Baek 1
Isabella Baek
Lab report
Yeast Genetics Complementation
Introduction
This experiment is aimed to identify a mutation in the metabolic pathway of
Saccharomyces cerevisiae using forward genetics and genetic screens in yeast plates.
Specifically the experiment is aimed to identify the mutation that causes canavanine resistance of
yeast cells. Saccharomyces cerevisiae was chosen as the model organism because they grow
rapidly, can quickly introduce mutations, and can either be haploid or diploid which makes it
efficient for scientists to study the relationship between genetic modification and the resulting
phenotypes. In addition, its genome has been extensively researched and sequenced and many of
its essential molecular functions are mirrored in humans as well.
This specific study deals with the arginine synthesis pathway and how the mutations in
the pathway that could negatively affect yeasts’ survival. The starting yeasts in the experiment
have deletion mutations in Arg8, Arg2, and Leu 2. Then, they are grown in minimal media with
ornithine and canavanine (mmoc) plates which, because of the presence of canavanine, should
kill the yeast cells. CAN1 gene is an Arginine permease that can transport L-canavanine across
the yeast cell membrane. However, in the presence of canavanine, wild type cells would
transport canavanine through CAN1 transmembrane protein instead of the protein they need for
survival, Arginine. This causes the wild type cells to die.
Yet, a mutation in the gene that encodes for CAN1 protein can make yeast cells resistant
to canavanine but unable to import arginine into cells. By plating yeast cells in canavanine, only
the cells that have a mutation in the CAN1 gene would survive in this environment. Then,
, Baek 2
through functional complementation of the surviving yeast colonies, yeasts that take up plasmids
that have selective markers that restore wildtype functions to the mutated yeast are selected. This
way, only yeasts that have restored wild type functions to their knocked out gene will be grown
for further analysis. Next, these colonies will go through illumina sequencing of their gDNA so
as to find the point mutation that is hypothesized to be responsible for canavanine resistance. In
addition, a single colony from a plate containing complete media lacking leucine but containing
arginine is taken for sanger sequencing. This sequencing data will positively identify what the
gene of the mutation is.
The experimental question is to test whether a mutation in the CAN1 gene causes yeast
cells to have resistance against L-canavanine toxicity. This experiment will use molecular
biology, functional complementation and genetic screening, whole-genome sequencing to find
the mutated sequence via illumina sequencing, and sanger sequencing of functional
complementation plasmids.It is hypothesized that the CAN1 gene is responsible for yeast cells
that have resistance to L-canavanine.
The results of this study suggest that the CAN1 gene is most likely to be responsible for
giving yeast resistance to canvanine because CAN1 was most often responsible for restoring the
wild type phenotype in mutant yeast cells. The specific mutations found through illumina
sequencing suggest that CAN1 protein function is most likely killed by nonsense mutations.
Materials and Methods
Isolation and Culture of Canavanine Resistance Yeast Strain
First step of this experiment was to grow a canavanine-resistance yeast strain by random
mutation on a plate that has canavanine. Tubes with the wild-type arg𝛥8 yeast were centrifuged
at 13,000 rpm for 10 seconds to form a pellet. The pellet was resuspended using 1mL of sterile
, Baek 3
water and pipetting up and down and re centrifuged. Next, we used 500 μL of sterile water to
resuspend the yeast cells to create a stock solution for serial dilution. Then, we did six serial
dilutions, 100, 10-1, 10-2, 10-3, 10-4, 10-5, containing 450 μL of sterile water and 50 μL of previous
dilution in each tube starting from having 450 μL of sterile water and 50 μL of stock solution in
the 10-1 tube. We then obtained 4 agar plates, two MMO and two MMOC plates. We plated 100
μL of 10-4,10-5 solution each on MMO plates using a glass spreader and sterilization. These
plated plates were incubated for a week at 27°C to 30°C for a week. Growth and observation
from the retrieved plates were documented and colony growth was noted. There were six to
seven colonies that grew on the MMOC plate of the 10-1 dilution and were restreaked onto
another MMOC (+canavanine) plate. The restreaking plate was divided into 8 sectors and one of
the sectors was conserved to plate the wild-type yeast strain as a control strain which is not
expected to survive in the MMOC plate. The plates were then labeled and incubated at 30°C
overnight. Next, a colony from the restreaked plate was selected and put into a tube that
contained 4mL YPD broth. The YPD tube was then incubated in a shaking incubator at 250 rpm
for 2 to 3 days at 30°C.
Culturing E.coli that Has a Yeast Genomic Library
We were provided a stock E.coli culture with cells that have plasmids with fragments of
the S. cerevisiae genome. We then used three microfuge tubes to create three serial dilutions of
the stock culture of 10-2, 10-4, and 10-6. Using 1000μL of saline solutions and 10μL of stock
solution to the 10-2 tube. Next, subsequent dilutions were made with 10μL of the previous tube
and 1000μL of saline solution. The dilutions were used to plate the two LB/ Kanamycin plates.
50μL of the 10-4 tube was each plate and spread with a sterile glass rod. These plates were then
labeled and incubated overnight at 37°C.
Isabella Baek
Lab report
Yeast Genetics Complementation
Introduction
This experiment is aimed to identify a mutation in the metabolic pathway of
Saccharomyces cerevisiae using forward genetics and genetic screens in yeast plates.
Specifically the experiment is aimed to identify the mutation that causes canavanine resistance of
yeast cells. Saccharomyces cerevisiae was chosen as the model organism because they grow
rapidly, can quickly introduce mutations, and can either be haploid or diploid which makes it
efficient for scientists to study the relationship between genetic modification and the resulting
phenotypes. In addition, its genome has been extensively researched and sequenced and many of
its essential molecular functions are mirrored in humans as well.
This specific study deals with the arginine synthesis pathway and how the mutations in
the pathway that could negatively affect yeasts’ survival. The starting yeasts in the experiment
have deletion mutations in Arg8, Arg2, and Leu 2. Then, they are grown in minimal media with
ornithine and canavanine (mmoc) plates which, because of the presence of canavanine, should
kill the yeast cells. CAN1 gene is an Arginine permease that can transport L-canavanine across
the yeast cell membrane. However, in the presence of canavanine, wild type cells would
transport canavanine through CAN1 transmembrane protein instead of the protein they need for
survival, Arginine. This causes the wild type cells to die.
Yet, a mutation in the gene that encodes for CAN1 protein can make yeast cells resistant
to canavanine but unable to import arginine into cells. By plating yeast cells in canavanine, only
the cells that have a mutation in the CAN1 gene would survive in this environment. Then,
, Baek 2
through functional complementation of the surviving yeast colonies, yeasts that take up plasmids
that have selective markers that restore wildtype functions to the mutated yeast are selected. This
way, only yeasts that have restored wild type functions to their knocked out gene will be grown
for further analysis. Next, these colonies will go through illumina sequencing of their gDNA so
as to find the point mutation that is hypothesized to be responsible for canavanine resistance. In
addition, a single colony from a plate containing complete media lacking leucine but containing
arginine is taken for sanger sequencing. This sequencing data will positively identify what the
gene of the mutation is.
The experimental question is to test whether a mutation in the CAN1 gene causes yeast
cells to have resistance against L-canavanine toxicity. This experiment will use molecular
biology, functional complementation and genetic screening, whole-genome sequencing to find
the mutated sequence via illumina sequencing, and sanger sequencing of functional
complementation plasmids.It is hypothesized that the CAN1 gene is responsible for yeast cells
that have resistance to L-canavanine.
The results of this study suggest that the CAN1 gene is most likely to be responsible for
giving yeast resistance to canvanine because CAN1 was most often responsible for restoring the
wild type phenotype in mutant yeast cells. The specific mutations found through illumina
sequencing suggest that CAN1 protein function is most likely killed by nonsense mutations.
Materials and Methods
Isolation and Culture of Canavanine Resistance Yeast Strain
First step of this experiment was to grow a canavanine-resistance yeast strain by random
mutation on a plate that has canavanine. Tubes with the wild-type arg𝛥8 yeast were centrifuged
at 13,000 rpm for 10 seconds to form a pellet. The pellet was resuspended using 1mL of sterile
, Baek 3
water and pipetting up and down and re centrifuged. Next, we used 500 μL of sterile water to
resuspend the yeast cells to create a stock solution for serial dilution. Then, we did six serial
dilutions, 100, 10-1, 10-2, 10-3, 10-4, 10-5, containing 450 μL of sterile water and 50 μL of previous
dilution in each tube starting from having 450 μL of sterile water and 50 μL of stock solution in
the 10-1 tube. We then obtained 4 agar plates, two MMO and two MMOC plates. We plated 100
μL of 10-4,10-5 solution each on MMO plates using a glass spreader and sterilization. These
plated plates were incubated for a week at 27°C to 30°C for a week. Growth and observation
from the retrieved plates were documented and colony growth was noted. There were six to
seven colonies that grew on the MMOC plate of the 10-1 dilution and were restreaked onto
another MMOC (+canavanine) plate. The restreaking plate was divided into 8 sectors and one of
the sectors was conserved to plate the wild-type yeast strain as a control strain which is not
expected to survive in the MMOC plate. The plates were then labeled and incubated at 30°C
overnight. Next, a colony from the restreaked plate was selected and put into a tube that
contained 4mL YPD broth. The YPD tube was then incubated in a shaking incubator at 250 rpm
for 2 to 3 days at 30°C.
Culturing E.coli that Has a Yeast Genomic Library
We were provided a stock E.coli culture with cells that have plasmids with fragments of
the S. cerevisiae genome. We then used three microfuge tubes to create three serial dilutions of
the stock culture of 10-2, 10-4, and 10-6. Using 1000μL of saline solutions and 10μL of stock
solution to the 10-2 tube. Next, subsequent dilutions were made with 10μL of the previous tube
and 1000μL of saline solution. The dilutions were used to plate the two LB/ Kanamycin plates.
50μL of the 10-4 tube was each plate and spread with a sterile glass rod. These plates were then
labeled and incubated overnight at 37°C.
