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Lecture 21

BIOL 5060 Lecture 21: 4:11_RecDNATech_Hoffman

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BIOL 5060

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4/11_recombinantDNATechnology_Hoffman 4/11/2017 3:01:00 PM What genes interact with my first gene of interest and how do they work together? … we left off with a temperature sensitive (ts) mutant strain- 3 Genes were cloned and we were able to identify a mutation in 1 of the 3 genes • GOI1- gene of interest 1 • HCS1- high copy suppressor one • HCS2- High copy suppressor two How can over-expressing other genes mask a defect in this gene? Possible mechanisms for HCS’s • Wild-type: GOI1—bound to HS1 o  together they stimulate HS2, which carries out the function • Mutant: goi1^ts mutation reduces the gene’s physical interaction with HS1 o Some goi^ts-HS1 are bound, other aren’t however reduces its expression less activation of HS2 defect o Does not eliminate o You can drive a reaction by increasing the concentration of the reactants without changing on/ off rate o If we over-express the mutant strain, we can restore more of the bound complex more stimulation of HS2 ▪  we are making more than is physiologically relevant driving the formation of the dimmer to restore function o high copy expression will bypass the need for the dimer ▪ HS2 doesn’t mean it has 0 activity in the absence of stimulation ▪ By over-expressing, it may create enough activity to bypass the need for the HS1-GOI dimmer 1 : Find the effect of deleting GOI1 • you can buy a strain w this gene deleted- wrong; the deletion isn’t necessarily in your strain background; might have a different consequence or no consequence •  we need to delete the gene of interest in our strain ourselves to make sure we are making the proper comparison o using homologous recombination in a diploid- in case gene is essential (meaning its needed for life under any condition) • Take 2 homologous chromosomes containing GOI with a selectable marker in between to tell if cells have embodied the marker and then tell if it happened by homologous recombination or not • Pre-PCR- we were totally dependent on the location of restriction sites; we had to use a restriction enzyme; Need a restriction enzyme that will cut within our ORF and not within the rest of the plasmid o PCR moved use away from this • VS. with ligate in a marker- we have a disruption rather than a deletion; still has the capacity to make some of the protein • Now, with PCR (post-PCR): we take a plasmid with a selectable marker and design oligonucleotides (LEU2) and we create sequences that will anneal to either side o We can add extra sequence to oligos (either side) o  PCR product A—LEU2—B o 25 bp in budding; 60 bp in fission yeast 2nd - Transform diploid to Leu+; how can we confirm homologous recombination? • We only care if it bound to the location of our GOI • PCR: Two strategies o 1) Using one internal and one flanking oligo ▪ Pro- sequences can be moderately close together to get an efficient PCR product ▪ Con- lack of product could be 1. Non-homologous insertion or 2. Technical problem o 2) two flanking oligos ▪ Pro- you know if it worked on a technical level ▪ Con- these tend to have to be
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