YG 2015 P5
Project 5: Gene replacement in yeast - generation of
knockout/knock-in strains
One of the biggest advantages of yeast over any other eukaryotic organism is that
gene replacements and deletions can be done is a relatively short amount of time (we will
try to do knockouts in four weeks!). The reason why yeast is such a good organism for
this manipulation is that it very efficiently repairs double strand DNA breaks. It
recognizes free ends of double stranded DNA and joins them to stretches of chromosomal
DNA which are homologous. Yeast can also do this with genes on a plasmid that do not
have free DNA ends, but the free ends enhance this process significantly. What is helpful
in this process is that the ends and the sequences close to the ends are most important for
the repair machinery to recognize where the repair should take place. Therefore, a marker
gene flanked by regions homologous to the gene we want to replace will be integrated
into the desired place in the genome relatively efficiently.
We will be using a procedure that is called one-step PCR-mediated gene
replacement. The advantage of this method is that it does not require any cloning steps.
The homologous ends that are required to target the marker gene cassette to the locus that
we want to knock out are usually introduced by PCR primers.
For this approach, we have designed long (60-70 bp) PCR primers that contain
short (17-20 bases) sequences at the 3’ ends of each oligo which are homologous to the
knockout cassette and act as primers for amplification of the knockout cassette from a
plasmid template. The rest (the 5’ ends) of the oligos (40-45 bases) are homologous to the
gene that we would like to replace. Usually this homologous region is just 5’ or 3’ to the
START and STOP codons of the open reading frame (ORF) of the target gene. The
resulting product of a polymerase chain reaction using these primers therefore contains
the marker gene (in this case the kanMX4 cassette, providing resistance to the antibiotic
geneticin) flanked by 40-45 base pairs of DNA homologous to the target gene. This linear
DNA is then used in a high efficiency transformation of yeast protocol to obtain
transformants that are resistant to the drug geneticin because the kanMX4 cassette has
integrated at the target locus and replaced the target gene. This approach is called the
short flanking homology PCR (SFH-PCR) method, because the ends of the PCR
products (containing the resistance cassettes) have relatively short homologous regions to
the target genes
1
YG 2015 P5
Every group will be using this method to knock out (KO) the yeast gene (every
group will have a different gene to knock out) in one of our yeast strains.
We will be generating four strains that will serve for different project.
A screen of the yeast genome for mitochondrial localized proteins has been
performed and two proteins of great interest for us have been identified and need further
analysis. RRP36 is a component of 90S preribosomes and it is involved in early
cleavages of the 35S pre-rRNA and in production of the 40S ribosomal subunit. HAS1
encodes an ATP-dependent RNA helicase and it is involved in the biogenesis of 40S and
60S ribosome subunits. These two proteins are involved in the ribosomal biogenesis, a
process well described in the cytosolic compartment but poorly documented in
mitochondria. We have in silico evidence that yeast may produce mitochondrial variants
of these proteins by initiating translation from a non-AUG start codon upstream of the
canonical translation initiation site. By performing the KO of RRP36 and HAS1 in our
yeast strains, you will be able to see the effect of the absence of a functional
Mitochondria Targeting Sequence (MTS) on the growth of the strain. Indeed the
knockout of the RRP36 and HAS1 won’t be possible without a functional copy of the
gene expressed in the cell (the strain is not viable without the gene products). As the
hypothetical MTS is localized at the upstream region of the initiation start codon, we
engineered a plasmid that contains the functional copy of the gene but with a point
mutation that leads for nonfunctional (putative) MTS. If our hypothesis is correct, the
protein should not be localized in the mitochondrial compartment anymore and a
respiratory deficiency phenotype should be observed.
MTS
ORF
Point mutation
The example is for the PCR mediated knockout of the RRP36 open reading frame.
Analagous method is used for HAS1 KO
2
YG 2015 P5
17-20 bases homologous to
5’/3’ ends of kanMX4
45 bases
homologous to
RRP36 locus (5’ of
START codon)
kanMX4
Template
plasmid
pFA6akanMX4
45 bases
homologous to
RRP36 locus (3’ of
STOP codon)
NOT TO SCALE!
PCR reaction
kanMX4
Template
plasmid
pFA6akan
MX4
The PCR reaction creates a knockout cassette flanked by short
homologous regions (40-45 bases on each side) to the target gene
3
YG 2015 P5
rrp36:kanMX4
knockout cassette
kanMX4
Homologous recombination
rrp36
Integration of kanMX4 into genome/replacement of target ORF in new
strain
rrp36::kanMX4
Homologous recombination results in integration of the kanMX4
cassette and replacement of the target gene. These transformants can be
selected for their resistance to geneticin.
4
YG 2015 P5
In this practice, you will learn
- how to prepare the PCR product containing a knockout cassette
- a high efficiency yeast transformation procedure
- how to make genomic yeast DNA
- how to verify a knockout/gene replacement by PCR
Procedure:
Day 1 (Tuesday 1st week): PCR-amplification of the hphMX4 cassette
with primers introduce homology to the RRP36/HAS1 locus
Each group will set up the following PCR-reaction in duplicate (we need a lot of
PCR product)
Assemble on ice!
PCR reaction to amplify the KANMX4 (Do the reaction in duplicate!!!)
X l template DNA (10ng, pFA6akanMX4, resistance cassette, plasmid #9)
1 l Primer A
1 l Primer B
1.0 l 10mM dNTP mix
10 l PCR buffer (Biorad iProof = Finnzymes Phusion)
0.5 l Biorad iProof
Bring up to a final volume of 50 l in H2O
Primer from project XXXXVIII
For RPP0 Primer A =#1 Primer B =#2
For HAS1 Primer A = #25 Primer B =#26
PCR-cycle conditions with pFA6akanMX4 template
30” @ 98oC
10” @ 98oC
20” @ 63oC
25”@ 72 oC
7’ @ 72 oC
24h @10 oC
Repeat 30 times
Program Geo in the PCR machine
5
YG 2015 P5
Day 2 (Wednesday 1st week): Cleanup/concentration of PCR product
-
For RRP36 and HAS1 knockouts (pFA6akanMX4 template)
Transfer PCR reaction to 1.5 ml Eppendorf tube.
Add 5 μl of 3M Na-Acetate followed by 120 μl of cold abs. ethanol , vortex and
centrifuge at top speed for 15 min.
Wash pellet with 70% ethanol, air-dry for 10 min, and resuspend pellet in 36 μl of TE.
Analyze 1 μl by agarose gel electrophoresis (0.7 % agarose/TBE; expected product size:
1.6 kb) and measure concentration with Nanodrop (2ul).
Use 33 μl (usually 1-5 μg of DNA) to transform Saccharomyces cerevisiae (see Day 3).
Day 3 (Tuesday 2nd week): high efficiency transformation in yeast
We will inoculate cultures of our W1536 5B as well as yeast strain (10ml) in the morning
of the day before.
Perform the high frequency yeast transformation according to the procedure from the
Yeast Transformation Page of the Gietz lab (separate file in the WIKI folder).
The yeast transformation home page (http://home.cc.umanitoba.ca/~gietz/). Select “The
best method”.
We will both take an OD600 reading (dilute your overnight culture 1:50 in YPD; use fresh
YPD as the reference), and count the cells (1:10 dilution). The Haemocytometer
described in the Gietz procedure is a Neubauer type haemocytometer. More information
on cell counting will be given during class.
Do two transformations (one with each PCR product). Add 5 ul of the precipitated PCR
product to a 1 x transformation mix (RRP36 or HAS1 knockout, pFA6akanMX4
template) (more if the reaction product concentration appeared to be low on the gel).
6
YG 2015 P5
Reagents
PEG 3500 50% w/v
LiAc 1.0 M
Boiled SS-carrier DNA
PCR product
Plasmid complementing
(RRP36 or HAS1) wild
type copy and the one
with the point mutation
Total
Number of Transformations
1
5 (6X)
10 (11X)
1440 µl
240 µl
2640 µl
36 µl
216 µl
396 µl
550 µl
50 µl
300 µl
33 µl
204 µl
374 µl
1ul
6ul
11ul
360 µl
2160 µl
3960 µl
After the heat shock, centrifuge the microfuge tubes at top speed for 30 sec and remove
the Transformation Mix with your pipette (Step 12 in the protocol). Using aseptic
technique, re-suspend the cells in 1 ml of YPD and add the cells to 4 ml of fresh YPD
(preferably pre-warmed to 30oC) in an Erlenmeyer flask, and incubate under agitation for
at least three hours at 30oC.
After incubation, spin down the cells in blue cap tubes at 3000-4100 g for 5 minutes.
Resupend the cells to a final volume of 200 ul in YPD. Make 1:10, 1:100 and 1:1000
dilutions in YPD (200 ul + 22 ul each time) and plate each of the dilutions and the rest of
the concentrated cells on YPD-geneticin (8 plates total per group).
Grow at 30 oC in incubator for several days
Day 4 : waiting….
Day 5 (Friday 2nd week): Pick geneticin resistant colonies, re-test
If we have done everything right, small colonies of geneticin resistant colonies
should have formed by Friday afternoon.
-
for the RRP36/HAS1 knockout pick several of these colonies(10-20) and streak
out on YPD geneticin and SCG plates for single colonies, as well as YPD control
plates. Number your candidates so you can follow them through the practical.
We will provide you the positive and negative controls for YPD geneticin and
SCG plates
-
Place in 30 oC in incubator over the weekend
7
YG 2015 P5
Day 6 (Tuesday 3rd week)): Identify/inoculate strongly geneticin
resistant
-
strongly geneticin resistant clones should have grown well by now on the plates
we streaked out last Friday. Choose your favorite candidates (each group 2) and
inoculate each in 10 ml YPD.
 o/n in shaker at 30 oC (plates in incubator)
Day 7 (Wednesday 3rd week): Yeast genomic DNA miniprep
Protocol for DNA Isolation from Yeast:
Charles S. Hoffman and Fred Winston, Gene, 57 (1987) 267-272
1. Harvest cells by centrifugation (4200xg, 5 min) and resuspend in 0.5 ml water,
transfer the cells to a 1.5 ml Eppendorf-tube and centrifuge again (13000 rpm, 2
min).
2. Decant the supernatant and vortex briefly the tube to resuspend the pellet in the
residual liquid.
3. Add 0.2 ml 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH=8.0, 1
mM EDTA. Add 0.2 ml phenol-chloroform-isoamyl alcohol (PCI) (25:24:1). Add
0.3 g acid-washed glass beads (0.45-0.52 nm diameter).
4. Vortex 3 to 4 min. Add 0.2 ml TE.
5. Centrifuge: 5 min, 13000 rpm. Transfer aqueous layer (with your DNA) to a new
Eppendorf-tube. Add 1.0 ml 100% Ethanol. Mix by inverting the tube.
6. Centrifuge ´: 2 min, 13000 rpm. Discard the supernatant and resuspend the pellet
(your DNA) in 0.4 ml TE plus 30 µg RNase. Incubate 5 min at 37C. Add 10 µl 4
M ammonium acetate and 1 ml 100% Ethanol. Mix by inverting the tube.
7. Centrifuge: 2 min, 13000 rpm. Dry the pellet (your DNA). Resuspend it in 50 µl
TE.
PCIA = TE-saturated phenol solution mixed 1:1 with chloroform-isoamylalcohol (CIA
24:1)
Day 8: Confirmation of knockout strain by analytical PCR
8
YG 2015 P5
There is a small possibility that the kanMX4 cassette have integrated elsewhere in
the genome than the RRP36orHAS1:: kanMX4 loci. The probability of this
happening is, however, large enough to be a problem (remember Murphy’s law!).
Testing the respiratory deficient phenotype of our isolates is already a good
control, but it is also necessary to have additional evidence that the integration
occurred in the right locus. In the olden days, this was done by Southern blotting
of restriction-digested genomic DNA, involving some radioactive work. These
days, it can be done by PCR, without the need to mess around with radioactive
isotopes. The idea is as following (example taken for the RPP0 gene): The
kanMX4cassette is about 0.7 kb larger than the RRP36 ORF. If we carry out a
PCR reaction with primers outside (5’ and 3’) of the integration site, we should
get different-sized PCR products if the knockout cassette has integrated in the
right place ( 2.4 kb for the rrp36::kanMX4 strain we started with).
Shown below is only the example for the rrp36 knockout:
rrp36::kanMX4
2.4 kb product
rrp36
1,8 kb product
The same principle is also used to confirm knockouts in higher Eukaryotes (e.g.mouse)!!!
We will also perform a PCR using the same 5’ primer specific for the rrp36 locus and a
3’ primer specific for the kanMX4 cassette. Only genomic DNA from strains that have
the kanMX4 cassette integrated in the proper locus will give a product:
9
YG 2015 P5
rrp36::kanMX4
kanMX4 specific primer anneals
Product (1.2 kb)
rrp36
kanMX4 specific primer
does not anneal  no
product
Carry out the PCRs as follows (for each prep):
(Assemble on ice)
PCR1 (will results in products – of differing sizes- in both the starting strain and the
strain with the gene replacement
~ 50 ng of genomic DNA
0.5 l Primer RRP36(or HAS1) up5’ (50μM) ,
0.5 l Primer RRP36 (or HAS1) down 3’ (50μM),
1 l of 10mM dNTP mix
10 l Buffer (Biorad iProof)
0.5 l Biorad iProof enzyme
final volume 50 l
30” @ 98oC
10” @ 98oC
20” @ 64oC
1’30” @72 oC
7’ @ 72 oC
24h @10 oC
10
YG 2015 P5
Repeat underlined part 25 times
PCR2 (should only give a product in the new strain in which the gene replacement
has occurred)
~ 50 ng of genomic DNA
0.5 l Primer RRP36 (or HAS1) up5’ (50μM)
0.5 l Primer kanMX4 3‘ (50μM) (IV #1))
1 l of 10mM dNTP mix
10 l Buffer (Biorad iProof)
0.5 l Biorad iProof enzyme
final volume 50 l
30” @ 98oC
10” @ 98oC
20” @ 64oC
1’30” @72 oC
7’ @ 72 oC
24h @ 10 oC
Repeat underlined part 25 times
We will supply you with DNA for wild type control reaction!
Day 9 (Wednesday 4th week)
Run out the reactions (30 l of each) on 0.7% Agarose/EtBr gel.
Expected sizes
RRP36 or HAS1 up and down primers:
RRP36 ~ 2kb
HAS1 ~2,3kb
rrp36::kanMX4 ~ 2.6 kb has1::kanMX4 ~ 2,3kb
with kanMX4 specific primer:
RRP36 – no product
HAS1 – no product
rrp36::kanMX4 ~ 1.6 kb
has1 rpp0::kanMX4 ~::kanMX4 ~ 1,7kb
If you succeeded, you just generated a knockout strain. Merci bien!
11
YG 2015 P5
Please inoculate your favorite candidate in 2ml of YPD and grow for two days at 30oC in
a shaker.
Day 10 (Friday 4th week):
Make stocks: transfer 0.9 ml of the YPD cultures and 0.9 ml of sterile 86% glycerol into
sterile cell culture tubes. Mix well. Flash freeze in liquid nitrogen.
Review of results
References:
Goldstein AL, McCusker JH (1999). Three new dominant drug resistance cassettes for
gene disruption in Saccharomyces cerevisiae. Yeast 15(14):1541-53.
http://microbiology.ucdavis.edu/heyer/protocols/kanMX%20PCRProtocol.pdf (18.1.2013)
12