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Key problem set 9 fa 2016

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from
(Amp)
mRNA ground up coral
materials
ive material toLab
label
nucleotides,
color
enzymes
to bind tomRNA
antibodies
amphicillin
materials to makecells
bacterial growth plates with
DNA from jellyfish cells
Ground up jellyfish
(Amp)
Lab
materials
Fin cells of a zebrafish
cells
The followingrestriction
DNA modifying
and JelliI,
biochemicals:
enzymes: enzymes
EcoRI, KpnI,
CoralVI, BanHI
amphicillin
materials to make bacterial growth plates with
(Amp)
Amp + Xgal
amphicillin
(Amp)
antibodies that wil
no antibio
Amp + Xgal
Ground
up jellyfish
Fluorescent
protein
DNA
from jellyfish
cells modifying
mRNA
from
DNAenzymes
from
Zebrafish
cells
The
following
DNA
and
biochemicals:
mRNA ground up coral
DNA from coral cells
no antibiotic
mRNA from
E. coli Bacter
to all antibioti
mRNA from
Fin cells of a zebrafish
other enzymes:
transcriptase,
DNA polymerase, RNA polymerase
restriction enzymes:
EcoRI, Ligase,
KpnI, reverse
JelliI, CoralVI,
BanHI
enzyme that adds sticky ends.
Lesson #9materials
Gene
toenzymes:
makeCloning
bacterial
growthreverse
plates
with
The
following DNA
enzymes
and biochemicals:
other
Ligase,
transcriptase,
DNAmodifying
polymerase,
RNA polymerase
no antibiotic
E. coli Bacteria
to all antibiotics
The following DNA modifying enzymes and biochemicals:
Ampnucleotides,
+ Xgal
radioactive
material to label
color enzymes to bind to antibodies
AGRO 315
Genetics
antibodies that will only
enzyme that adds
sticky ends.
E. coli Bacteria susceptible
restriction enzymes:
EcoRI, KpnI, JelliI, CoralVI, BanHI LAB MATERIALS
amphicillin
restriction enzymes: EcoRI,
KpnI, JelliI, CoralVI, BanHI Fluorescent protein from
to all antibiotics
(Amp)
mRNA
DNA
jellyfish
cells
other
enzymes:
Ligase, reverse transcriptase,
DNA
polymerase,
RNAcells
polymerase color enzymes
mRNA
radioactive
material
tofrom
label
nucleotides,
toZebrafish
bind tofrom
antibodies
mRNA ground
up coral
Labfrom
materials
DNA
Zebrafish
cells
antibodies
that willGround
only biu
mRNA
ground
up coralDNA from
enzyme that adds
stickymaterials
ends.
Other
Ground
up jellyfish
cells polymerase,
other
enzymes:
Ligase, reverse
transcriptase,
DNA
RNA
polymerase
The following
DNAfrom
modifying
enzymes
and biochemicals:
Fluorescent
protein
from co
DNADNA
fromfrom
coral,
jelly
fish
mRNA
ground
up
Antibodies
that
will
only
pBluescript
plasmid
coral
cellsto label nucleotides, color
cells
radioactive
material
enzymes to bind to antibodies
antibodies that will only bind to the red
enzyme
that
adds
sticky
ends.
or zebrafish cells
coral,
jelly
fish
or
zebrafish
bind
to
coral
red
membranes that proteins and
Fluorescent protein from coral
E. coli Bacteria susceptible
s and
yfish cells
hine
NA ground up coral
s
restriction enzymes: EcoRI, KpnI, JelliI, CoralVI, BanHI
Otherfrom
materialsnucleotides
fluorescent
mRNA
stick to
Other
materials
Ground
up jellyfish
mRNA from
protein
to all antibiotics
Fin cells of a zebrafish
mRNA
from
plasm
other enzymes: Ligase, reverse
transcriptase, DNA
polymerase,
RNAnucleotides,
polymerase
radioactive
material
to label
color enzymespBluescript
to bind to antibodies
membranes
that proteins
and an
membranes
that
proteins
electroporation
enzymeand
that addsmachine
sticky ends.
mRNA fromnucleotides stick to
Fin cells of a zebrafish
nucleotides stick to
Fin cells of a zebrafish
radioactive
material to label nucleotides, color enzymes to bind to antibodies
electroporation
a gene
E.ancoli
bacteriamachine
susceptible
togun Membrane
that proteins and Electroporation machine
zapper!!
pBluescript plasmid
all
antibiotics
zapper!!
angun
electroporation machine nucleotides stick to
a gene
ates with
ntibiotic
DNA from Zebrafish cells
DNA from coral cells
materials to make bacterial growth plates with
Other materials
cd player,
DNA from jellyfish cells
Lab
materials
cds,
amphicillin
no antibiotic
Other materials
cd player,
country western
a gene
gun
country
western
cds,
cds cells
rock cds
DNA
from
coral
membranesrock
that proteins
and
Amp + Xgal(Amp)
zapper!!
membranes that proteins and
nucleotides stick to
pBluescript plasmid
antibodies that
will onlywith
bind to the red
Recombinant
plasmid
no antibiotic
Fluorescent
protein
from
coral
the cloned gene inserted
Amp + Xgal
Recombinant
mRNA
fro
Recombinant
plasm
DNA from Zebrafish cells
pBluescript
plasmid
mRNA ground
up coral
zapper!!
Recombinant plasmid with an
insert that is a clone of the jellyfish
green fluorescent protein gene
cells
insert thatGround
is a clone
insert
that
isuppraj
green fluorescent
greenfrom
fluoresc
mRNA
Recombinant
plasmid
nucleotides stick to
cd player,
Fin cells of a zebrafish
The
DNA modifying enzymes and biochemicals:
countryanwestern
cds, following
insert that is a clone of
electroporation
machine
an
electroporation
machine
rockBacteria
cds susceptible
pBluescript plasmid
prote
E. coli Enzymes/chemicals:
Other
Restriction Enzymes:
Gene gun
Plates with bacterialgreen fluorescent
a gene gun restriction enzymes:
E. coli Ba
EcoRI, KpnI, JelliI, CoralVI, BanHI
to all antibiotics
-Ligase
growth media options:
materials to
make gun
bacterial growth plates
with
zapper!!
a gene
merase
to all anti
no antibiotic
cd player,
-Reverse
transcriptase
EcoRI
ing enzymes and
biochemicals:
Recombinant plasmid with an
Amp
+
Xgal
zapper!!
other
enzymes:
Ligase,
reverse
transcriptase,
DNA
polymerase,
RNA
polymerase
country western
cds,
insert that is a clone of the jellyfish
-DNA polymerase
KpnI
cd player,
rock cds enzyme
amphicillin
green fluorescent protein gene
that
adds sticky
ies
antibodies-RNA
that will
only bind to the
red
polymerase
JelliIends.
(Amp)country western cds,
E.
coli
Bacteria
susceptible
KpnI, JelliI,
CoralVI,
Fluorescent
protein fromBanHI
coral
-Sticky-end adding enzyme
CoralVI
rock
cds BanHI
radioactive
material
to label nucleotides, color enzymes
to bind to 1.
antibodies
to all antibiotics
-Radioactive material
to
Amp (amphicillin) antibodies that will
The following DNA modifying enzymes and biochemicals:
label
nucleotides
2. Amp + Xgal Fluorescent protein
se transcriptase, DNA polymerase, RNA polymerase
-Colored enzyme to bind restriction enzymes: EcoRI, KpnI, JelliI, CoralVI, BanHI
3. No antibiotic
E. coli Bact
s.
antibodies
Other materials
to all antibi
other enzymes:
Ligase,
reverse
transcriptase,
polymerase,
RNA polymerase
1. Assume you are doing
a project
where
you
need toDNA
obtain
a genomic
clone of the
pBluescript plasmid
membranes
that to
proteins
and
nucleotides, red
color
enzymes
bind
to
antibodies
enzyme
that adds sticky ends. antibodies that will only bind to the red
fluorescent
protein
(RFP)
from coral and a cDNA clone of the green fluorescent
nucleotides stick to
protein
(GFP)
gene ofradioactive
a jellyfish.
Fluorescent
proteintofrom
Recombinant
plasmid
with an
material to label nucleotides,
color enzymes
bind tocoral
antibodies
insert that is a clone of the jellyfish
an electroporation
green fluorescent
protein gene machine
antibodies that will on
Fluorescent protein fro
a. What item above is your starting material for your library that will contain the RFPpBluescript plasm
Other
materials
clone?
a genegene
gun
DNA from coral cells
zapper!!
cd player,membranes that proteins and
nucleotides stick to
country
western
cds,
b. What
item
above is your starting material for your library that will contain the
rock cds
GFP
gene clone?
an electroporation
machine
mRNA from jellyfish
a gene gun
Recombinant p
insert that is a c
green fluoresce
pBluescript plasmid
pBluescript
plasmid
zapper!!
c. What
restriction enzyme will you use to cut your pBluescript plasmid?
cd player,
BamHI,
EcoRI,
or KpnI
country western
cds,(it must cut in the Lac-Z region)
zapper!!
rock cds
Recombinant
plasmidDNA
withfrom
an the
d. What enzymes will you need in order to make
double stranded
insert that is a clone of the jellyfish
starting material for your GFP library?
green fluorescent protein gene
Reverse transcriptase, DNA polymerase
e. Will the lab strain of E. coli you start with be susceptible or resistant to the AMP
antibiotic (this will be before transformation)?
Susceptible
Recombinant plas
insert that is a clo
green fluorescent
f. After you transform your E. coli, how could you tell the difference between
bacteria that are transformed and those that are not transformed?
Transformed bacteria will be able to grow in the media with antibiotics because the
plasmid carries a gene for resistance.
g. When will you use ligase in your gene cloning work?
To ‘seal in’ the DNA that you inserted into the plasmid.
h. How will you transform your bacteria?
Electroporation (or heat shock)
i.
Assume that jelly fish have 10,000 genes in total, but they express ~1,000 genes
in most of the specific tissues. Which type of gene library would require the most
bacterial colonies to isolate a clone of your gene?
Genomic library
j.
Assume the petri dishes below contain colonies growing on Amp/Xgal obtained
from transforming amp sensitive bacteria with the plasmids you made in #1.
Circle all of the colonies you would keep if you wanted to obtain a clone for your
Genomic gene library.
All the white colonies
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?
All the colonies would be white. You would know the colonies had plasmids, but not
if they contained added pieces of DNA in those plasmids.
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?
It would be a lawn of bacteria. All the bacteria would be able to grow (transformed or
not) and all the colonies would be white.
2. Assume that after cloning the GFP you form a company that makes the plasmid
below. Arabinose sugar positively regulates the expression of GFP.
Arabinose sugar
After transformation you plate some transformed and some untransformed bacteria on
the media types shown below.
1
2
3
4
A. Match the outcome below with the plate number above
_2_ will contain green glowing colonies
_3_ will contain no bacteria growth
_4_ will be a continual lawn or bacteria where colonies grew together
_1_ will contain only white colonies of bacteria that do not glow green
araC
Transcription
Factor
protein
Arabinose
sugar
B. Arrange these steps in the proper order to describe the function of the araC and the
GFP genes on this cloning vector. Hint: GFP is under negative regulation.
_3_a. araC transcription factor protein binds to GFP promoter
_1_b. araC gene is transcribed
_4_c. arabinose sugar binds to the araC transcription factor protein
_5_d. araC protein falls off the GFP promoter
_2_e. araC mRNA is translated
_6_f. GFP is transcribed, GFP mRNA is translated, GFP glows green inside the
E. coli cell
C. If you were going to use the pGLO plasmid above as a cloning vector and use the
restriction enzyme HindIII, where would you want the HindIII restriction enzyme cutting
site?
___a. Amp gene
_X_b. GFP gene
____c. araC gene
D. Which colonies would you select from the plates to make your gene library using the
vector?
___ a. green glowing colonies
_X_b. colonies that DID NOT glow green
_____E. 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
3. If you sequenced a gene cloned from your GFP library would you be able to
determine the amino acid sequence of the protein encoded by this gene? Why?
Yes, the gene can be read in codons to determine the amino acid sequence because
each codon codes for only one type of amino acid.
4. How about your RFP gene library? Remember, both jellyfish and coral are
eukaryotes.
No, because it is a genomic clone. This means the introns have not been spliced out
and we have no way to know what areas are introns and what are exons. The bacteria
itself is incapable of splicing out the introns.
5. 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.
Yes, the DNA would still be inserted and it would still interrupt the reporter gene on the
plasmid. The plasmid would still give the bacteria resistance. You could not, however,
screen the library with an antibody because the E. coli would not make the proper
protein.
A
B
6. Make sure you view the animation “Screening a DNA Library” to help you answer the
following questions.
a. Which molecules above would we be trying to detect if this library was being screened
with the antibody that binds to the RFP from coral.
__X_ protein
___DNA sequence
b. Which molecules above would we be trying to detect if this library was being screened
By sequencing followed by database analysis?
___ protein
_X__DNA sequence
c. Which molecules would we be trying to detect if this library was being screened by PCR
followed by gel electrophoresis?
___ protein
_X__DNA sequence
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