BICH/GENE 432 MOLECULAR GENETICS

Laboratory Exercise #1.1 Lithium Acetate Transformation of Yeast with

Linearized pRS406 plasmids containing set1 mutants
INTRODUCTION
The purpose of todays lab is to introduce a mutant copy of the set1 gene into the genome of S.
cerevisiae. In order to achieve this, a lithium acetate transformation of yeast protocol adapted
from the protocol used in the Bryk lab will be completed using StuI digested pRS406-set1-Y967F
and pRS406-set1-Y967A plasmids OR pRS406 and pRS406-SET1 plasmids as integrating DNA.
To successfully understand this strain construction experiment, two important aspects must be
considered: (1) the design and function of the plasmid as integrating DNA and (2) the strain
background as it relates to this project. These are important aspects because this knowledge is
fundamental to understanding how we are going to generate strains of S. cereivisiae to develop a
screen where differences in growth of set1 mutants compared to SET1 and set1 can easily be
detected on specialized growth media. These strains are important for an on-going project, with
strains generated this semester being particularly important as they allow for differences in
growth to be assessed that is dependent on (1) Set1 activity and also (2) if Set1 activity can be
attributed to H3K4 methylation specifically.. To date, growt of Set1 mutant strains have been
compared with HIS3 reporter in the silent region of the rDNA vs the active euchromatic region
of ChrXII outside of the rDNA, and where HIS3 reporter is in its endogenous location of ChrXV.
We are now asking the question with HIS3 in its endogenous location, is the phenotype on schis+3AT due to Set1 activity with the substrate Lys3 of Histone3 (H3K4).
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StuI digested pRS406-set1-Y967F/A, pRS406-SET1, pRS406

The plasmid for this project is shown as Figure 1 below. There are a few critical components
within this plasmid that must be understood. The first is the cloning site which is called the
MCS multiple cloning site located within the lacZ gene. This is the site whereby genes can be
cloned into the plasmid using restriction enzymes to digest insert DNA and the empty plasmid to
produce homologous ends that can align and be ligated. In the pRS406-set1-Y967F/A plasmids
we are using, the gene encoding either Y967F or Y967A set1 was cloned into the plasmid using
the SacII and XhoI restriction sites. A double digest of both the set1 gene and the pRS406
plasmid was performed, and the resulting fragments were ligated together to generate the
pRS406-set1-Y967F/A plasmids.
There is also a URA3 gene. It is imperative the plasmid have this gene, as this is the portion of
the plasmid that has homology to the genomic DNA of the yeast strain we are working with to
allow for homologous recombination to occur. It can be seen that a StuI site is located within the
URA3 gene. When the circular plasmid is digested with StuI, it is linearized whereby a portion of
the URA3 gene is located on either end. It is at these sites that homology exists between the
plasmid and our yeast strain that allows for the process of homologous recombination and thus
insertion of our plasmid into our yeast strains genome.

Figure 1: Vector map of pRS406.

PLEASE NOTE A COPY OF THIS PLASMID AND THE DIAGRAMED LOCATION OF WHERE THE
set1GENE IS LOCATED NEEDS TO BE INCLUDED IN YOUR NOTEBOOK PRIOR TO START OF
CLASS!

S. cerevisiae Background Strain Information MBY3030

In constructing strains for a specific purpose of study, it must always be considered what
background the strain has that you have picked to use as the parent strain. In our case, there are
three important pieces of information that must be known about the parent strain MBY3030 that
we are using to integrate our linearized plasmid into for study of Set1 function as it relates to
growth differences on selective media. Here is the genotype for strain MBY3030:
MAT a HIS3+ (hht1hhf1)::LEU2(hht2hhf2)::KANMX4 lys2-128 trp163 ura3-52
set1::KANMX4 with plasmid phht2K4R-HHF2CEN

Important Alleles to Recognize:

ura3-52 is auxotrophic, but this gene fragment is important because it allows for
us to select for transformants (by plating on scura media). The entire sequence of
the URA3 gene is present, but interrupted by the presence of a TY element. This is

important because we will be using URA3 gene sequences for homologous

recombination to integrate SET1 or set1 single amino acid substitution mutants.
set1 indicates that there is no SET1 gene present in this strain, thus no Set1
protein can be made by the yeast. Therefore, whatever set1 gene we insert into the
genome using homologous recombination, will be the only source of Set1
produced in the cells. In our specific strains, the gene KANMX4 has replaced the
SET1 gene (hence set1::KANMX4 portion of the genotype).
(hht1hhf1)::LEU2(hht2hhf2)::KANMX4 both genomic copies of H3 and H4
have been knocked out, thus the plasmid phht2K4R-HHF2CEN supplies H3 and
H4, where H3 contains the K4R mutation (H3K4R).

Generating set1 mutant strains for study

A specific type of genetic recombination, known as homologous recombination, results when

two pieces of DNA contain sequence information that is similar enough (homologous) to allow
for the exchange (recombination) of nucleotide sequences (Figure 2). This process has many
important functions, including producing new combinations of DNA sequences, an important
process in genetic variability.

Figure 2: Homologous Recombination

Homologous recombination occurs in organisms within all three domains of life. Today, we will
be using the naturally occurring homologous recombination process in yeast to introduce a
mutated gene. Yeast offers a simplistic model system that allows for the study and use of
recombination for scientific purposes. On the following page is the abstract from a 1981 article
published in PNAS, indicating the rates of recombination of different types of transforming
DNA.
Yeast transformation: a model system for the study of recombination.
T L Orr-Weaver, J W Szostak, and R J Rothstein
Proc Natl Acad Sci U S A. 1981 October; 78(10): 63546358.
Abstract
DNA molecules that integrate into yeast chromosomes during yeast transformation do so by
homologous recombination. We have studied the way in which circular and linear molecules
recombine with homologous chromosomal sequences. We show that DNA ends are highly
recombinogenic and interact directly with homologous sequences. Circular hybrid plasmids can
integrate by a single reciprocal crossover, but only at a low frequency. Restriction enzyme
digestion within a region homologous to yeast chromosomal DNA greatly enhances the
efficiency of integration. Furthermore, if two restriction cuts are made within the same
homologous sequence, thereby removing an internal segment of DNA, the resulting deletedlinear molecules are still able to transform at a high frequency. Surprisingly, the integration
of these gapped-linear molecules results in replacement of the missing segment using
chromosomal information. The final structure is identical to that obtained from integration of a
circular molecule.

In todays lab, we will be making use of the process of homologous recombination whereby the
linearized pRS406 plasmids encoding either of the set1 genes Y967F or Y967A, SET1, or
empty vector for set1, will be used as transforming DNA to integrate into the chromosome of
yeast cells that are set1. We purposefully constructed this plasmid in a manner that allows for
easy integration into the yeast genome by ensuring that the URA3 gene sequences surrounding
the set1 gene are homologous to the region of the chromosome where the target gene is being
integrated.
Note in the abstract above, the statements shown in bold. These statements indicate that
linearizing a circular plasmid using restriction digest enhances the rate of recombination such
that it occurs at a high frequency. Thus, the pRS406-set1 plasmids that are gifts from the Mary
Bryk lab (Dept of Biochemistry, Texas A&M Univ) have been digested with StuI to ensure the
linearized plasmids contain the necessary sequence of the URA3 gene on either end that is
homologous with the ura3-52 gene contained within the yeast genome. This is critical because it
is at these sites of homology that recombination will occur. Thus, the StuI linearized plasmid is
efficient transforming DNA, like that mentioned in the abstract above, and can readily integrate
into our set1ura3-52 strain (MBY3030). Further, our digesting the pRS406 set1 plasmid
produces a linearized plasmid with identical nucleotide sequences of the URA3 gene that allows
for integration of our mutant set1 gene and a functional copy of the URA3 gene (Figure 3). By
completing this transformation, a yeast strain is engineered that has a mutated set1 gene, either
encoding Y967F or Y967A, WT Set1 or no Set1, and a now functional copy of URA3 that allows
for marker checks so that strains that are URA+ also contain the set1 gene. This is how we will
select cells that have been successfully transformed (thus containing the set1 gene) and those
which were not successfully transformed (thus are still set1 and ura3-52). The transformed
strains are required for future plate assays whereby we look at growth differences between wild
type and set1 strains as a basis for comparing point mutations in set1 strains to determine the
effect of that point mutation on Set1 activity. Thus set1 strains that grow more like wild type
indicate the point mutation had little to no effect on Set1 activity, whereas set1 strains that grow
like set1 are indicative of a point mutation having a more significant effect on Set1 activity.

Figure 3: Homologous recombination of set1 ura3-52 yeast strain transformed with linearized pRS406-set1
plasmid with functional URA3 gene.

Easy Lithium Acetate/Heat Shock Yeast Transformation

This protocol is good for integrating plasmids into yeast genomes.
Materials:
pRS406-set1Y967F and pRS406-set1Y967A mutant plasmids (150 ng/L each)
OR
pRS406 () and pRS406-SET1 plasmids (150 ng/L each)
carrier DNA (10 ng/L)
40% PEG 3350/1xTE/0.1 M LiAc buffer
Strain MBY3030 overnight saturated cultures
Water bath set at 42 C
Synthetic complete plates lacking uracil (sc-ura)
Protocol:
1. Grow an overnight culture of the yeast to be used in transformation reaction in YPDT.
We will be using MBY3030.
2. 1.5 mL of saturated O/N culture will be given in three different epi-tube that will be
labeled with each of the Set1 genotypes (Y967F and Y967A, or WT and ) and a tube
labeled negative control
**Students will begin here
3. Centrifuge for 2 min at 5000 rpm. Please make sure that epi-tubes are balanced in the
desk top centrifuge. There are more groups than there are desk top centrifuges,
please share. Also make sure that the plastic lid is in place prior to beginning
centrifugation (if you hear a horrible noise, the lid is likely not on or the centrifuge
is not balanced).
4. Pour off supernatant, careful not to disturb cell pellet.
5. Resuspend cell pellet in 1.5 mL of dH2O. dH2O is given to you in a snap cap tube.
6. Centrifuge for 2 min at 5000 rpm and then pour off supernatant, careful not to disturb cell
pellet.
7. Resuspend cell pellet in 500 L of 40% PEG 3350/1xTE/0.1 M LiAc. This buffer will be
provided in an epi tube labeled buffer.
8. Incubate cells in an epitube rack in 30 C incubator for 30 mins.
9. Get reactions from incubator. Take careful note of strain labels and plasmid labels at this
point.
10. Add 5 L of 150 ng/L plasmid DNA to each cell suspension. You will be adding
pRS406-set1-Y967F or pRS406-set1-Y967A to two of the labeled epi-tubes OR you will
be adding pRS406-SET1 or pRS406. Please be sure to add the correct plasmid to the
properly labeled cell suspension epitube it is important that you keep track of
strains. Make sure to switch tips between adding plasmids to cultures to not cross
contaminate.
a. Calculate the final amount in ng of plasmid in the transformation reaction for each
mutant strain being constructed: ___________

b. Why is no plasmid added to the negative control? What does this mean in terms
of plating at the end of the transformation lab?
11. Carrier DNA aids in uptake of plasmid by chemically treated cells. The concentration of
carrier DNA is 10 ng/L, and 5 L will be added to each of the epi-tubes containing cell
suspension.
a. Calculate the final amount in ng of carrier DNA in the transformation reaction for
each cell suspension: ___________
12. Mix reaction by inverting or flicking tubes.
13. Incubate at 42C for 15 min. This is the heat shock step, it stresses the chemically treated
cells to facilitate the take up exogenous DNA from their surroundings.
14. While the transformation is incubating:
a. Label epitubes based on strain and add 1.4 mL of sterile dH2O to each epitubed
for dilution of cells. (We have three transformation reactions, you will need
three tubes and to keep track of which reaction is which.)
b. Label three sc-ura plates on agar side for each transformation reaction. This
should include
i. Strain info
ii. Date
iii. Group ID
15. Remove transformation reaction from water bath, mix contents by inverting the epitube
4-6 times, pipette 100 L from each transformation reaction and add to appropriate
epitube containing 1.4 mL dH2O. There should be three dilutions one for each mutant
strain and one for the negative control. Be sure to keep track of labels.
16. Mix each reaction by inverting epitube 4-6 times.
17. Using a pipette, add 100 L of dH2O using pipette and dispense reaction in center of
properly labeled plate.
18. Remove 20 L from transformation reaction using pipette and dispense reaction in center
of properly labeled plate where spot of 100 L of water is located. Make sure you
switch tips between adding cultures otherwise you have contaminated your newly
constructed strains! Using a sterile spreader (aka hockey stick), spread the reaction well.
Be sure to cover the entire plate surface. *Poor spreading of reaction will make colony
selection difficult, if not impossible so use good technique. The larger volume of
water and reaction should aid in the spreading of the culture over the plate.
19. Repeat this for each reaction using a new sterile spreader (hockey stick) each time.
20. Set plates on table by incubator agar side up. TAs will put all plates in incubator at end of
lab.
21. Incubate plate at 30C for 2-3 days to see colonies.

Transformation Solutions:
10 x TE: 100 mM Tris HCl, pH8.0, 10mM EDTA. Autoclave.
1 M Li Acetate: Autoclave.
50% PEG: 50 g PEG 3350 in water, 100 ml final volume. Autoclave.
40% PEG 3350/1 x TE/ 0.1M LiAc: 8 ml 50% PEG, 1 ml 10 x TE, 1 ml 1 M LiAc. Make up
fresh each time.

CLEAN-UP
- Empty all desk-top biohazard bags by pouring contents into large biohazard waste containers
at end of bench (be sure to keep desk top bags, just empty contents)
- Empty all unused buffers in sink and discard snap cap tubes into biohazard waste containers
at end of bench
- Make sure all plates are properly labeled and placed on bench by incubator. This includes
labeling the agar side of the plates with your group ID and pertinent strain information (TAs
will be the ones to actually put the plates in the incubator)
- Tubes may be thrown in the large biohazard waste containers
- Wipe down your bench area with 70% Ethanol and paper towels, then throw gloves and
paper towel waste products in the biohazard waste containers at end of bench
- Wash your hands before leaving the laboratory