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 ubiquitin-

activating 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 gDNAFinal Concentration

1 Reaction Stock Concentration

3XReaction Mix

1.0 microgram gDNA

1.0 microgram 1.0 microgram/microliter

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

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Working Stock for cDNAFinal 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

#1 (AvrII)

___________ 0.214µL 1.5µL

Restriction Enzyme

#2 (XhoI)

___________ 0.5µL 3.5µL

100 X BSA 1 X BSA 0.2µL 1.4µL

Water ___________ 14.0857µL 98.6µL

Total (Final

Volume is 20µL)

17µL (plus 3µL of

plasmid DNA)

119µL

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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

#1 (HindIII)

___________ 0.5µL 1.5µL

Restriction Enzyme

#2 (NheI)

___________ 0.5µL 3.5µL

100 X BSA 1 X BSA 0.2µL 1.4µL

Water ___________ 13.8µL 98.6µL

Total (Final

Volume is 20µL)

17µL (plus 3µL of

plasmid DNA)

119µL

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

Concentration

Sample 1 Sample 2

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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