Download Genetics Day 1 interpretation question

Genetics Day 1 interpretation question
1. Today you performed transposon mutagenesis.
a) What is a transposon?
A transposon is a piece of DNA that can move around the genome of cells that carry it.
b) What is required for a transposon to move around the genome?
The transposon must have thee appropriate DNA sequence at its ends, these are inverted
repeats that are recognized by transposase. The enzyme transposase is also required for
movement of the transposon.
c) The transposon is delivered to your cells by using phage. Usually, when phage infect
bacterial cells, they lyse and therefore kill the bacterial cells. Will the phage we are
using kill our bacterial cells? Why or why not?
The phage that is used in the mutagenesis (lamba 1205) is itself a mutant. This phage has been
modified such that the genes that encode the proteins for lysis (killing of the host cell) have been
deleted.
d) When a transposon jumps into the bacterial chromosome, it confers a new property
to the bacterial cell, and at the same time, it takes away a property of the bacterial cell.
• What new property is added in each cell?
In our case, the transposon carries DNA encoding two different proteins, but only
the kanamycin resistance gene is complete, i.e., it has a promoter, a start codon, and
the coding region. It encodes a protein that allows cells to survive in the presence of
kanamycin, thus all cells that have the transposon will be kanamycin resistant
• What property is taken away in each cell?
Because the transposon can integrate randomly into the genome, we can not answer
this question specifically. However, each gene that is disrupted by the transposon
has its own particular function, and the property that is lost is the property that the
disrupted gene governs
2. The E. coli strain that you used for your mutagenesis experiment (pNK/BW140) is
phenotypically Ara + and Lac-.
a) What does it mean for a strain to be Lac-? What indicator plates would you use to
distinguish a Lac- strain from a Lac+ strain? What result (on these plates) would tell you
that you had a Lac- strain?
Lac- cells have lost the ability to grow on media where the sole carbon source is lactose. If you
plate Lac+ cells and Lac- cells on media where the sole carbon source is lactose, only the Lac+
cells will grow.
b) What does it mean for a strain to be LacZ-? What indicator plates would you use to
distinguish a LacZ- strain from a LacZ+ strain? What result (on these plates) would tell
you that you had a LacZ- strain?
lacZ- cells have lost the function of the enzyme β-galactosidase. Cells that are LacZ- can not
grow on media where the sole carbon source is lactose, and will be white on media containing
Xgal. If you plate LacZ+ cells on media contain X-gal, the colonies will turn blue.
c) What does it mean for a strain to be Ara+? If you did not have Mac plates, but had
minimal media plates, rich media plates and minimal + ara plates, how could you use to
distinguish an Ara- strain from an Ara+ strain?
Ara+ cells can utilize arabinose so can grow on media that has arabinose as the sole carbon
source. Ara- cells have lost the ability to grow on media where the sole carbon source is
arabinose. If you plate Ara+ cells and Ara- cells on media where the sole carbon source is
arabinose, only the ara+ cells will grow.
d) Which of the following two statements is true? Justify your answer briefly. (You
may assume that there is only one copy of the lac genes in these strains.)
A Lac- strain must always be LacZA LacZ- strain must always be LacA lac- strain can be missing lacZ, lacy, or lacA, any of which will make the cell Lac-. A LacZcell is missing the function of β-galactosidase so will definitely be Lac-.
3. You decided to repeat the mutagenesis you performed in lab today, but realize that
you have run out of pNK/BW140 cells. Instead, you perform four separate
mutagenesis experiments (experiments #1-4) using the strains listed below:
a) Relative to the pNK/BW140 experiment, how many total colonies do you expect to
see on the Mac Ara Kan plates in each experiment? (Same? More? Fewer?) Justify
your answers briefly.
b) Relative to the pNK/BW140 experiment, how many white colonies do you expect to
see on the Mac Ara Kan plates in each experiment? (Same? More? Fewer?) Justify your
answers briefly.
c) Relative to the pNK/BW140 experiment, how many blue colonies do you expect to
see on the LB Xgal Kan plate in each experiment? (Same? More? Fewer?) Justify your
answers briefly.
1
Starting
Strain
KBS1
Relevant Phenotypes
total colonies?
white colonies?
blue colonies?
TetS, KanS, Ara+, Lac-
Fewer. No transposase so
no mutagenesis, so no
Kan resistance
Fewer. No
transposase so no
mutagenesis
Fewer. No
transposase so no
mutagenesis
2
pNK/KBS2
TetR, KanS, Ara-, Lac-
same
3
pNK/KBS3
TetR, KanR, Ara+, Lac-
More. Strain begins as
KanR, so no selection
4
pNK/KBS4
TetR, KanS, Ara+, Lac+
same
More. Strain
begins as AraBecause there is no
selection, won’t see
any of the mutants
(too many cells
present to
distinguish
individual
colonies)
same
same
Because there is
no selection,
won’t see any of
the mutants.
More. Strain
begins as Lac+.