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Lecture 22

BIOL 5060 Lecture 22: 4:18_RecDNATech_Hoffman

by OneClass1714247 , Spring 2017
8 Pages
43 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
22

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4/18_RecombinantDNATech_Hoffman 4/18/2017 3:01:00 PM
Permissive genes
Plasmid shuffle- goal is to generate and identify conditional alleles of a gene
of interest
Deletion allele in a haploid cell is dead at all temperatures
Strain with conditional allele is dead at some temperatures and
alive at others
Conditional allele- two types,
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o general temperature sensitive- where the restrictive
temperature is elevated relative to permissive
has to do with protein folding stability
o Cold sensitive- restrictive temperature is lower than
permissive
More rare phenotype
has to do with whether the protein is acting a complex
or not; has to do with complex formation
mutation the reduces the interaction becomes more
sensitive at low temperature
this library has all the alleles of our gene of interest
if we sporulate we have 2 dead for every tetrad but 2 live Leu-
progeny in each tetrad
o 4 are held within larger cell called an ascus
If we have our wildtype form on another plasmid- some progeny
with wildtype will get plasmid and some progeny with the deletion
will get plasmid
o If you sporulate, you get some Leu+ Ura+ progeny
o The wildtype haploid has to be a URA3 marked plasmid
o Because URA3 is counterselectable- What does this mean?
Why is the counter selection for URA3 essential *** vvvv
Transform with library of (HCS1∆ with HCS1+ haploid) HCS1
alleles with a 3rd selectable marker (Leu2 is our first URA3 is our
second) we can make our 3rd TRP1
o we have HCS1+ plasmid in a TRP1 vector
o why doesn’t it have to be an expression vector? TRP1 comes
from yeast- has a promoter that is recognized by the
organism- the insert will supply the promoter
o 2 ways to make mutant alleles- pass it through cell 1red? Or
through a PCR approach**
take advantage of yeast and homologous recombination
to create mostly wild-types but also some mutations
that would effect function- PCR
o Take PCR product and co-transform it with the cloning vector
o Transform to Trp+
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find more resources at oneclass.com
o Now you have deletion on chromosome, wildtype allele on
URA3 plasmid, and questionable allele on Trp plasmid
o Introduce uracil on growth medium
now rather than looking at one big cell, lets look at a plate of
colonies…
o on Trp plate- original plate as four colonies, 1) a wild-type,
2) one with no insert, 3) a ts allele and 4) a cs allele
Finally getting back to the question of WHY IT IS
COUNTERSELECTABLE?
o We are going to replicate plate to 5FOA at 37 degrees (pretty
high temp), 30 degrees (nice temp), and at 20 degrees (cold
for yeast)
o URA3 was essential for life before we added Trp, but now that
we added Trp, is URA3 still essential for life? ^^^ the 5FOA
will kill everything with URA3 and only keep those that have
the TRP1 alone
o Using counter-selection, you only see the things that have
lost it (URA3)… (we are seeking ts alleles)
o 1) The wild-type colony (trp) will grow at all temperatures
o 2) The allele with no insert won’t grow in any temperatures
o 3) The ts allele will grow in 20 and 30 degree plates
o 4) The cs allele will grow in 30 and 37 degree conditions alone
o ***This allows us to examine growth of cells from a mixed
colony of transformants
o At the end, we have identified the ts allele because it has kept
alleles alive at low temperatures but not at high temperatures
o Gives us strains that allow us to work with mutants, but
mutants that are alive under only certain conditions….
Alternative approach: Clone wild-type HCS1+ ORF only into expression
vector that uses a regulated promoter. Study the effect of “turning off”
expression.
Taking advantage of expression vectors to show levels of
expression; cells will be alive at higher levels expression of the
promoter and then we can turn off the promoter and see what
happens
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Description
4/18_RecombinantDNATech_Hoffman 4/18/2017 3:01:00 PM Permissive genes Plasmid shuffle- goal is to generate and identify conditional alleles of a gene of interest • Deletion allele in a haploid cell is dead at all temperatures • Strain with conditional allele is dead at some temperatures and alive at others • Conditional allele- two types, o general temperature sensitive- where the restrictive temperature is elevated relative to permissive ▪ has to do with protein folding stability o Cold sensitive- restrictive temperature is lower than permissive ▪ More rare phenotype ▪ has to do with whether the protein is acting a complex or not; has to do with complex formation ▪ mutation the reduces the interaction becomes more sensitive at low temperature • this library has all the alleles of our gene of interest • if we sporulate we have 2 dead for every tetrad but 2 live Leu- progeny in each tetrad o 4 are held within larger cell called an ascus • If we have our wildtype form on another plasmid- some progeny with wildtype will get plasmid and some progeny with the deletion will get plasmid o If you sporulate, you get some Leu+ Ura+ progeny o The wildtype haploid has to be a URA3 marked plasmid o Because URA3 is counterselectable- What does this mean? Why is the counter selection for URA3 essential *** vvvv •  Transform with library of (HCS1∆ with HCS1+ haploid) HCS1 alleles with a 3rdselectable marker (Leu2 is our first URA3 is our rd second) we can make our 3 TRP1 o we have HCS1+ plasmid in a TRP1 vector o why doesn’t it have to be an expression vector? TRP1 comes from yeast- has a promoter that is recognized by the organism- the insert will supply the promoter o 2 ways to make mutant alleles- pass it through cell 1red? Or through a PCR approach** ▪ take advantage of yeast and homologous recombination to create mostly wild-types but also some mutations that would effect function- PCR o Take PCR product and co-transform it with the cloning vector o  Transform to Trp+ o Now you have deletion on chromosome, wildtype allele on URA3 plasmid, and questionable allele on Trp plasmid o  Introduce uracil on growth medium • now rather than looking at one big cell, lets look at a plate of colonies… o on – Trp plate- original plate as four colonies, 1) a wild-type, 2) one with no insert, 3) a ts allele and 4) a cs allele • Finally getting back to the question of WHY IT IS COUNTERSELECTABLE? o We are going to replicate plate to 5FOA at 37 degrees (pretty high temp), 30 degrees (nice temp), and at 20 degrees (cold for yeast) o URA3 was essential for life before we added Trp, but now that we added Trp, is URA3 still essential for life? ^^^ the 5FOA will kill everything with URA3 and only keep those that have the TRP1 alone o Using counter-selection, you only see the things that have lost it (URA3)… (we are seeking ts alleles) o 1) The wild-type colony (trp) will grow at all temperatures o 2) The allele with no insert won’t grow in any temperatures o 3) The ts allele will grow in 20 and 30 degree plates o 4) The cs allele will grow in 30 and 37 degree conditions alone o ***Th
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