YG 2015 P4
Project 4: Plasmid shuffle
The RFT1 gene of S. cerevisiae is an ER (endoplasmic reticulum) protein,
involved in flipping lipid-linked sugar moieties from the outer to the inner leaflet of the
ER during the generation of sugar chains later used in the glycosylation of proteins. A
former PhD student isolated a mutant form of it during our screen for a hydroxyacyl-CoA
dehydratase involved in fatty acid elongation in the ER. Unfortunately, the Rft1 protein is
not involved in fatty acid elongation, but the work has interesting implications for the
determining of the sugar chain flipping mechanism. The strains and plasmids we created
during this project are nice tools to demonstrate a few techniques and principles
frequently used in yeast genetics. The student has generated an artificial “flippase”
(ELO3-hydratase2 or ELO3-H2) that can substitute for RFT1function (but not very well).
RFT1 is essential, which means cells cannot grow without a functional copy of
this gene. This causes a problem when creating the knockout strain: how do you create a
knockout and still get viable cells? This problem can be solved by transforming the cells
with a plasmid that carries the RFT1 gene and then performing the knockout by replacing
the RFT1 gene with the KANMX cassette that confers resistance e.g. to the antibiotic
geneticin.
After this step, however, we are stuck with a strain that is now forced to maintain
the plasmid carrying RFT1 in order to survive. How is this helpful then? Well, we are
now able to “shuffle in” other plasmids carrying various forms of RFT1, for example
mutant forms or forms that are expressed from promoters that enable us to control
expression levels. We will try to shuffle in the latter type of plasmid, as well as a plasmid
with RFT1 expression controlled from its native promoter. We have a strain ready for you
where the rft1 deletion is complemented with a multicopy plasmid carrying the ELO3-H2
construct under the control of the GAL1 promoter. This means that expression from this
plasmid can be controlled by the carbon source: repressed under glucose, derepressed
under raffinose (and several other carbon sources) and activated under galactose.
It is easy to monitor loss of the initial plasmid in this strain: the strain we used has
the ade2, ade3 double mutation, and the initial plasmid (pTSV30 ) carrying ELO3-H2
(also under control of the GAL promoter) contains the ADE3 gene (as well as a LEU2
marker). The rft1 strain carrying this plasmid needs a derepressing carbon source
(anything other than glucose), as it needs the plasmid expressing the ELO3-H2 construct.
Therefore, it maintains also the ADE3 marker on the plasmid, which makes it
genotypically mutant in ade2 only. These ade2 mutant strains have a characteristic red
color.
If another plasmid carrying RFT1, but not the ADE3 marker, is introduce in these
cells (in this case YCp33GAL1- RFT1, with RFT1 under GAL promoter control, or
YCp33RFT1p-t under its own regulatory elements), they now can lose the initial plasmid
(pTSV30 ELO3-H2). This process results in “sectored” colonies, where white sectors in
the otherwise red colonies originate from cells that lost the pTSV30 ELO3-H2 plasmid.
In this project, you will transform these both plasmids into the rft1/ pTSV30
ELO3-H2 strain, shuffle out the pTSV30 ELO3-H2 plasmid and then examine the effect
of carbon source on the growth of these transformants.
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YG 2015 P4
If everything works out, you’ll learn in this practice:
1.)
2.)
3.)
4.)
5.)
How to perform a simple one-step transformation procedure in yeast
How to identify sectored colonies
how to carry out a colony-colour based plasmid shuffle
how to carry out a growth assay using a serial dilution spotting technique
a bit about carbon source control of expression/glucose repression/gal
induction
Day 1: Transformation of the rft1/pTSV30 ELO3-H2 strain
We have inoculated the rft1/ pTSV30 ELO3-H2 strain for you yesterday. The
plasmids you are using are:
YCp33RFT1p-t (RFT1 controlled from native regulatory sequences on a single copy
URA3 plasmid)
YCp33 GAL-RFT1 (GAL promoter, single copy)
And as control plasmid transformation:
YCplac33
Use the following procedure (each group does two sest of transformations):
Transformation of reporter gene plasmid into yeast
Method:
D.-C. Chen, B.-C. Yang, and T.-T. Kuo. (1992). One step transformation of yeast in
stationary phase. Curr.Genet. 21: 83-84
Materials:
One step buffer
0.2M Lithium Acetate
40% PEG (Polyethylene Glycol 3350)
100mM DTT (Dithiothreitol) (add fresh from 1M stock)
Single -stranded salmon or herring sperm DNA
10mg/ml sheared and boiled salmon or herring sperm DNA in TE buffer
When you make up the one step buffer, only add the LiAc and PEG. Take into account
the volume of DNA and DTT that will be added to make the entire solution.
For Example:
For a 100 ul transformation, you add 10ul DTT and (typically) about 5ul total DNA
(ssDNA plus plasmid DNA). The volume of the Buffer stock added per transformation is
85 ul. Therefore, if you make up Buffer for 100ml final volume, bring up the LiAc and
PEG to only 85ml in water and autoclave.
Transformation
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for each transformation planned, pellet 400 ul of cells of the overnight culture in a
microfuge tube (5-10s at full speed) and remove the supernatant
Resuspend the cell pellet to a final volume of 100ul one step buffer, taking into
account the DNA and DTT to be added
85 ul one step buffer stock
10 ul 1M DTT
4 ul ssDNA (boiled for 5 minutes and put on ice)
1ul (or more) plasmid DNA (plasmids as above in separate transformations, three
transformations per group total)
if high transformation frequency is not required, the ssDNA may be omitted
-
Incubate at 42oC for 30 minutes
Plate onto selective media (SCGal/Glu-uracil) and grow for several days at 30oC.
Day 2: Waiting….
Day 3 (Tuesday 2nd week): Picking the colonies, restreaking
By today, the transformants should be large enough to be picked. You will
probably be able to see sectors and whites, and we will pick two colonies (preferably
white, or sectoring) of each the transformants (also red colonies from the control!; 6x2
colonies per group) and streak them on YPGal/Glu (Galactose 2% (Glucose 0.05%) for
single colonies. The glucose helps with the growth (yeast really does not like to grow on
galactose), but does not repress in these low concentrations. ALL plates go back in the
30oC incubator.
We’ll keep the transformation plates for a bit longer to get really good color
development.
Day 4: Waiting
Day 5 (Friday 2nd week):
Restreaking of (preferably) white or sectoring colonies from the Tuesday streaks
on YPGal/Glu for single colonies (also restreak the control colonies – they will be red).
From the previous incubation, cells that have lost the pTSV30ELO3-H2 plasmid will
have accumulated.
Grow for four days in the 30oC incubator. We will have sectoring and white colonies
on Tuesday on our plates.
Day 6 (Tuesday 3rd week): isolation of white colonies (+ red controls):
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YG 2015 P4
If everything worked out, we should have at least a few pure white colonies on the
YP- galactose/low glucose plates with the YCp33 GAL-RFT1 or YCp33RFT1p-t
transformants. These have now lost the pTSV30 ELO3-H2 plasmid. Pick one
representative of each and inoculate each into 5 ml YPRaf/Glu (2% Raffinose, 0.05%
glucose) liquid medium.
There should be no sectoring colonies on the other plates with the control transformants
(sometimes, a few odd white ones will appear).
Inoculate the control strain – transformed with YCplac33 – in 5 ml YPGal/Glu, as it
barely grows on Raffinose at all.
Grow o/n in the 30oC shaker
Day 7 (Wednesday 3rd week): Spotting assay
Depending on the density of our overnight cultures, we will either dilute them and
let them grow 3-4 hours more or use the cultures straight for the spotting assay (if the
density is really low).
For the dilution of overnight cultures with a high OD, inoculate the new cultures to a
final OD600 of ~ 0.25 in 5 ml YPRaf/Glu media and put them back into the incubator for
a few more hours.
Spotting assay:
1. Take out 1 ml of each culture
2. Measure OD600
3. Take out the equivalent of 1ml cells at an OD600 of 0.5. You will have to
normalize for different culture densities. For example, if your culture is at an
OD600 of 0.72, take out 0.5 : 0.72 = 0.69 ml. If you sample is at an OD600 of 0.36,
take out 0.5 : 0.36 = 1.39 ml (and so on). It is important to use equivalent
amounts of cells in this assay, and therefore this normalizing step is
absolutely required!
4. Spin down the cell samples in microfuge tube for 10 seconds
5. Remove the media and resuspend in 1ml sterile H2O
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6. Make serial dilutions in
H2O:
100 l cells
100 l cells
900 l
H2O
900 l
H2O
1:10 X tube
900 l sterile H2O
+ 100 l cells
from 1 X tube
1X tube (OD600
= 0.5)
100 l cells
900 l
H2O
1:100 X tube
900 l sterile H2O
+ 100 l cells
from 1:10 X tube
1:1000 X tube
900 l sterile H2O +
100 l cells from
1:100 X tube
7. Spot 2 l cells from each sample on YPGal/Glu and YPD 4 % plates, left to right
in descending concentration (2 sets of plates = 4 plates per
group)
YP-Gal plate
1x
1:10x
YPD plate
1:100 x 1:1000x
1x
1:10x
1:100 x 1:1000x
YCp33 RFT1p-t
YCp33 GAL-RFT1
YCplac33
8. “Spot” by pushing out the 2 l cell drop from your pipette tip (it will still hang
there) and touching the agar with the drop (but not the tip!!). The adhesion force
of the agar will pull off the drop and this will result in a nice round spot later on if
you don’t wiggle too much
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Tip: It will be useful if you pre-mark the positions on which you are planning to
spot the drops beforehand on the back of the plate with a fine felt pen. You will
see this mark through the agar and can use it as an aim to get an even spotting
pattern!
Let the plates absorb the liquid (doesn’t take more than 15 minutes) and then transfer
plates into the 30oC incubator. Grow several days.
We will monitor the growth and may put the plates into the fridge for you when the
growth is good enough. This is to prevent colonies from the YCp33GAL-RFT1
transformants from coming up on the YPD plates – repression is often not 100% in
all cells, so colonies come up after a while, especially if the glucose around the cell is
used up.
Day 8 (Tuesday 4th week): Scoring of Growth
Describe the differences in growth between the different samples if there is any.
Document the differences by taking pictures of the plates.
Day 9: Nothing to do…
Day 10 (Friday 4th week):
Review of results
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