Download Lesson12 sp2012

Lesson #12 Gene cloning
Genetics Agronomy 315
Questions due Monday, April 9th, 2012 11:59 pm
Lab materials
DNA from jellyfish cells
DNA from Zebrafish cells
DNA from coral cells
mRNA from
mRNA ground up coral
cells
Ground up jellyfish
mRNA from
Fin cells of a zebrafish
materials to make bacterial growth plates with
Amp + Xgal
no antibiotic
amphicillin
(Amp)
The following DNA modifying enzymes and biochemicals:
restriction enzymes: EcoRI, KpnI, JelliI, CoralVI, BanHI
E. coli Bacteria susceptible
to all antibiotics
other enzymes: Ligase, reverse transcriptase, DNA polymerase, RNA polymerase
enzyme that adds sticky ends.
radioactive material to label nucleotides,
color enzymes to bind to antibodies
antibodies that will only bind to the red
Fluorescent protein from coral
Other materials
membranes that proteins and
nucleotides stick to
an electroporation machine
pBluescript plasmid
a gene gun
zapper!!
cd player,
country western cds,
rock cds
Recombinant plasmid with an
insert that is a clone of the jellyfish
green fluorescent protein gene
1. Assume you are doing this to obtain a genomic clone of the red
fluorescent protein (RFP) from coral. You are also trying to obtain a
cDNA clone of the green fluorescent protein (GFP) gene from jellyfish.
a. What item above is your starting material for your library that will contain
the RFP gene clone?
b. What item above is your starting material for your library that will contain
the GFP gene clone?
c. What restriction enzyme will you use to cut your plasmid?
d. What enzymes will you need to make double stranded DNA from the
starting material for your GFP library?
e. Will the lab strain of E. coli you start with be susceptible to the AMP
antibiotic or resistant to the antibiotic (this will be before transformation)?
f. After you transform your E. coli, how could you tell the difference between
bacteria that are transformed and those that are not transformed?
g. When will you use ligase in your gene cloning work?
h. How will you transform your bacteria?
i.
Assume that jelly fish or coral have 10,000 genes and they express 1000
genes in most of the specific tissues they have in their bodies. Which
gene library will you need to make the largest in terms of number of
bacteria colonies in order to be likely to contain a clone of your gene?
Assume the petri dishes below contain colonies growing on Amp/Xgal obtained
from transforming amp sensitive bacteria with the plasmids you made in #1 .
j.
Circle all of the colonies you would keep if you wanted to obtain a clone
for your Genomic gene library.
k. How many petri dishes like those shown above should you plan to work
with if you want your library to obtain the desired cloned gene?
____ 2
___ 12
____ 100
____ 1000
___ 1,000,000
l. What would your Petri dish look like if you had forgotten to put X-gal into the
media but plated the same number of bacteria onto the plate?
m. What would your Petri dish look like if you had forgotten to put amp and X-gal
into the media but plated the same number of bacteria onto the plate?
Assume that after cloning the GFP, you form a company that makes the
following plasmid to use in educational kits to teach students the gene cloning
process. The plasmid used in this experiment is…
2. Arabinose sugar positively regulates the expression of GFP.
Procedure: After transformation
1
2
3
4
3a. . Match the outcome below with the plate number above
___ will contain green glowing colonies
___ will contain no bacteria growth
___ will be a continual lawn or bacteria where colonies grew together
___ will contain only white colonies of bacteria that do not glow green
3b. Assume you want to make genetically engineered zebra fish that glow
either green or red. You will use the gene you are cloning in your oral question
(#1)
1. replace the promoter that is a part of your gene clone with a mouse cancer
cell promoter
2. replace the promoter that is a part of your gene clone with a fish fin
promoter.
3. add a fish fin promoter to your cloned gene
4. add a mouse cancer cell promoter to your cloned gene.
5. breed your transgenic zebra fish with non transgenic zebra fish to produce
your new product for the pet owner market.
6. follow the fate of these cancer cells in your lab mice by observing the red
or green fluorescing cells.
7. electroporate the clone and modified gene into a fertilized zebra fish egg
8. inject the cloned and modified gene into a fertilized zebra fish egg
9. combine the cloned and modified gene with a virus that invades fish cells
and introduce this virus to zebra fish eggs.
10. combine the cloned and modified gene with a virus that invades cancer
cells and introduce these cells into laboratory mice.
11. put the eggs in the right environment for development to fry.
a. From the list of steps below, select the four that would be done to
perform Genetic Engineering
List their order below:
First _____ then _____ then ______ followed by ______
b. Assume that cancer researchers use your cloned gene to follow the
growth and spread of cancer cells in lab mice. Select the steps that
would be done to perform this gene therapy procedure.
List their order below:
First _____ then _____ followed by ______
3.c. Explain why even though we are transferring the jellyfish or coral gene to
mice or fish and expecting them to make the fluorescent protein, we do not need
to transfer ribosomes or tRNAs from jellyfish or coral to the genetically
engineered fish or mice.
_____3.d. Assume that you do an in vitro translation experiment with the
following materials. Ribosomes from coral, tRNAs from jellyfish, mRNAs from
zebra fish, amino acids from mice. The proteins made in vitro should be the
same proteins found in the cells of..
a. coral
b. jellyfish c. mice d. Zebra fish
4. If you sequenced a gene clone from your GFP library would you be able
to determine the amino acid sequence of the protein encoded by this
gene?
5. How about your RFP gene library? Remember, both jellyfish and coral
are eukaryotes.
6. If the genetic code was not universal but all organisms used DNA as their
genetic material, would the gene cloning process we are using in the problem set
still work to help us identify bacteria that have a jellyfish or a coral gene?
Assume the AMP resistance gene on our cloning plasmid uses the E.coli gene
code. Explain.
A
B
7. Make sure you view the animation “Screening a DNA Library” to help you answer the
following questions.
a. Which molecules above that we would be trying to detect if this library was being
screened with the antibody that binds to the RFP from coral.
___A
___B
b. Label the molecules above that we would be trying to detect if this library was
being screened with a cloned gene that was similar to the clone we were trying
to find our DNA library.
___A
___B
c. Screening a library with an antibody is similar what other molecular test we have
learned about?
___ lateral flow strip test
___ PCR
d. Screening a library with a similar cloned gene is based on the concept of
“complementary sequences” . What other molecular test we have learned about
that is based on this concept as well?
___ lateral flow strip test
___ PCR