cct2 gene in tetrahymena thermophilafaculty.jsd.claremont.edu/ewiley/files/r96n0.pdfbms110 honors,...

BMS110 Honors, MSU Fall 2011

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CCT2 Gene in Tetrahymena Thermophila

By: Jessica Schuler and Jackie Bradley

Fall 2011

Abstract:

The goal of this research was to study the TCP1 Beta gene (CCT2 gene in Tetrahymena

Thermophila) in order to gain a better understanding of the function of that gene in Tetrahymena. The

CCT2 gene is very prominent in the cytosol of Tetrahymena because it helps produce and fold

microtubules that from the cilia and cytoskeleton of the cells. Since this gene is so important in

Tetrahymena, it is necessary to perform research on it in order to learn more about this in Tetrahymena

in general. First in our research experiments, we isolated the CCT2 gene in the DNA of Tetrahymena.

We then copied it using Polymerase Chain Reactions and specially formed primers that bonded to the

strands of the DNA in order to make copies of the strands. Next, we separated and purified the DNA

fragments using Agarose Gel Electrophoresis, and found out if the predicted gene sequence and

numbers of introns and exons were accurate or not. We cloned the CCT2 gene, and inserted it into E.

Coli to see if the DNA was successfully cloned. After constructing a plasmid map, we could see the

restriction enzyme sites in our plasmid, and predict where the gene would be inserted into the E. Coli

cells. We ran another Agarose Gel Electrophoresis on the new plasmids with the restriction enzymes,

and found that our gene was probably inserted into two of the 12 total colonies. Finally, we

cryopreserved the cells that showed positive results in the last gel electrophoresis so that later classes

can continue research on the CCT2 gene. Hopefully, the research will ultimately lead to comparing and

contrasting the function of CCT2 in Home sapiens versus Tetrahymena.


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Introduction:

The gene CCT2 plays a very important role in Tetrahymena. It is involved in the process of

folding the actin and tubulin microfilaments that make up many structures of the cell, but most

importantly, the cilia. Tetrahymena is the perfect specimen to do research for the CCT2 gene on

because it has over 17 different arrays of microtubules.(Soares et al, 1997) Since CCT2 plays such a huge

role in the production of microfilaments, then it easy to see this gene mostly anywhere in the cytosol of

the cell at any given time. It is found in the cytosol of eukaryotic cells, and was first discovered in 1979

in a mouse on chromosome 17. (Kubota et al, 1995) It is made up of 535 amino acids. Several studies

have shown that when the CCT2 gene was removed from the cell, it could no longer produce cilia, and

therefore the cell died within two cell cycles. “We have reported that in Tetrahymena, the expression of

CCT chaperonin subunit genes is up-regulated during cilia regeneration following deciliation.”(Seixas et

al, 2010) This proves that the CCT2 gene plays a very important role in cilia production because large

amounts of it were found whenever the cell needed to make more cilia. The cilia of the cell are

especially important in Tetrahymena because that is its way of locomotion.

CCT2 is a type of chaperonin in the cell that protects newly formed proteins from the crowds of

other molecules around them. It helps to fold actin and tubulin so that the hydrophobic regions are not

as exposed to deformities. (Brackley & Grantham, 2009) CCT2 is part of a family of eight other CCT

genes. All eight of these CCT genes are arranged in a ring like structure and act as a center for bonding

for its substrates. (Brackley & Grantham, 2009) These genes all act as chaperones to other proteins and

help to fold the microfilaments in the cell in order to make up the cytoskeleton. (Soares et al, 1997)

As described above, the CCT2 gene is a very important factor of Tetrahymena and other species.

That is why we will perform extensive research in order to fully understand the structure of the gene,


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the approximate size of it, and its location in the cell. We will also attempt to clone it in the end, and

see whether the new cells display the same functions as the original ones.

Methods/Procedure

Bioinformatics:

In order to begin studying the TCP1 beta gene in Homo sapiens, we used a number of online databases

where different researches have posted their discoveries and results of experiments concerning our

gene. The first step was to find the amino acid sequence of TCP1 beta in Homo sapiens. In order to do

this, we used the NCBI database (http://www.ncbi.nlm.nih.gov/). Next, we had to figure out if there was

a homolog of TCP1 beta in Tetrahymena by using the Tetrahymena Genome Database Wiki

(http://www.ciliate.org/). We copied the protein sequence into the BLAST database and compared it

with other sequences that are close to the one we found for TCP1 beta. The closest match was

Ttherm_00149340 with an e value of 1.1e-143. We then found the protein sequence, coding sequence,

and genomic sequence for this homolog on the TGD Wiki page. Next, we compared the coding

sequence with the genomic sequence using http://proline.bic.nus.edu.sg/mgalign/mgalignit.html. The

result was that the name of the homologous gene of TCP1 Beta in Tetrahymena is the CCT2 gene. Refer

to complete lab protocol for more detailed instructions. (Smith 2011)

Tetrahymena Genomic DNA Isolation:

The next step was to isolate the genomic DNA from a Tetrahymena culture in order for it to be used as a

template to make many copies of it later. We made two samples in order to be safe and see which one

was better in the end. In order to do this, we put a culture of Tetrahymena in a centrifuge so that it

eventually formed a pellet in the bottom of the tube. We then suspended the pellet using Urea Lysis

http://www.ncbi.nlm.nih.gov/

http://www.ciliate.org/

http://proline.bic.nus.edu.sg/mgalign/mgalignit.html


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Buffer and added 700 μL of phenol:chloroform:isoamyl alcohol in order to extract the lysate from the

cells. After this, we added NaCl and isopropyl alcohol to the extracted lysate, which precipitated the

DNA after centrifuging. Next, we added 70% ethanol in order to dissolve the salts out of the DNA pellet.

We poured off all of the supernatant and suspended the pellet one more time in Tris-EDTA (TE) buffer.

Finally, we added RNase A and incubated the tube at 37°C. We now had a mixture that contained the

genomic DNA of Tetrahymena cells.

Our next step was to determine how pure our sample of DNA actually was. In order to do this, we

placed 1 μL of each of the samples of our DNA in the Nano Drop machine. We read the absorbance and

concentrations from the Nano Drop 2000, and concluded that the DNA was indeed pure enough to use.

We continued the experiments with DNA Sample 1, since it was the most pure.(Smith 2011)

Polymerase Chain Reaction (PCR):

PCR reactions involve three different steps: denaturing the DNA, annealing of the primers, and

polymerase extension. The purpose of PCR is to make many copies of the DNA using the isolated

genomic DNA and oligonucleotide primers that we ordered from Integrated DNA Technologies.

CCT2-TF

5’-

CACCCTCGAGAGCATGGATATGGTTGGTGTAAG

CCT2-TR

5’-

AGAGCCTAGGTCACATTCTATCTCTTCTTCTAGG

Figure 1: Primer sequences. The primers are little bits of DNA that will make up the new copied sequences of the

genomic DNA. They were ordered from Integrated DNA Technologies. We inserted the TF primer at the beginning

of the genomic sequence, and TR primer at the end of the sequence.

First, we hydrated the primers and diluted them to 200 μL by adding 144 μL of water to the TF primer

and 156.5 μL of water to the TR primer. Then, we diluted them down even more to have 200 μL of a 20

μM working stock. We made two 150 μL master mixes (one with gDNA and one with cDNA (+H4)). Each

mix also contained other reactants which include: 3 μL of DNA sample, 1.5 μL of 0.2 μM TF Primer, 1.5


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μL of 0.2 μM TR Primer, 1.5 μL of Phusion polymerase, 30 μL of 1X GC buffer, 30μL of 1.0 M Betaine, 3 μL

of 0.2 mM dNTPs, and 79.5 μL of sterile distilled water. However, when we added the gDNA to the

master mix, we forgot to dilute it to 1.0μg/μL. Therefore, the gDNA was fully concentrated in each

reaction. After the master mixes were made, we separated the mixture into six different 50μL tubes (3

gDNA and 3 cDNA). We then calculated the annealing temperatures for the reactions which turned out

to be 54.5°C, 56.5°C, and 58.5°C, but since the whole class was using the same thermocycler, our

temperatures became 53.3°C, 55.7°C, and 58.0°C. Next, we used a thermocycler to cycle the reactions

through the different temperatures for different amounts of time. Refer to the complete lab protocol

for specific times and temperatures.(Smith 2011)

Agarose Gel Electrophoresis:

Since we now had many copies of the DNA, the next step was to run the DNA through Agarose Gel

Electrophoresis. This process is used to separate the DNA using friction and electrical forces as it runs

through the gel. Since DNA is negatively charged due to the phosphates, it will run to the positively

charged end of the electrophoretic chamber. For 70 ml of 1% agarose gel, we used 0.7 g of agarose

instead 0.5 g because we had a bigger chamber due to sharing it with another group. We then added 70

ml of 1X TAE, covered it, and microwaved it for 1 minute and 15 seconds. Next, we added 0.7 μL of 10

mg/ml Ethidium Bromide. In order to set up the chamber, we placed 1 comb with 14 teeth in the empty

chamber, then poured the mixture into the casting tray and waited for it to solidify. We filled the sides

of the chamber with 1X TAE until it covered the gel, and then took the comb out so that there were 14

wells. Lanes 1-7 were ours, so we loaded lane 1 with 1 kb ladder, lane 2 with C1, lane 3 with G1, lane 4

with C2, lane 5 with G2, lane 6 with C3, and lane 7 with G3. Each C stands for cDNA and G stands for

gDNA. Each lane also had drop of 10X sample dye with it. Next, we ran 120 volts of power through the

gel for 30 minutes, and then moved it up to 140 volts for the rest of the time. As the DNA was


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migrating, the xylene cyanol dye showed the DNA at 4000 base pairs, while the bromphenol blue dye

showed it at 500 base pairs. We stopped the gel when the bromphenol blue band was halfway down

the gel.(Smith 2011)

TOPO Cloning and E. Coli Transformation:

Dr. Smith ran our cDNA on agarose gel again to make sure it worked properly, so from now on, we used

this DNA. In order to start the cloning process, we made a mixture of 0.5 μL of our PCR product, 1 μL of

salt solution, 1 μL of the TOPO vector, and 3.5 μL of sterile water. The next step was to add the mixture

to E. Coli cells. We then put it on ice for 30 minutes so that our DNA could get inside the E .coli cells and

hopefully reproduce rapidly using our DNA as a template. Next, we heat shocked it for 30 seconds so

that the pores of the cells would close, and the DNA could not escape. We then added SOC Medium to

make it grow faster and shook it in a shaking incubator at 37°C for 1 hour. 200 μL of the solution was

spread on an LB plate that contained 50 μg/μL of kanamycin, and about 50 μL was spread on a different

LB plate with the kanamycin. They all contained sterile beads in order to mix them. Reference the lab

protocol for more detailed instructions.(Smith 2011)

Construction of Plasmid Map and Restrictive Enzyme Digestion Design:

We were now ready to design a plasmid map on the computer in order to see where our DNA would fit

into the plasmid, identify restriction enzymes, and eventually be able to see if our gene was clone in the

E. Coli. To start, we added the primer sequences to our gene sequences that we found in the

bioinformatics report. We replaced the ATG start codon at the beginning of our sequence with the TF

primer sequence and typed the TR primer sequence from 5’ to 3’ end at the end of our gene sequence.

We then inserted our gene sequence into the plasmid map. Next, we located different restriction

enzymes in the plasmid that would confirm that our gene was actually inserted in the plasmid. We

chose AvrII and XhoI (digest #1) and EcoRV and BglII (digest #2). Next, we predicted the size that these


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genes would show when run on agarose gel. The restriction enzymes will cut the plasmid at the place

where they exist in the plasmid, so we will be able to tell if our gel results match our predictions.(Smith

2011)

Plasmid Purification and Restriction Enzyme Digest

In order to isolate the plasmid from the E. Coli cells, we added 350 μL of Sucrose Lysis Buffer and 25 μL

of lysozyme solution, and heat shocked it at 99°C for I minute to break open the cell walls. After

centrifuging, we discarded the pellet that contained all of the rest of the cell parts, besides the DNA. We

then precipitated the DNA by adding 40 μL of 3M NaOAc and 220 μL of isopropanol to the solution, and

centrifuged it for 10 minutes. Next, we discarded the supernatant and washed the pellet with 1000 μL

of 70% ethanol, and let it dry. After we resuspended the pellet in 50 μL of Tris-EDTA (TE) buffer, we

were ready to begin the restriction enzyme digest. The cocktail for digest 1 (AvrII/Xho1) required 14 μL

of Buffer 2, 1.4 μL of BSA, 3.5 μL of Xho1, 1.5 μL of AvrII, and 98.6 μL of sterile water. We only used 1.5

μL ofAvrII because we ran out of it in class, so we just added 2 more μL of water instead. The cocktail

for digest 2 (BglII/EcoRV) required 14 μL of Buffer 3, 1.4 μL of BSA, 3.5 μL of BglII, 3.5 μL of EcoRV, and

96.6 μL of sterile water. We added 17 μL of the cocktail and 3 μL of the plasmid to 6 different tubes for

each digest, and incubated at 37°C. Next, we used Agarose Gel Electrophoresis to confirm if our DNA

was inserted into the plasmid or not. We will know it was inserted if the band sizes on the gel come out

to be the predicted sizes from the previous lab. The predicted band sizes for digest 1 (AvrII/Xho1) were

2590 bp and 1654 bp. The predicted band sizes for digest 2 (BglII/EcoRV) were 2686 bp, 954 bp, and 574

bp.(Smith 2011)

Cryopreservation of E. Coli containing plasmid with gene

Based on the results from the restriction enzyme gel electrophoresis, we picked the plasmids from the

wells that showed the correct sizes as the ones that we would freeze down. First, we placed each of the


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corresponding colonies into 2 mL of LB-Kan media and put them in the shaking incubator at 37°C in

order to make the colonies replicate more. The next day, the media was clouded with many colonies.

We added 700 μL of the cells to a cryopreservation tube containing 700 μL of sterile 50% Glycerol, and

froze them at -80°C so that all of the metabolic processes will stop, and the cells will be preserved for

further research. We chose Glycerol instead of water because the ice crystals from water would

damage the cell by penetrating the cell wall. (Smith)

Results

In order to find the homolog of TCP1 Beta in Tetrahymena, we first needed to find the protein sequence.

This sequence is shown in Figure 2. It starts with Methionine because that is the amino acid for ATG,

which is the start codon of all gene sequences. There are 20 possible amino acids which each stand for a

different codon that make up the base pairs of DNA.

>TTHERM_00149340(protein)

MSMDMVGVRFSKIHNKNEASEEKGEMARLSSFVGAIAVADLVKTTLGPKGMDKILRPTGA

GPKADTITVTNDGATILRSMYIDNPAAKILIEISKTQDEEVGDGTTTVAVLAGELLREGE

KLVNQKIHPQHIINGWRKARDCAQHTLNLIARDNSKNEEKFREDLLNIARTTLSSKLLTQ

DKEHFARLAVEAVLRLRGSGNLDQIQIIKKSGGSIRDSYLADGLILEKSITVGCPKRLEN

AKIMVANTPMDYDKIKIYGTKVKVESMDKIAEIETAEKEKMKAKVEKILAYEPTVFINRQ

LIYNYPEQMLADKGLMVIEHADFDGIERLSAATGAEILSTFDNPERAEKIMGRCKLIEEI

IIGEDKFIQFSGCEKGEACTIVLRGASTHIIDEADRSLHDALCVLITTVKNSKVVYGGGN

SEIQMALAVEKLTQSVKGKQALAIEAYARALRQLPTIIADNGGYDAPELVQALKVEIAEG

STSAGLDMMNGEVADMEKLGVTECLRVKEQALLSASEAAELILRVDSIIRCAPRRRDRM

Figure 2: Protein Sequence of CCT2 gene homolog. This sequence shows the order of the amino acids that make

up the gene sequence of CCT2. This sequence contains 535 amino acids. The TTHERM number is shown above the

sequence, which identifies which homolog this sequence refers to.

Next, we translated the protein from amino acids into DNA. This allowed us to see the gene sequence

which contains the introns, which are the parts of the DNA that are taken out when coding; exons,

which are the components of the coding DNA (cDNA); EST’s, which are the required parts of the

sequence to make the gene; a start codon (ATG) which represents the start of the gene, and the place


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where the amino acids will begin building; and the TF and TR primers that are placed at the beginning

and end of the sequence, and are used in PCR reactions.

Next, we researched how close this CCT2 homolog in Tetrahymena was to TCP1 Beta in Homo sapiens.

The results showed that the coverage of alignment was 100.00. This means that the gene sequences

matched up perfectly.

The next step was to isolate the DNA from the Tetrahymena cells and purify it using a number of

solutions and salts. It was imperative that the DNA was clean because if it had other cell material in it,

CCT2(Need to flip sequence) ATGAGCATGGATATGGTTGGTGTAAGATTTTCAAAAATTCACAATAAGTAAAAGCTAACTAACTATATTTT

TTATAATTAAATTAACAGAATGTCTTGCCTCAAGTTCTTAGGAACGAGGCCTCTGAAGAAAAGGGTGAAAT

GGCTAGACTTGTAATTTTTATTTTTGCTTTTATCCTTATCAATTAATTAATTATGTTAATTTACATTCTAC

TTCAAAAATTAACTTTAATAAATTTTAAATCTAGTCATCTTTTGTCGGAGCTATTGCTGTTGCAGATTTAG

TTAAAACCACTTTAGGTCCTAAGGGTATGGTAATGAATTAATGCTTTTCGATTTAAGCTAGTTTGATTCTA

ATTTTTTTTTATTACTCTAATAGGATAAAATTCTTAGACCTACTGGCGCTGGTCCTAAGGCTGACACTATC

ACTGTCACTAATGATGGTGCTACCATTTTGAGATCTATGTATATTGATAATCCCGCTGCTAAGATTTTAAT

TGGTAATTTACATATTTTTATTGCAACAGAAAGTCTATAGCTAATTTGCTTTTCAGTTTATAAACATGCCT

TAATCAAGTTTGTTTAGCGAGTTATTAAATAAATGCATTAACATTGCAAAGGCGATTAAAAATAAAATTTT

AAGGATTAAAGTAGCACGCATCAGAAAATAGGATTATCTATTTAAAAGTTTTAAGTTTATTTTCCAGTATG

ATAAGTGAAGAATTAGTGAAATGTTTTAAAGTTAGTTTTCAAAAATCTAACAATATAATTGAGAATATCAT

AAAGAAATTATTCTAAACACCTATATTCAACACATTATATAAATTGGTTGAGATAACTTATCTTTGAGCAC

TACACTATACTTCCTTAACTACCATACTGAAAATAAAACAAGCACATAATGAATATCTTTCTTTGTTTGCA

TTTTAGCATAGTTTTAAAAAAATTTAATTCTAATAAAAATAAATAAATGGTATCTAAAAAATTTATTGATA

ATAATTAGAAATTTCTAAGACTTAAGACGAAGAAGTTGGTGATGGTACCACTACTGTTGCTGTCTTAGCTG

GTGAATTATTAAGAGAAGGTGAAAAATTGGTTAACCAAAAGATTCACCCTTAACACATCATCAATGGTTGG

AGAAAGGCTAGAGACTGTGCTCAACACACTCTTAATTTAATTGCTAGAGATAACTCCAAGAATGAAGAAAA

GTTCAGAGAAGATTTACTTAACATTGCAAGAACTACTCTTTCTTCCAAGCTTTTGACTTAAGACAAGGAAC

ACTTTGCTAGATTAGCTGTCGAAGCTGTTCTCAGATTAAGAGGTTCTGGTAATCTTGATCAAATCTAAATC

ATTAAAAAGTCTGGTGGTTCTATCAGAGATTCTTACCTTGCTGACGGTCTTATTTTAGAAAAGTCCATTAC

TGTCGGTTGCCCCAAGAGACTTGAAAATGCAAAGTAATTATTTTATTTATTCATTCATTCATTCTTACATA

TATTCAACAAAGGATATATAGCTTTATCTTGAATTTATTCAAATTCTAATCAATAAAATTATTATTAAAAA

AATAACTTATTTATTTATTTAACTTAATCTCAAAAGGATTATGGTTGCTAACACTCCTATGGATTACGACA

AGATTAAGATTTACGGTACCAAGGTTAAAGTAGAAAGCATGGATAAAATTGCTGAAATTGAAACTGCTGAA

AAGGAAAAAATGAAGGCTAAGGTCGAAAAGATTCTTGCTTACGAACCCACTGTCTTCATTAACAGATAATT

GATTTATAACTATCCTGAATAAATGTTAGCTGATAAAGGTTTGATGGTTATTGAACACGCTGATTTCGATG

GTATTGAAAGACTTTCCGCTGCTACCGGTGCTGAAATTCTCTCTACTTTCGATAATCCTGAAAGAGCTGAA

AAGATTATGGGTAGATGCAAACTTATTGAAGAAATTATTATTGGTGAAGACAAGTTCATTCAATTCTCTGG

ATGTGAAAAGGGTGAAGCTTGCACTATTGTCTTGAGAGGTGCTTCTACTCACATTATTGACGAAGCTGATA

GATCTCTCCACGATGCACTTTGTGTTCTTATTACTACAGTTAAGAACTCTAAGGTTGTCTATGGTGGTGGT

AACTCTGAAATCCAAATGGCTCTTGCTGTTGAAAAGCTTACTTAATCTGTTAAGGGCAAATAAGTAATAAA

TTTTTTTATTTTTATATATTTTAATAATATAATTCTGCTTATTAAAATACTTTTGTTTCCAAGTGATTTTG

TATTTCAATAACTTTTATTTTATAATTTAGGCACTCGCCATTGAAGCATATGCTAGAGCTTTAAGATAATT

ACCTACTATTATAGCTGATAATGGTGGTTATGATGCTCCTGAATTAGTTTAAGCTTTGAAGGTCGAAATCG

CTGAAGGTTCTACTTCCGCTGGTCTTGATATGATGAACGGTGAAGTTGCTGATATGGAAAAGCTCGGTGTT

ACTGTAAAAATTTTTATTTAAAAAATTAATATTTATCTAAATTATTTATTTTAAAATCTAAAGGAATGTTT

AAGAGTTAAGGAATAGGCTTTATTATCTGCTTCAGAAGCTGCTGAACTTATTCTTAGAGTTGATAGCATTA

TCAGATGTGCTCCTAGAAGAAGAGATAGAATGTGA

Figure 3: Genomic Sequence of CCT2. Nucleotide sequence of the Tetrahymena CCT2 (TTHERM_00149340). Start codon is in bold green, and stop codon is in bold purple. Exons that are used to make protein are in red and the introns that are removed are in black. Underlined sequence represents the ESTs for the gene. Highlighted regions represent the area that the PCR primers were constructed from (Yellow- Forward Primer-CCT2 TF; Blue- Reverse Primer-CCT2 TR)


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then the cloning process would not work as well. Once it was clean, we used the Nanodrop 2000 to test

the purity by measuring the absorbance of light through the sample. Table 1 shows the results of the

Nanodrop readings.

DNA Sample 1 DNA Sample 2

A260nm 65.441 61.261

A280nm 29.374 30.157

A260nm/ A280nm 2.23 2.03

Concentration 3.2721 3.0630

Table 1: Results of Nano Drop 2000. DNA Sample 1 had the higher ratio of absorbance readings, which meant that

it was the most pure. It also had a higher concentration of DNA, therefore we decided to use DNA sample 1 from

this point on.

The minimum value of A260nm/ A280nm value in order for the sample to be considered pure is 1.8.

Since our highest value was 2.23, we concluded that DNA Sample 1 was the most pure, and that is the

one that we used for the rest of the experiments.

Now that we knew our sample of DNA was pure enough, we ran PCR reactions in order to replicate the

DNA using the primers shown in Figure 1. We constructed a master mix of solutions combined with our

cDNA and gDNA samples. However, when adding the gDNA, we forgot to dilute the sample from its

original concentration shown in Table 1 to 1.0000 M. We put the samples in a Thermocycler at 53.3°C,

55.7°C, and 58.0°C. The PCR reactions made copies of our DNA by attaching the primers to make new

sequences.

Now it was time to run these PCR samples on Agarose Gel Electrophoresis in order to separate the DNA

using electrical forces and friction. Figure 4 shows the results of the gels, which revealed that only the

cDNA worked in the gel. This was probably because we did not dilute the gDNA in the PCR reactions.


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However, the cDNA size was very accurate. We predicted that the size would be 1620 base pairs, and as

you can see, the results showed that this was indeed correct. This means that the predicted amounts of

introns and exons turned out to be accurate in the genomic sequence. You can also see a few smaller

bands under the bright bands in the gel. These are primer dimers, are pieces of the added primers

bonded together to from small strands of DNA. Since there were not many of them, the DNA did not

need to be purified again.

Once we had run the Agarose Gel Electrophoresis the first time, we determine that the C1 reaction was

the brightest with the least amount of primer dimers. Since our gDNA did not work, Dr. Smith ran the

gel again with both the cDNA and gDNA in order to see if it was our DNA that was defective, or if the

mistake was because of some other error. Since both cDNA and the gDNA worked in this gel, the

mistake must have been because we did not dilute the gDNA in the PCR reactions. This gel is also shown

in Figure 4.

Figure 4: PCR of CCT2 genomic and cDNA. A) 1% agarose gel of PCR of genomic and cDNA at 53.3°C. B) PCR of

CCT2 genomic and cDNA at 53.3°C, 55.7°C, 58.0°C. (L) 1.0 Kb ladder; (G) genomic Tetrahymena DNA template; (C)

cDNA from UV treated Tetrahymena cells.

L C G C G C G

B) 53.3°C 55.7°C 58.0°C

4 3 2 1.5 1 0.5

Size (kb)

A) L G C


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After the Gel Electrophoresis, we knew that we had just our gene in the DNA. Our next step was to

clone the DNA by mixing our PCR product with salt, water, and a TOPO vector that was ordered in a

cloning kit.

Next, we mixed that cocktail with E. Coli cells, and heat shocked it so that the cell walls of the bacteria

would disintegrate, and our DNA could migrate into the cells. We then spread 50 μL of the mixture on

one KB plate, and 200 μL on another KB plate. Figure 5 shows the results of the E. Coli mixture that was

spread on LB plates containing kanamycin. It shows that the E. Coli formed 13 colonies on the plate with

50 μL and 181 colonies on the plate with 200 μL. However, we did not know if our gene was inserted

into the plasmids of the cells until we performed gel electrophoresis on the cells to see if the band were

the predicted sizes.

Figure 5: Results of TOPO Cloning. After one day, LB plate A) contained 50 μL of our E. Coli sample and grew to 13 colonies. LB plate B) contained 200 μL of our E. Coli sample and grew to 181 colonies.

A) B)


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Before we could predict the gel sizes, we designed a map of the plasmid where the DNA was inserted.

On this map, we marked the positions of the restriction enzymes on the plasmid. These enzymes are

designed to cut the plasmid at these spots where they are located. This will allow us to see if the DNA

was inserted into the plasmid or not by seeing if the DNA is cut at the sizes that we predicted. The gDNA

Plasmid Map is shown in Figure 6 and the cDNA Plasmid Map is shown in Figure 7.

Figure 6: Plasmid Map of gDNA. Xho1/Avr11 are at the beginning and end, and Bgl11/EcoRV are present to mark if DNA was successfully inserted into the plasmid. Magenta represents the Kanamycin Resistant Cassette (part of the plasmid that breaks down Kanamycin). Yellow is the PUC ori (origin of replication). Brown striped represents the LR recombinase site (gene is inserted between both brown sites). Green represents our DNA sequence. Black represents the introns.


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We chose AvrII and XhoI (digest #1) because they were at the beginning and end of where our gene was

inserted into the plasmid map. We also chose EcoRV and BglII (digest #2) because they were located

both in our DNA and in the rest of the plasmid. The predicted band sizes for the cDNA in Digest 1

(AvrII/Xho1) were 2590 bp and 1654 bp. The predicted band sizes for the cDNA in digest 2 (BglII/EcoRV)

were 2686 bp, 954 bp, and 574 bp.

Figure 7: Plasmid Map of cDNA. Xho1/Avr11 are at the beginning and end, and Bgl11/EcoRV are present to mark if DNA was successfully inserted into the plasmid. Magenta represents the Kanamycin Resistant Cassette (part of the plasmid that breaks down Kanamycin). Yellow is the PUC ori (origin of replication). Brown striped represents the LR recombinase site (gene is inserted between both brown sites). Green represents our DNA sequence.


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Next, it was time to isolate the DNA from the E. Coli cells and run them on Agarose Gel

Electrophoresis in order to know for sure if the DNA was cloned or not. The results of this gel

are shown in Figure 8.

The gel shows that in Digest 1, only wells 3 and 4 showed the 2 predicted band sizes of 2590 bp

and 1624 bp. Well 6 shows a very bright band, which may mean that the gene was cloned in

that colony. In Digest 2, wells 1, 3, 4, and 6 show the 2686 band, but none of them show the

954 and 574 band. This could either be because we were at the bottom of the gel and it did not

run long enough to see the smaller bands, or because the restriction enzyme placement

predictions were wrong, and that there were only supposed to be 2 sites instead of 3. When

the sizes of the 2 smaller bands are added up, you get 1528, and there is a band here in almost

1 2 3 4 5 6 L 1 2 3 4 5 6

A) B) B)

Figure 8: Restriction Enzyme Gel. L represents 1 kb ladder. Side A) represents digest 1 using Avr11/Xho1. The predicted band sizes for this digest were 2590 bp and 1624 bp. Well 6 shows the 2590 band, but not the 1624 band. Wells 3 and 4 show both bands, but were faint. Side B) represents digest 2 using Bgl11/EcoRV. The predicted band sizes for this digest were 2686, 954, and 574 bp. None of the wells showed all of the bands well enough to prove the existence of the restriction enzymes.

Size (kb)

1.0

1.5

2

5

3 4

6

8 6 8 10


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all of the wells. This would still prove that our gene was cloned, it would just mean that there

was not another restriction enzyme where we thought it would be.

The last step of this experiment was cryopreservation. This is a process of freezing down the

DNA that was cloned. We froze the DNA from wells 3,4, and 6 from Digest 1 shown in Figure 8.

We chose these because they showed the correct band sizes, which means the DNA was

cloned. Future classes can now study our gene, and put it back into Tetrahymena in order to

track it and see what functions it performs in the cells. They can use our research to learn more

about CCT2 and perform many more experiments using our genes that we have cloned.

Discussions/ Conclusions

In this project, we studied whether an important gene that exists in Homo sapiens (TCP1 beta)

also exists in the species Tetrahymena Thermophila. We found that the gene CCT2 in Tetrahymena is

extremely similar to the TCP1 Beta gene in Homo sapiens. The E-value for the homolog CCT2 with

TTHERM_00149340 was 1.1e-143. The farther away from 1 this number is, the more similar the two

sequences are to each other. This meant that CCT2 and TCP1 Beta basically had the exact same gene

sequence. We also found that CCT2 has 8 exons and 7 introns, as shown in Figure 3. This is a lot of

introns and exons for a gene to have, which makes it more complicated to identify and clone.

Once we found a homolog (CCT2), we isolated this gene and copied it many times with PCR

reactions using the primers found in Figure 1. We then cloned it using TOPO vectors. After we cloned it,

we inserted it into E. Coli so that it would rapidly grow and produce more colonies with our cloned DNA.

We also predicted the exact location that our gene would be inserted into the plasmid, and the

restriction enzymes in the plasmid that will tell us if the DNA is actually present in the plasmid or not.


17

After performing another Gel Electrophoresis, we learned that the gene was indeed cloned in several of

our colonies. We knew this because the gel band sizes, shown in Figure 8, were the predicted sizes of

where the restriction enzymes were located in the plasmid maps in Figures 6 and 7. We also learned

that the placement of the restriction enzymes on the plasmid map was slightly wrong. There should

have only been two sites of BglII and EcoRV, instead of 3. This will help later researchers to fully

understand the CCT2 gene. Lastly, we froze down the cells that were cloned so that more research can

be performed on them in upper level classes.

We only ran into one major problem along the way that affected the results of the labs that

followed. This problem was that we did not dilute the gDNA when adding to the PCR reactions, and it

affected the agarose gel electrophoresis in the fact that it did not show up in the final picture. However,

this problem was fixed because Dr. Smith ran a new gel with new PCR reactions. These new reactions

contained the correct dilution of gDNA, therefore, the bands showed up on the gel picture.

One thing that I think that could be improved in this research would be to provide more

information on each procedure, such as PCR reactions and the electrophoresis gel method, so that we

could fully understand what was actually going on in the cells during these parts of the experiment.

Another improvement would be to further investigate the functions of each gene in our original species

versus the functions of the same gene in the Tetrahymena. This would help us to better understand the

importance of each gene and how it relates to species that we are more familiar with.

Despite the few problems with this research project, it helped me to understand how complex

genes are, and how important they are in any given species. One of the most interesting parts was the

fact that we could clone our gene and insert it into a different species in order for it to grow and

multiply to even more cells containing the DNA of our gene. This makes me wonder about how close we

are to cloning human beings, along with many other different animals. Also, the chemical side of this


18

project was intriguing because of the way that the primers were able to bond to the right DNA strands in

order to make copies, and how the DNA automatically migrate down the gel to the positive end of the

chamber because the phosphates make it negatively charged. This process brought the structures that I

have read about in Biology books for years, such as DNA and enzymes, into reality because I saw

firsthand how these structures work.

Though we have completed a main portion of research, there are still many important things to

be discovered about CCT2. For example, it would be interesting to insert our cloned gene back into

Tetrahymena and track it as it performs functions in the cells. The next class after us will be able to

watch the CCT2 gene as it travels in Tetrahymena Thermophila and works to fold proteins for the cilia to

function properly. They will also understand how it relates to some of the other genes researched in the

class such as CCT3. They can observe the 8 ring structure that forms between all of the CCT genes, and

watch as it protects newly formed proteins from being broken down by water. From this research, they

will be able to compare the specific functions of CCT2 to TCP1 Beta in Homo sapiens.


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References

Brackley, K. I., & Grantham, J. (2009). Activities of the chaperonin containing TCP-1 (CCT): implications

for cell cycle progression and cytoskeletal organisation. Cell Stress and Chaperones, 14:23–31.

Kubota, H., Hynes, G., & Willison, K. (1995). The chaperonin containing t-complex polypeptide 1 (TCP-1)

Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. 3-16.

Seixas, C., Cruto, T., Tavares, A., Gaertig, J., & Soares, H. (2010). CCTa and CCTd Chaperonin Subunits Are

Essential and Required for Cilia Assembly and Maintenance in Tetrahymena. PLoS ONE, 1-13.

Smith, J. J. (2011). BMS 110 Honors Lab Protocols.

Soares, H., Cyrne, L., Casalou, C., Ehmann, B., & Rodrigues-Pousada, C. (1997). The third member of the

Tetrahymena CCT subunit gene family, TpCCTa, encodes a component of the hetero-oligomeric

chaperonin complex. Biochem, 21-29.

cct2 gene in tetrahymena thermophilafaculty.jsd.claremont.edu/ewiley/files/r96n0.pdfbms110 honors,...

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