of this plasmid into a cell) the ligation works to achieve this proper alignment but occasionally the ligation fails and the plasmid DNA anneals back on itself. We need a way to find only the colonies with successful ligation that are expressing our gene of interest off the synthetic vector plasmid.<br> | This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, it is likely that the hundreds of thousands of bacteria making up that colony are genetically identical daughters of a single cell. This allows us to take bacteria from a single colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your agar with ampicillin all have the plasmid responsible for amp resistance, that does not mean that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that may have a plasmid without the gene of interest. That can happen when a vector plasmid is "empty". Our plasmid, pPD129.36 ''lsy-2'', was genetically modified from a purchased base plasmid that was genetically synthesized by a pharmaceutical company. Remember that researchers need to be able to study any gene of interest; therefore, base plasmids are created that allow insertion of any gene of interest in proper alignment with synthetic gene promoters so that the gene of interest can be expressed as desired in a bacterial or a yeast model system. The first step in this process after acquiring a base plasmid such as pPD129.36 is to isolate small segments of chromosomal DNA that contains your gene. Then you must make lots of copies of it by PCR after designing primers that will copy just your gene, and then ligate the gene from the PCR product into the vector plasmid so that it is in proper alignment with an effective synthetic promoter. If all goes well, after transformation (uptake of this plasmid into a cell) the ligation works to achieve this proper alignment but occasionally the ligation fails and the plasmid DNA anneals back on itself. We need a way to find only the colonies with successful ligation that are expressing our gene of interest off the synthetic vector plasmid.<br> | ||
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To achieve this goal we are going to do a '''colony PCR'''. Instead of adding purified ''lsy-2'' gene fragments as template DNA in a PCR reaction with primers specific for your gene, you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test both well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid.<br> | To achieve this goal we are going to do a '''colony PCR'''. Instead of adding purified ''lsy-2'' gene fragments as template DNA in a PCR reaction with primers specific for your gene, you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test both well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid.<br> | ||
