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Agro/ANSC/Biol/Hort/Gene 305
Fall, 2016
Homework #4 (Due back on Dec 2, 2016)
Name:
Section :
Q1.
Describe the important features of cloning vectors. Explain the purpose of
selectable marker genes in cloning experiments.
Cloning vector must have the following features:
i.
ii.
iii.
Origin of replication – this will allow the vector to replicate
independent of the chromosome inside the host cell.
Selectable marker gene – usually an antibiotic resistance genes –
Following transformation into host cells, the cells when grown on media
containing the corresponding antibiotic will allow only those host cells
to grow that contain the antibiotic resistance gene, an indicator of the
presence of the vector.
A unique restriction site preferably in the middle of the lacZ gene.
When induced using an analog of allolactose (IPTG) the -galactosidase
can break down X-Gal (equivalent of lactose) and produce a blue
product. If a piece of DNA (gene of interest), is inserted in the restriction
site in the middle of the lacZ gene, the gene does not either produce a
product or a non functional product and thus cannot breakdown the XGal and produce the blue product. Thus, when transformed cell are
plated on X-Gal and IPTG, two kinds of colonies are seen on the plate,
blue and white. The white colonies have your gene of interest and the
blue colonies have the intact vector with no inserted DNA.
Q2. In a stepwise manner describe the steps in cloning a gene. Your answer should
also include how you would identify a bacterial colony that contains your gene of
interest.
i.
ii.
Treat the genomic DNA containing the gene of interest with the same
restriction enzyme as the unique restriction enzyme set. Mix the two
sources of DNA, add ligase and competent host cells. The transformed
cells from the genomic library that contains some cells that have the
gene of interest.
The library is plated in a media containing the antibiotic and subjected
to colony hybridization using a tagged (radioactive) probe. The probe is
a fragment of DNA containing sequences that are complementary to the
sequence of the gene (complete or partial). This involves lifting the DNA
from the colonies on to membrane (replica plating) and using the
membrane for hybridization with the probe. Following incubation with
the probe, the membrane is washed to remove all non hybridizing probe
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and exposed to X-ray film. The hybridizing colony will expose the X-ray
film as a spot above it and will show up as black spots on the film. These
spots will be matched up with the master plate and the colony
containing the gene will be isolated
Q3. Some vectors used in cloning experiments contain bacterial promoters that are
adjacent to unique cloning sites. This makes it possible to insert a gene sequence next to
the bacterial promoter and express the gene in bacterial cells. These are called expression
vectors. If you wanted to express a eukaryotic gene in bacterial cells, would you clone a
genomic DNA or cDNA into the expression vector? Explain your choice. What other
manipulations have to be done to ensure that the expression vector will allow the
translation of the eukaryotic mRNA in the bacterial cells?
For expression in a bacteria, we have to use the cDNA rather than genomic DNA
because bacteria cannot process introns.
The Shine-Dalgarno sequence has to be engineered downstream of the promoter for
recruitment of the ribosomes.
Q4. Starting with a sample of RNA that contains the mRNA for the -globin gene,
explain how you would create many copies of the -globin cDNA using reverse
transcriptase PCR.
Isolate RNA from blood cells and mixed with reverse transcriptase, a primer that binds
to the 3’UTR of the -globin RNA and dNTPs. This generates a single stranded cDNA,
which can be used as a template to produce the second strand using a second primer
that binds to a region in the 3’UTR of the cDNA, with Taq polymerase. The process of
PCR can be done with the double stranded DNA for multiple cycles to produce multiple
copies of the cDNA
Q5. In Southern, Northern and Western blotting, what is the purpose of
electrophoresis?
Electrophoresis is a used to fractionate DNA fragments (for Southern blots),
different RNA species (northern blot) and proteins according to their size
(western blot).
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Q6. To produce transgenic plants, plant tissue is exposed to Agrobacterium
tumefaciens containing a gene of interest, and then grown in media containing
kanamycin, carbenicillin and plant growth hormones. Explore the purpose of each
of the three agents. What would happen if you left out kanamycin?
Plant tissue is exposed to Agrobacterium tumfaciens containing your gene of interest
along with a kanamycin resistance gene in the TDNA for a period of time, during
which time the TDNA is transferred to the plant cells. The plant cells are then
incubated in a media containing carbenicillin, kanamycin and plant hormones.
The carbenicillin kills the A. tumefaciens which has already transferred
its TDNA to the plants cells
Presence of Kanamycin the media allows only those plant cells to grow
that has the Kanamycin resistance gene. This would imply that your
gene of interest is also in those cells
The plant hormones allow the plant cells to regenerate into whole
plants.
Q7. What is the strategy for expressing human genes in a domestic animal’s milk?
A human gene encoding the medicine is engineered behind the promoter of a
mammary gland specific gene (lactoglobulin) and the vector containing this gene
construct is injected into a sheep’s oocyte. The transgene (human gene behind the
lactoglobulin gene promoter) is then integrated into the sheep’s genome. The
fertilized oocyte is then implanted into a female sheep, which then gives birth to a
transgenic sheep. The milk from this sheep will have the human protein.
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