Download Solutions to Genetics Day 6 Interpretation Questions

Solutions to Genetics Day 6 Interpretation Questions
1. Interpret your data from Genetics Day 5. Some issues to consider…
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Are the Ara- and KanR phenotypes linked in your mutant strains?
Are the phenotypes consistent among the four transductants from the same mutant
strain?
Did your LacZ phenotype remain consistent before and after transduction?
Are there any unusual observations?
Assuming that the KanR and Ara- phenotypes are linked, you should expect that your transductants
will have the same phenotype(s) as the “white” mutant from which the P1 transducing lysate was
made. This is because, upon transduction, there is genetic exchange between the “donor” and
“recipient” strains—and a resulting change in phenotype. For example, if mutant B1W1 was
genotypically araA::lacZ+, kan, you would expect that the transductants would have the following
phenotypes:
LB
G
LB Kan
G
Mac Ara Kan
G, white
LB Xgal Kan
G, white/light blue
LB Ara Xgal Kan
G, dark blue
If observations were inconsistent with their expectations, you should consider the possibility that
their Ara- and KanR phenotypes were not linked.
2. Consider wild type λ phage, the modified λ phage that carried the mini TN10 transposon,
the P1 phage we used to make lysates and the P1 phage found in the transducing lysates.
Address the following:
• Is each lytic, lysogenic, both or neither?
a) Wildtype λ phage is both lytic and lysogneic
b) The modified λ phage is neither lytic nor lysogneic under the conditions used in the lab.
c) The P1 phage used to make lysates was lytic.
d) P1 phage found in the transducing lysates was a mixed population, so some were like the P1 used
to make the lysates and were thus lytic. Others had packaged bacterial DNA and were neither lytic
nor lysogneic.
Of the phage stocks used in the lab, which contained a homogenous population of
phage?
The wild type λ phage and the modified λ phage that carried the mini TN10 transposon
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• Which of these carry E. coli DNA?
The modified λ phage that carried the mini TN10 transposon had part of the lacZ gene and some of
the P1 phage found in the transducing lysates will have packaged bacterial DNA. Also: ( *Not
required) a tiny % the P1 phage used to infect the mutants will have host DNA)
Of the phage stocks used in the lab, which did you use at high MOI? Which did you
use at low MOI? Explain why.
a) The modified λ phage was used at low MOI because we wanted any cell that received a transposon
to receive 1 and only 1 transposon. This creates a population of mutant cells that each likely carry a
single insertion mutation.
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b) The P1 phage used to make lysates was used at relatively high MOI because each phage was
identical and the overall goal for the experiment was to infect all the cells, hope to get the rare phage
that packages host DNA and lyse the cells. More of the P1 phage does not interfere with this goal.
c) The P1 phage found in the transducing lysates was used at a very low MOI. It consisted of a
mixed population where the vast majority of the phage were like the P1 used to make the lysates and
were thus lytic. Only a very small percent of the phage in this population had packaged bacterial
DNA and only a few of those carry the transposon. We are selecting for the rare recipient cell that
was infected with a phage carrying the transposon. If a second phage were to infect this cell, greater
than 99% of the time it would be lytic phage and thus the rare tranductant that we are hoping to
find would be killed.
3. During the module, your experiments relied upon random insertion of DNA into the
bacterial genome and non-random insertion of DNA into the bacterial genome.
• On which days did each occur?
On day 1 our experiment relied upon random insertion of DNA.
On day 4 our experiment relied upon non-random insertion of DNA.
• What was the goal of each?
On day 1 our experiment relied upon random insertion of DNA. The goal was to make a population
of cells that each had a different transposon insertion mutation.
On day 4, the goal was to move the gene carrying the insertion mutation into a new bacterial strain.
How was the random insertion of DNA into the bacterial genome accomplished? Name
one thing that could have prevented this from occurring.
We used a modified λ phage that carried the mini-TN10 transposon to deliver the DNA to the
bacterial cell. This phage was no longer lytic or lysogenic and the INT sites of the phage DNA were
removed to prevent specific insertions. The gene encoding transposase was carried on a plasmid in
the starting strain and expression of enzyme transposase was induced to allow for transposition of
the mini-TN10 from the phage DNA into the host DNA.
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Any change that prevents transposase from being expressed, any change that interferes with phage
adsorption, any change that allowed the λ phage to form a lysogene or lyse the cells could have
prevented random insertion from occurring.
How was the non-random insertion of DNA into the bacterial genome accomplished?
Name one thing that could have prevented this from occurring.
The P1 phage used to make transducing lysates. The rare phage in this lysate will carry the gene
that is disrupted by the transposon. This phage will deliver this DNA into the recipient cell. The
DNA flanking the transposon is identical to DNA in the recipient cell’s genome and this allows
homolgous recombination to occur.
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No flanking DNA that is holomolgous, Poor lysis during the preparation of the transducing lysates,
too high a MOI when using the transducing lysates, other answers acceptable.