Inaugural Run Second Half Labs – Focus on Group Projects & Pooled Data –
Assessment and Study Guide
Fundamental Principle: Determine whether a mutation inactivates or impairs the
function of a gene. Negative selection in the plasmid shuffle technique resolves the basic
problem of not being able to know whether a gene is essential or not since, under normal
circumstances if you inactivate the gene, you kill the cell, and you never “see” the desired
clone. That is, you can’t distinguish between a knockout experiment that inactivates a
gene and kills as cell from one where the inactivation failed to occur do to a technical
difficulty (such as poor transformation efficiency).
New Technique: Plasmid Shuffle, counter-selection
Experimental Goal: To determine whether the prp43H218A mutation resulted in a lethal
event.
Experimental Design: Clone a functional copy of Prp43 in a URA3-based plasmid and
the prp43H218A mutant allele on a TRP1-based plasmid and place in a genetic
background where the chromosomal copy of the essential PRP43 gene is deletion. Select
the plasmids based on the nutritional markers (e.g., TRP1 and URA3) then score for
prp43H218A activity on 5’FOA media. 5’FOA is an anti-metabolite toxic in the presence
of the URA3 encoded activity and therefore selects for cells that have spontaneously lost
the URA3-based plasmid (and the WT PRP43 gene) during mitotic division.
Class Outcome: Outstanding This experiment worked flawlessly and we were able to
demonstrate that the prp43H218A mutation, while producing an enzyme with only ~30%
normal ATPase activity, is viable. We also used the 5’FOA negative selection to show
that loss the SNR19-YCplac33 plasmid restored the temperature sensitivity of the snr19-1
ts mutant.
Fundamental Principle: Protein-protein interaction can be monitored in living cells
based on the bi-molecular reconstitution of the Gal4 transcription factor
New Technique: Yeast Two Hybrid Assay
Experimental Goal: Map the Prp43-binding surface on the Pxr1 protein
Experimental Design: Create a full-length Prp43-Gal4 DNA binding domain fusion on
the pAS2 plasmid and a series of Pxr1 peptide-Gal4 transactivation domain fusions on
pACT. Introduce into a yeast strain bearing a Gal4-regulated HIS3 gene plus mutations
in the trp1 and leu2 genes. Select the double transformants based on the TRP1 and LEU2
plasmid markers then score for reporter gene transactivation on medium lacking uracil
(and containing 3 aminotriasole).
Class Outcome: Outstanding This experiment worked flawlessly and we were able to
localize the Prp43 binding surface to a 50 amino acid region of Pxr1.
Fundamental Principle: Use of an antibody to score for changes in protein abundance
New Technique: Western blot
Experimental Goal: Determine whether the C. elegans RNAi knockdown experiments
resulted in decreased target protein levels.
Experimental Design: We harvested C. elegans from our control, Dyn1, Dpy1 and
Erm1 knockdown animals. Extracted the protein, and probed a western transfer with
antibodies specific for Dyn1 and Erm1. An alkaline phosphatase development system
was used.
Class Outcome: Marginal The protein extraction, gel electrophoresis and transfer
worked well. The BCIP/NBT developed membrane showed only weak protein bands
with one of the antibodies and it was not possible to firmly conclude whether the target
proteins were reduced (although we know, based upon the clear developmental
phenotypes, that the RNAi constructs were effective). Next year increase the amount of
protein loaded on each gel and decrease the antibody dilution to favor the visualization of
weak bands.
Fundamental Principles: Changes in mRNA expression/stability and processing can be
inferred by careful measurements of RNA abundance and structure.
New Techniques: Random-prime probe preparation of dsDNA probe, rtPCR, RNase
mapping
Experimental Goal: Determine whether the selective gene activation in the prp43H218A
mutant (compared to WT yeast) and the presence of previously unreported introns and
alternate 3’ splice site use in both the WT and the prp43H218A yeast strains predicted by
our Illumina deep sequencing experiments were valid. Also, to identify which of two
yeast strains contained an overexpressor gene for SNR19.
Experimental Design: We used RNA from the PRP43 and the prp43H218A mutant for
a northern blot that we probed with a random labeled experimental and control probe.
Prepare cDNA from the same DNA sample and use gene-specific primers to score for the
novel introns and alternate splicing. Use an RNaseA mapping approach to score for
mRNA abundance.
Class Outcome: Very Good The rtPCR experiment worked great and we were able to
detect both the novel introns and the predicted alternative 3’ splice site usage in the target
genes. The random primer labeling worked great as did the ADE3 control hybridization.
Background rRNA binding with the second probe prevented us from scoring for change
signal for YNR073C RNA. A second probe that hybridized to the TY1 transposon
worked well, however, are we were able to confirm the ~2-fold enhancement of TY1
signal in the prp43H218A mutant background. Also, the RNaseA protection experiment
worked fantastic & we were able to clearly distinguish which of the coded strains
overexpressed SNR19.
Fundamental Principles: Random mutagenesis can be used to score for gene function
on a plasmid.
New Techniques: Hydroxylamine mutagenesis of plasmid DNA, XL1-Red (mutator
strain) mutagenesis, replica plating
Experimental Goal: Determine the effectiveness of hydroxylamine treatment or XL1Red propagation in generating mutations in the tetracycline resistance gene of pBR322.
Experimental Design: Treat pBR322 for variable lengths of time with hydroxylamine,
transform the treated DNA into TG1 E. coli and then score for 1) plasmid inactivation
and 2) changes in the number of AmpR, TetR and AmpR, TetS transformants. Similarly,
propagate pBR322 in TG1 (control) or XL1-Red (experimental) E. coli, extract plasmid,
transform TG1 with the control & experimental DNAs and score for strain-specific
differences in the number of AmpR, TetR and AmpR, TetS transformants.
Class Outcome: Good It appears that residual pyrophosphate in the control and
experimental DNA strongly inhibited transformation, preventing us from determining the
mutagenesis rate. Next year, try using molecular exclusion chromatography to remove
the pyrophosphate prior to E. coli transformation. The transformation and recovery of
plasmid DNA from the XL1-Red strain worked well. The XL1 mutagenesis worked well
with groups reporting from 6 to 16% mutants (ave ~11% 104/900). In TG1 the rate was
much lower (8/1056 =0.76% - one high report of 6/27). Next year, dry ampicillin plates
two days at 37C before replica plating and have students practice technique to avoid
smearing of colonies
Fundamental Principle: Epitope tags offer value in the isolation and characterization of
protein abundance.
New Techniques: Yeast protein isolation, chemiluminescence using a horseradish
peroxidase conjugated antibody
Experimental Goal: Determine relative abundance of a set of yeast proteins each
expressed from the native promoter and tagged with an identical C-terminal calmodulin
binding domain-protein A fusion.
Experimental Design: Break the TAP-tagged yeast strains using glass beads, resolve
the yeast proteins by denaturing SDS gel electrophoresis, and probe a membrane transfer
with the anti-horse radish peroxidate conjugated horseradish peroxidase antibody.
Develop using the Pico-Western (Pierce) chemiluminescence system & with alkaline
phosphatase
Class Outcome: Encouraging. The protein extraction worked well and the AP reaction
the developed showed multiple strain-specific tagged proteins consistent with the
experimental goals. The HRP development was less satisfying in that only a single band
was observed – the highly abundant Prp43. Given this, we will want to purchase fresh
PAP antibody for next year as the most likely problem is that this antibody or the HRPassociated enzyme has gone bad. Also, there was evidence of protein degradation in the
yeast prep, next year include the serine protease inhibitor, phenylmethanesulfonylfluoride
(PMSF), in the extraction buffer.
To prepare for the Exam
Re-read all of the labs and all assigned reading, review the posted slides from the
Friday talks. For the labs, ask yourself – what did we do and why did we do it?
Understand the goals and experimental steps involved – including the reagents used. Be
able to “design an experiment” using these same techniques to answer a novel question.
Bring questions to class before the exam and asked for clarification of any questions you
have during the exam.
Note 1: For the final exam, at least one question will be derived from each of the
required reading assignments and from the topics relevant to our visits to the DNA
microarray and DNA sequencing core facilities.
Note 2: Your notebook must be turned in for grading at the beginning of lab on
Wednesday November 7, 2011. You will lose one point on the notebook grade for each
day that it is late.
Other Topics:
Plasmid host range and copy number, other cloning vectors (λ, BAC, YAC, cosmid)
TAP-tags for relative protein abundance measurements, protein localization, and protein
complex selection
cDNA and Genomic DNA libraries
Deep sequencing methodologies (Ion Torrent, 454, Illumina, Sanger capillary)
Nanostring technology
Synthetic lethality, dosage suppression, extragenic suppression