Difference between revisions of "20.109(S11):Prepare DNA for cloning (Day3)"
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==Introduction== | ==Introduction== | ||
− | Last time you designed a modification to the P<sub>lux-λ</sub> promoter in order to weaken its basal expression in the absence of AHL — you hope. To test your idea, we will have to make a new DNA construct, pED-IPTG-YFD, where YFD is "your favourite design." Depending on how modest a change you made, there could be multiple ways to implement it; for example, a few DNA basepairs can readily be mutated by a PCR-like approach called site-directed mutagenesis. In our case, we will clone a modified P<sub>lux-λ</sub> insert into the pED-IPTG backbone. We are outsourcing insert construction to a company specializing in DNA synthesis. Recall that you designed two strands, each with a restriction site overhang complementary to the cognate one on the backbone. Before the strands can be ligated into the backbone, they must be annealed together into double-stranded DNA. This can be done by heating the DNA above its melting temperature (94-95 ° C) and then letting it slowly cool - very similar to how we denatured RNA and cooled it, except this time we want two strands to anneal rather than to form independent secondary structures. | + | Last time you designed a modification to the P<sub>lux-λ</sub> promoter in order to weaken its basal expression in the absence of AHL — you hope. To test your idea, we will have to make a new DNA construct, pED-IPTG-YFD, where YFD is "your favourite design." Depending on how modest a change you made, there could be multiple ways to implement it; for example, a few DNA basepairs can readily be mutated by a PCR-like approach called site-directed mutagenesis. In our case, we will clone a modified P<sub>lux-λ</sub> insert into the pED-IPTG backbone. We are outsourcing insert construction to a company specializing in DNA synthesis (Integrated DNA Technologies, or IDT). Recall that you designed two strands, each with a restriction site overhang complementary to the cognate one on the backbone. Before the strands can be ligated into the backbone, they must be annealed together into double-stranded DNA. This can be done by heating the DNA above its melting temperature (94-95 °C) and then letting it slowly cool - very similar to how we denatured RNA and cooled it in Module 1, except this time we want two strands to anneal rather than to form independent secondary structures. |
− | The backbone must also be prepared for cloning, first by restriction digest, and then by gel purification. (''What could happen if we didn't purify the large backbone fragment of interest from the rest of the digestion mixture?'') As you will see today, one of the enzymes that we are using for cloning is not very good at cutting plasmids. (Lesson: always read ''all the way through'' the product notes!) Thus, we have prepared pED-IPTG-INS for you that was digested overnight with ''XmaI'' and ''BamHI''. Because this plasmid is large (a little over 10 Kbp), you will run it on a relatively low weight percent gel (0.7 %) and use a kit especially designed for long DNA to purify it. | + | The backbone must also be prepared for cloning, first by restriction digest, and then by gel purification. (''What could happen if we didn't purify the large backbone fragment of interest from the rest of the digestion mixture?'') As you will see today, one of the enzymes that we are using for cloning is not very good at cutting plasmids. (Lesson: always read ''all the way through'' the product notes!) Thus, we have prepared pED-IPTG-INS for you that was digested overnight with ''XmaI'' and ''BamHI''. Because this plasmid is large (a little over 10 Kbp), you will run it on a relatively low weight percent gel (0.7 %) and use a kit especially designed for long DNA to purify it. As with the kit we used in Module 1, silica particles are used in concert with a chaotropic salt and pH variation to bind, wash, and elute DNA in turn. This time the particles are loose rather than in a column format. While the gel runs, you will set up control digests to understand how they work and why we had to do this one for you in the interests of time. |
− | Next time you will ligate the backbone and insert together, and then transform the new construct into bacteria. You will also continue gathering data about the 'broken' model system, namely the transfer function that maps IPTG concentration to Miller units. Combining this transfer function with the analogous one for the edge detector (that maps light to Miller units) will allow us to relate the IPTG- and light-sensitive systems to each other. | + | Next time you will ligate the backbone and insert together, and then transform the new construct into bacteria. You will also continue gathering data about the 'broken' model system, namely the transfer function that maps IPTG concentration to Miller units. Combining this transfer function with the analogous one for the edge detector (that maps light intensity to Miller units) will allow us to relate the IPTG- and light-sensitive systems to each other. For the mapping to work well, you should treat all the different cell strains as consistently as possible. Thus, you will set up cells carrying the transfer function plasmid for overnight culture from an OD of 0.0025, just like you did for the complete IPTG-sensitive system. |
− | Now is also a good time to add to our understanding of P<sub>lux-λ</sub> (specifically the'' lux'' part, as we covered λ last time) and P'' | + | Now is also a good time to add to our understanding of P<sub>lux-λ</sub> (specifically the'' lux'' part, as we covered λ last time) and P''ompC''. In doing so, we will learn about two fascinating pieces of prokaryote biology: two-component signaling systems and quorum sensing. |
− | Bacteria | + | Bacteria don t exactly have a reputation as great communicators! However, it turns out that these single-celled organisms do |