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, FOA 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 the Polymerase Chain to quantify the abundance of a
particular nucleic acid.
New Technique: Realtime PCR
Experimental Goal: Determine the quantity of target DNA in a sample of unknown
DNA concentration.
Experimental Design: Conduct realtime PCR to set up a standard curve of SQS1 DNA
abundance vs threshold cycle using the double strand specific SYBR green dye. Use this
standard curve to determine the DNA concentration of your “unknown” sample of SQS1
DNA.
Class Outcome: Outstanding. The standard curves completed by the students were well
within acceptable limits and allowed each student to successfully define the target DNA
abundance.
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 A
mapping
Experimental Goals: 1) Determine whether the presence of previously unreported
introns and alternate 3’ splice site use in yeast predicted by our Illumina deep sequencing
experiments were valid. 2) Determine whether the differences in gene expression
predicted by Ion Torrent sequencing of PRP43 (wildtype) and the prp43H218A mutant
mRNAs were valid. 3) Identify which of two yeast strains contained an overexpressor
gene for SNR19.
Experimental Design: We prepared cDNA from the same DNA sample and use genespecific primers to score for the novel introns and alternate splicing by rtPCR. We used
RNA from the PRP43 and the prp43H218A mutant for a northern blot that we probed
with a uniformly labeled DNA probes against RPL17B, RPS10B, and DFR1. Use an
RNaseA mapping approach to score for mRNA abundance.
Class Outcomes: Strong technical success. cDNA synthesis and PCR worked
extremely well and we were able to validate three of four predicted changes in splicing
patterns for the yeast mRNA. The random-prime labeling worked flawlessly with every
student getting great labeling. The northern analysis revealed greatly impaired splicing in
the prp43H218A mutant compared to wildtype but did not support the 4-fold differences
in mRNA abundance predicted by the Ion Torrent sequencing. The RNase A mapping
would well for the majority of student groups and we were able to successfully identify
the overexpressor as strain “B”.
Fundamental Principles: Random mutagenesis can be used to score for gene function
on a plasmid.
New Techniques: Hydroxylamine mutagenesis of plasmid DNA, 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 with hydroxylamine, transform the treated DNA
into CJ236 E. coli and select on ampicillin plates. Control treatment was with TE rather
than hydroxylamine. Next score the number of AmpR, TetR and AmpR, TetS
transformants in the control and experimental cultures to learn if HA acted as a mutagen.
Class Outcomes: in progress.
Fundamental Principle: Epitope tags offer value in the isolation and characterization of
protein abundance.
New Techniques: Yeast protein isolation by chitin-agarose chromatography, chemical
lysis of E. coli, use of a T7 coupled expression system in ER2566 bacteria, intein
cleavage; chemiluminescence using a horseradish peroxidase conjugated antibody. This
same blot was also used to probe with an alkaline phosphatase-conjugated antibody.
Experimental Goals: 1) One-step purification of a recombinant protein (Spp382 1-121)
expressed in E. coli. 2) Determine relative abundance of a set of yeast proteins that
include a wildtype untagged negative control and three strains each expressed from the
native promoter and tagged with an identical C-terminal calmodulin binding domainprotein A fusion (Tda1, Sup35, Pgk1).
Experimental Designs: 1) Use the Impact system from NEB to recover express a
recombinant protein in bacteria, lyse with B-Per reagent, select Spp382-121 on chitin
agarose then release via intein cleavage with DTT. 2) 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 peroxidase conjugated horseradish
peroxidase antibody. Develop using the Pico-Western (Pierce) chemiluminescence
system & with alkaline phosphatase (as an alternative detection scheme).
Class Outcomes: 1) Outstanding. Class recovery of the Spp382 (1-121) peptide was
great in terms of yield and purity – with the only contaminant being a small amount of
the intein-CBD peptide released from the beads. 2) in progress.
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. In addition, one question will be taken
from the 2012 mid-term exam and one question will be taken from the 2011 final exam.
Note 2: Your notebook must be turned in for grading at the exam at 1:00 PM on
Wednesday December 12, 2012.
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