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Lecture 21

BIOL 5060 Lecture 21: 4:11_RecDNATech_Hoffman

8 Pages
23 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
21

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4/11_recombinantDNATechnology_Hoffman4/11/2017 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: GOI1bound 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
1st: 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)
find more resources at oneclass.com
find more resources at oneclass.com
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 ALEU2B
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 much larger products;
maybe harder to get
3rd- after we successfully identified a diploid strain where this interaction
occurred, we will sporulate and Dissect tetrads
diploid (2n) cell will go through meiosis and produce and Ascus with
4 spores (n)
2:2 segregation plate it on reach medium (supports all strains)
and separate out the spores of the tetrads
o we can look at the products of a single meiotic event
find more resources at oneclass.com
find more resources at oneclass.com
2 progeny should have the deletion and 2 progeny should have the
wild type…. If this gene is essential we will see only 2 progeny
germinate and form colonies
o the leu phenotype of the things that grow(there must be a
leu mutation a the leu allele)
o plate on a medium lacking leucine we will still see some
growth because there are some diploid cells (not everything
has sporulated) but we will see no Leu+ progeny If leu is
essential!!
o If Leu is not essential… we will still see Leu+ progeny growth
and we can identify which were the mutant strains with the
deletion allele
4th- identify the phenotype of the complete loss of this gene
HCS1 is essential and hcs2 doesn’t confer any detectable
phenotype- you delete a gene and there is no effect
What’s our goal? What’s the method by which we achieve this?
We have an over-expression, that masks the effect of the deletion
We can use the new reagents we are creating to learn more about
the pathway- the function of the proteins that are working together
to carry out a process
One outcome of the deletion, and corresponding technique:
o Can we find ts allele of HCS1?
o GOI1- alleles= wt, goi1^ts, goi1∆
o HCS1 allele= we, ____, hcs1∆
o It would die at high temperature considering the allele is
essential?
o Use plasmid shuffle
Another outcome of the deletion, and another corresponding
technique:
o Are there other genes who activities allow survival of HCS2∆
cells? Is there something else going on that the cells can
sustain a deletion of this gene without dying?
There might be a second copy of this gene or a closely
related gene that carries out this function
There might be a second pathway
find more resources at oneclass.com
find more resources at oneclass.com

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Description
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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