BMS110 Honors, MSU Fall 2011
Cloning of UBA4 from Tetrahymena thermophila
Mackenzie Seyer and Jereme Sebastian
Fall 2011
Abstract
The gene UBA5 is most commonly found in mice (Mus musculus). We worked
with UBA4, a homolog of this gene which is found in Tetrahymena. It is an ubiquitinactivating enzyme-like protein and is an E1-like enzyme that plays a minor role in
urmylation which is the folding of protein sequences. Ubiquitin is a small modifier
protein that is joined to substrates to target them for degradation (Goehring et al, 2003).
Matching these ubiquitin-like proteins with other proteins and enzymes can help with
cell and DNA repair. Our main goal for these series of labs was to clone our gene many
times and place it in a plasmid, which would be frozen for future BMS classes and other
scientists to use.
Introduction
UBA5 is a gene found in Mus musculus (the house mouse). UBA4, the gene we
worked with, is a homolog of UBA5 and is found in Tetrahymena thermophila. The
ubiquitin-like protein Urm1 can be linked to other proteins through a mechanism
involving the ubiquitin E1-like enzyme Uba4 (Pedrioli et al, 2008). UBA4 is in the UBA
gene family. The UBA gene family provides instructions for making proteins called
ubiquitin-like modifier activating enzymes. Ubiquitin is a protein used for protein
recycling and is used to tag proteins for destruction/modification which affect other
proteins’ activity, interactions with other proteins, and locations within the cell.
Activation of ubiquitin-like proteins (Ubls) influence some body processes such as the
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immune system and embryonic development. Mutations in humans can cause the
ubiquitin system to malfunction which can lead to cancer, diabetes and several other
diseases. It can also cause X-linked infantile spinal muscular atrophy which can be
fatal (Genetics Home Reference, 2011). It is still unknown what mutations can cause in
UBA4 in Tetrahymena. Linking of Ubl proteins to other proteins has a role in many
functions in eukaryotic cells. Many of these functions play a role in protein trafficking,
DNA repair, genome stability, and the regulations of gene expression (Hugo et al,
2011).
The main goal of all of our labs is to clone our gene through Polymerase Chain
Reaction. To do this we had to carry out a series of labs. First we had to isolate our
DNA to use as a template. Next we created a Polymerase Chain Reaction to copy our
UBA4 gene. Then we ran a gel electrophoresis to see if our DNA was present. We
then created a plasmid map to clone our PCR-amplified gene sequence into the vector.
Lastly we isolated the plasmid, placed our gene in the plasmid vector, and confirmed if it
was the correct plasmid in our PCR product by running a gel electrophoresis. If we can
get our gene to clone we will then freeze it through cryopreservation. The higher level
BMS classes can then use our cloned gene to put into a Tetrahymena cell. They will
put a tracking protein (Green Fluorescing Protein) on the gene in order to see how it
functions in the Tetrahymena cell.
Methods/Procedures
Bioinformatics
We began by researching our gene UBA5. We first went to the NCBI homepage:
http://www.ncbi.nlm.nih.gov/ to find the amino acid sequence of our protein. We found
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our protein size (which is the number of amino acids) to be 403. Then to determine if
there was a Tetrahymena homolog we went to the Tetrahymena Genome Database
Wiki: http://www.ciliate.org/. We found four homologs the best homolog being UBA4
TTHERM_00554610 because it had the best and smallest e-value (3e-66). To obtain
the protein sequence of our Tetrahymena homolog we went back to the TGD Wiki page
for our specific Homolog and looked under where it said >TTHERM_# (protein). In
order to obtain the nucleotide sequence for our protein homolog we looked under where
it said >TTHERM_# (coding). Our entire genomic sequence was obtained through the
genome browser. Finally we compared our Tetrahymena homolog coding sequence
with the genomic sequence by aligning it on the MGAlignIt homepage
http://proline.bic.nus.edu.sg/mgalign/mgalignit.html. <Complete protocol in BMS110
Honors Lab 3: Bioinformatics and Molecular Computational Tools, Fall 2011> (Smith,
2011)
Tetrahymena Genomic DNA Isolation
To isolate our genomic DNA we centrifuged 1.4 mL of the Tetrahymena culture
for 1 minute at 10,000 rpm. We then added 700µL of Urea Lysis Buffer to resuspend
the DNA pellet and phenol-extracted the lysate by adding 600µL of
phenol:chloroform:isoamyl. Then we added 150 µL of 5M sodium chloride and,
precipitated the DNA by adding isopropyl alcohol to the extracted lysate. We then
centrifuged the mixture again for 10 minutes. Then we added 500 µL of 70% ethanol,
centrifuged it again for 3 minutes at maximum speed, and then removed the ethanol
with a pipette. We then allowed the pellet to air dry. Lastly we added Tris-EDTA buffer
and 1µL of RNase A and incubated the mixture at 37°C for 15 minutes. To quantify our
genomic DNA we used a Nanodrop and placed 1.0µL of our genomic DNA straight on it.
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<Complete protocol in BMS110 Honors Lab 4: Tetrahymena Genomic DNA Isolation,
Fall 2011> (Smith, 2011)
Polymerase Chain Reaction
The water used to resuspend the primers had already been added by a student
in a previous lab. The amounts of water added were 132.5µL to the forward primer and
168.5µL to the reverse primer (Mitchell, 2010). We used his resuspended primers to
add to our working stock. We made the working stock (seen below) which was divided
equally into 3 tubes for gDNA and 3 tubes for cDNA. We then placed the tubes in the
thermocycler at the appropriate annealing temperatures. Our temperatures were 52.5,
54.5, and 56.5°C however in the thermocycler we placed them at 52.5, 54.4, and
56.8°C. <Complete protocol in BMS110 Honors Lab 5: Polymerase Chain Reaction, Fall
2011> (Smith 2011)
Working Stock for gDNA
Final
Concentration
1 Reaction
Stock
Concentration
1.0 microgram
gDNA
1.0 microgram
1.0
3.0 microliters
microgram/microliter
0.2 micromolar TF
.50 microliters
20 micromolar
1.5 microliters
0.2 micromolar TR
.50 microliters
20 micromolar
1.5 microliters
1.0 unit (U) Phusion
Polymerase
.50 microliters
2 U/microliter
1.5 microliters
1XGC Buffer (1.5
millimolar MgCl2)
10.0 microliters
5X
30.0 microliters
1.0 molar Betaine
10.0 microliters
5.0 molar
30.0 microliters
.20 millimolar
dNTPs
1.0 microliters
10.0 millimolar
3.0 microliters
Sterile Distilled
Water
26.5 microliters
______________
79.5 microliters
Final Volume
50.0 microliters
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3XReaction Mix
150.0 microliters
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BMS110 Honors, MSU Fall 2011
Working Stock for cDNA
Final
Concentration
1 Reaction
Stock
Concentration
3XReaction Mix
cDNA (1:10 dilution)
1.0 microgram
______________
3.0 microliters
0.2 micromolar TF
.50 microliters
20 micromolar
1.5 microliters
0.2 micromolar TR
.50 microliters
20 micromolar
1.5 microliters
1.0 unit (U) Phusion
Polymerase
.50 microliters
2 U/microliter
1.5 microliters
1XGC Buffer (1.5
millimolar MgCl2)
10.0 microliters
5X
30.0 microliters
1.0 molar Betaine
10.0 microliters
5.0 molar
30.0 microliters
.20 millimolar
dNTPs
1.0 microliters
10.0 millimolar
3.0 microliters
Sterile Distilled
Water
26.5 microliters
______________
79.5 microliters
Final Volume
50.0 microliters
150.0 microliters
Agarose Gel Electrophoresis
We made an agarose gel to see if our gene was present in our DNA. We made
the gel by adding 1% agarose to 50 mL of 1X TAE and heating this up to dissolve it.
When the agarose solution was no longer hot we added 1/100,000 of Ethidium Bromide
and swirled the flask to mix it. We then placed a comb in the casting tray to create the
wells. We poured our agarose solution containing Ethidium Bromide into the casting
tray and allowed it to solidify. Once the gel was made we prepared the samples by
adding a small dot of 1µL of 10X sample dye to the samples. We then loaded 10µL of
our samples mixed with the dye along with 20µL of a 1 kb ladder into the wells of the
gel. We turned on the power supply to run at constant volts (90-120 volts), and when
the bromphenol blue band was ¾ down the gel, we turned off the power supply and
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took a picture of our gel using the UV light box. <Complete protocol inBMS110 Honors
Lab 6: Agarose Gel Electrophoresis, Fall 2011> (Smith, 2011).
TOPO Cloning and E. coli Transformation
To make our reaction we added .5µL of our PCR product, 1µL of salt solution,
3.5 µL of sterile water, and 1µL of the TOPO vector. We then incubated the reaction at
room temperature for 30 minutes. Then we mixed 2µL of our TOPO cloning reaction
with E. coli and incubated the mixture on ice for 27 minutes. After that we heat shocked
the cells for 30 seconds in a water bath at 42°C and then put them back on ice for one
minute. Then 250µL of SOC Medium was added to the mixture and the tube was
placed in a shaking incubator at 37°C for 43 minutes. Once that was finished, we
spread 200µL of the mixture on one 50µg/µL kanamycin plates. We spread the
remaining 50µL on another kanamycin plate. We then mixed the plates used sterile
glass beads and shaking the plates gently. The plates were then incubated overnight in
at 37°C and were put in the cold room the next day. <Complete protocol in BMS110
Honors Lab 7: TOPO Cloning and E. coli Transformation, Fall 2011> (Smith, 2011).
Plasmid Map and Restriction Enzyme Digestion Design
To make our plasmid maps, we used the pENTR/TOPO-D plasmid map
Gene Construction Kit document that contained the sequence of the plasmid used to
clone our PCR product. Using the Gene Construction Kit we were able to make a
plasmid map for our cDNA that showed sequences in our gene that would be joined by
specific restriction enzymes. Based on this map, we determined that the best restriction
enzymes would be Nhe I and Hind III as well as Xho I and Avr II. We also made a
sample gel picture for the bands and their sizes with the restriction enzymes. These
sample gels predicted our band sizes would be 2590 and 1155 for the XhoI and AvrII
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restriction enzymes and 2883 and 596 for the HindIII and NheI restriction enzymes.
These predicted band sizes would be used in Lab 10 to determine if our restriction
enzyme digests were successful. <Complete protocol in BMS110 Honors Lab 8:
Plasmid Map and Restriction Enzyme Digestion Design, Fall 2011> (Smith, 2011).
Plasmid Purification and Restriction Enzyme Digest
In order to determine if the bacteria that grew on our plates from Lab 7 contained
our PCR product, we needed to isolate the plasmid and confirm it was the correct
plasmid. To do this we inoculated six 2 mL cultures of LB liquid media tubes with six
colonies from our plates from Lab 7. We used a wooden stick to pick up the colony and
dot it onto a LB-KAN plate labeled 1-6. We then swirled the stick in 2 mL LB-Kan liquid
media. The next day we transferred 1.5 mL of each culture into microcentrifuge tubes
using a plastic transfer pipette. We then centrifuged on maximum speed for 2 minutes.
Then we poured off the supernatant and added 350µL of Sucrose Lysis Buffer to
resuspend the pellet. Then 25µL of lysozyme solution was added and the mixture was
incubated at room temperature for 5 minutes. It was then heated at 99°C for one
minute in a boiling water bath. Then it was centrifuged for 15 minutes at maximum
speed. The pellet was then removed with a toothpick and discarded and the
supernatant was saved. 40µL of 3M NaOAc and 220µL of isopropanol was then added
to the supernatant and mixed by inverting. Then the mixture was incubated at room
temperature for 5 minutes then centrifuged for 10 minutes at maximum speed. The
supernatant was then poured off and discarded. The plasmid pellet was washed with
1000µL of 70% ethanol and centrifuged for 2 minutes. The supernatant was then
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removed and the pellet was allowed to dry. Then 50µL of Tris-EDTA (TE) buffer was
added and the pellet was resuspended.
The following week we ran a 1% agarose gel electrophoresis (using the same
procedure from Lab 6) containing our plasmid and restriction enzyme digests. To do
this we made a working stock for each pair of restriction enzymes (XhoI and AvrII;
HindIII and NheI). The working stock for both XhoI/AvrII and HindIII/NheI can be seen
below. Note that only 1.5µL of AvrII was used because Dr. Smith ran out. This
increased the amount of water used. The stocks were incubated at 37°C. The results
from this gel would help us determine which colony containing our plasmid we would
choose to freeze. <Complete protocol in BMS110 Honors Lab 10: Plasmid Purification
and Restriction Enzyme Digest, Fall 2011> (Smith, 2011)
Working Stock for XhoI/AvrII Restriction Enzymes
Stock
Final
1 Reaction
7 Reactions
10 X Buffer2
1 X Buffer
2.0µL
14.0µL
Restriction Enzyme
___________
0.214µL
1.5µL
___________
0.5µL
3.5µL
100 X BSA
1 X BSA
0.2µL
1.4µL
Water
___________
14.0857µL
98.6µL
Total (Final
17µL (plus 3µL of
119µL
Volume is 20µL)
plasmid DNA)
#1 (AvrII)
Restriction Enzyme
#2 (XhoI)
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Working Stock for HindIII/NheI Restriction Enzymes
Stock
Final
1 Reaction
7 Reactions
10 X Buffer2
1 X Buffer
2.0µL
14.0µL
Restriction Enzyme
___________
0.5µL
1.5µL
___________
0.5µL
3.5µL
100 X BSA
1 X BSA
0.2µL
1.4µL
Water
___________
13.8µL
98.6µL
Total (Final
17µL (plus 3µL of
119µL
Volume is 20µL)
plasmid DNA)
#1 (HindIII)
Restriction Enzyme
#2 (NheI)
Cryopreservation of E. coli containing plasmid with gene
By looking at our results from Lab 10 and using the predicted band sizes from
Lab 8, we were able to determine which colonies (1-6) worked the best and contained
our plasmid. We picked the colony that worked the best from the plate with a wooden
stick and swirled it in a 2mL tube of LB-KAN liquid media and placed the tube in a
shaking incubator. The next day we obtained one 2mL cryopreservation tube
containing 700µL of sterile 50% Glycerol. We added 700µL of our bacterial cells. After
labeling the tube with our clone number (which was colony 5) and the plasmid name we
gave it to Dr. Smith to be cryopreserved and used by future BMS classes. <Complete
protocol in BMS110 Honors Lab 14: Cryopreservation of E. coli containing plasmid with
gene, Fall 2011> (Smith, 2011).
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Results
We first began our project by researching UBA5 and finding the best homolog in
Tetrahymena which we found was UBA4. We then found its genomic sequence seen in
figure 1 and coding sequence. The next week we isolated our DNA from the rest of the
DNA in Tetrahymena, quantified our DNA, and found it to be pure. The quantifications
can be seen in figure 2. Since our DNA was pure enough we could then carry out a
Polymerase Chain Reaction in the next lab which would copy our DNA quickly. We then
ran our PCR product in a gel electrophoresis. Our very first gel produced no results so
we had to redo labs 5 and 6 using different primers. Dr. Smith also ran an extra gel for
us that produced better results. He also ran one for the whole class using our best
annealing temperatures. Results are seen in figure 3. From the results we decided to
use cDNA to clone. We then created a TOPO cloning reaction with our gene. This
produced colonies from which we picked 6 of to use for Labs 10 and 14. After inserting
our plasmid into the vector seen in figure 4, we ran a gel electrophoresis containing our
plasmid and restriction enzyme digests seen in figure 5. Using the predicted band sizes
from Lab 8, we determined from figure 5 that colony 5 produced the best and correct
results. We then chose colony 5 because it contained our gene UBA4 to cryopreserve
for future BMS classes to use
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DNA Concentration
A260/280 DNA
Sample 1
Sample 2
Concentration
Concentration
3.5579 microgram/microliter 5.2706 microgram/microliter
A260
71.159 nanometers
105.413 nanometers
A280
31.202 nanometers
47.594 nanometers
A260/280
2.28
2.21
Figure 2: Concentration of DNA Table. This table shows the DNA concentration from Lab 4 using the Nanodrop.
The A260/280 concentration for both samples was greater than 1.8 showing they were pure samples.
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Discussion/Conclusion
For the most part our labs went well. However, we had to redo labs 5 and 6
because our first gel produced no results. We believe this was a problem from the
primers. After redoing this, our gel showed odd results. It showed 3 bands that looked
to be cDNA from the sizes however 2 of the bands were in gDNA wells therefore we
think we may have loaded this wrong. From then on we were successful in our labs.
From performing these labs we were able to successfully clone our UBA4 gene
to cryopreserve for future BMS classes to use. They will put a Green Fluorescing
Protein on it to track its functions in Tetrahymena. We are happy to at least have one
colony that we cryopreserved and hope it comes in use. This has been a great
experience, one that I have never been through before, and I think it will come in great
use for future BMS classes. The only thing I would have changed would be taking my
time so my lab partner and I didn’t have to redo things.
References
Genetics Home Reference. (2011, November). Retrieved December 2011, from UBA
Gene Family: http://ghr.nlm.nih.gov/geneFamily/uba
Goehring, A., Rivers, D., & Sprague, G. J. (2003). Urmylation: a ubiquitin-like pathway
that functions during invasive growth and budding in yeast. Mol Biol Cell,
v.14(11): 4329-41.
Miranda, H. V., Nembhard, N., Su, D., Hepowit, N., Krause, D. J., Pritz, J. R., et al.
(2011). E1- and ubiquitin-like proteins provide a direct link between protein
conjugation and sulfur transfer in archaea. PNAS, v.108(11): 4417-4422.
Mitchell, T. G. (2010). UBA5/UBA4 Homolog in Tetrahymena Thermophila Cloning.
Retrieved December 2011, from Ciliate Genomic Consortium:
http://tet.jsd.claremont.edu/files/r73n0.pdf
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Pedrioli, P. G., Leidel, S., & and Hofmann, K. (2008). Urm1 at the crossroads of
modification. EMBO reports, v.9(12) 1196-1202.
Smith, J.J (2011). BMS110-999: Lab Protocol. Springfield, MO.
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