1
Cloning SIRT6 Homolog, THD11,
in Tetrahymena thermophila
Chase Neff and Vanessa Lea
Fall 2009
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Contents
Abstract ......................................................................................................................................................... 2
Introduction ................................................................................................................................................... 3
Methods/Procedure ....................................................................................................................................... 4
Lab 3-Bioinformatics ................................................................................................................................ 4
Lab 4-Genomic DNA Isolation ................................................................................................................. 5
Quantification of Genomic DNA .......................................................................................................... 6
Lab 5-Polymerase Chain Reaction ........................................................................................................... 6
Lab 6-Agarose Gel Electrophoresis.......................................................................................................... 7
Lab 7-Cloning PCR Product ..................................................................................................................... 7
Removal of Primer Dimers ................................................................................................................... 7
Quantification of PCR Product ............................................................................................................. 8
TOPO Cloning ...................................................................................................................................... 8
Lab 8-Plasmid Mapping ........................................................................................................................... 8
Results ......................................................................................................................................................... 10
Lab 3-Bioinformatics .............................................................................................................................. 10
Lab 4-Quantification of Isolated Genomic DNA .................................................................................... 14
Lab 5-Polymerase Chain Reaction ......................................................................................................... 14
Lab 6-Agarose Gel Electrophoresis........................................................................................................ 15
Lab 7-Cloning PCR Product ................................................................................................................... 15
Lab 8-Plasmid Mapping ......................................................................................................................... 16
Written Results ............................................................................................................................................ 18
Discussion/Conclusion ................................................................................................................................ 19
References ................................................................................................................................................... 20
Abstract
In this experiment we cloned the gene THD11 from the ciliate, Tetrahymena thermophila,
into Escherichia coli plasmid. THD11 is a homolog gene for the protein SIRT6 in Homo sapiens.
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Not much is known about SIRT6; its function in humans has not yet been determined though
other Sirtuins are known to repair DNA, play a part in chromosomal stability, and in longevity in
Saccharomyces cerevisiae(Michan and Sinclair 2007). One function of SIRT6 is its part in
promoting normal DNA repair in cells; this is exciting because studying it could potentially help
us find a way to prevent or slow down many progeroid degenerative syndromes, along with
cancer. To learn more about the mammalian protein SIRT6 we used a homolog gene from the
organism Tetrahymena thermophila. T. thermophila is a commonly studied unicellular model
organism. It is found in most bodies of fresh water and has one of the fastest reproduction rates
among eukaryotes, making it ideal for our experiment. This experiment will give us a better
understanding of the SIRT6 homolog and get us closer to understanding a possible link to cancer
cells. We will be cloning the genes from Tetrahymena thermophila, and then storing them in
plasmid vectors so that they can be further studied and researched.
Introduction
SIRT6 is of the Sirtuin family and is a nuclear, chromatin-associated protein that
promotes resistance to DNA damage and suppresses genomic instability. Sirtuins get there name
from being silent information regulators (SIR) and are a broadly expressed throughout cells. At
the protein level, SIRT6 is readily detectable with the highest levels in muscle, brain, and the
heart(Liszt et al 2005). Sirtuins have been found to extend yeast life span by inhibiting
recombination in the rDNA repeats (Mostoslavasky et al 2006). Sirtuins are also thought to play
a role as a histone deacetylase. Histone deacetylases are enzymes that catalyze the removal of
acetyl groups from lysine residues in both histone and non-histone proteins. They play a key role
in the regulation of gene transcription and many other biological processes involving chromatin.
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Recent studies suggest that histone deacetylases are critically involved in cell-cycle regulation,
cell proliferation, differentiation, and in the development of human cancer. Histone deacetylase
inhibitors are currently being exploited as potential anti-cancer agents (Sengupta and Seto 2004).
SIRT6 is also a potential mediator of caloric restriction, the only non-genetic method that
consistently increases maximal lifespan in mammals (Michan and Sinclair 2007). Studying
SIRT6 will help us better understand its’ functions, histone deacetylases, and more about age
related illnesses.
The first step in this experiment was to identify an appropriate gene homolog in T.
thermophila for the gene SIRT6 and to find its protein sequence, coding sequence, as well as its
genomic sequence using online databases. This allowed us to find an appropriate homolog along
with the necessary forward and reverse primers. We then isolated the T. thermophila genomic
DNA and ran it through Polymerase Chain Reaction in order to produce a larger quantity of the
DNA. Once this was completed we ran the coding and genomic DNA through Agarose gel
electrophoresis in order to determine if the PCR was successful. Finally we cloned the isolated
gene THD11 into the E. coli plasmid vector and then used a plasmid construction program to
map our plasmid so that we could find the appropriate enzymes to use for digesting. Once we
made the digestive enzyme cocktail and cut the plasmid into three bands we ran it through
Agarose gel electrophoresis to confirm the presence of the SIRT6 homolog, THD11.
Methods/Procedure
Lab 3-Bioinformatics
(Reference Lab 3 handout) First thing we did in this lab was find the amino acid sequence
of the gene SIRT6 using the protein database at http://www.ncbi.nlm.nih.gov/ and then the T.
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thermophila homolog gene was found using the Tetrahymena thermophila genome database at
http://playground.bradley.edu/~rpunia/TGD/. Using the Playground website we found each of
the IPI (Human) and SGD protein homologs and their e-values for each of the top three tTHERM
numbers. Next, this same database was used to find the protein sequence, coding sequence, and
the genomic sequence of the best T. thermophila homolog. Then we used the website,
http://origin.bic.nus.edu.sg/mgalign/mgalignit.html to compare the T. thermophila homolog coding
sequence with the genomic sequence in order to find the introns and exons. Finally, we went
back to the NCBI website to compare protein sequences of the Homo sapien and T. thermophila
homologs.
Lab 4-Genomic DNA Isolation
(Reference lab 4 handout) We started by using a plastic transfer pipette to place 1.4 mL
of Tetrahymena culture into a microcentrifuge tube. We collected the cells by centrifuging the
culture for 1 minute at 10,000 rpm and then removed the supernatant with a pipette. Next we
added 700 µL of Urea Lysis Buffer and completely re-suspended the cell pellet. Then we
proceeded to phenol-extracted the lysate by adding 600 µL of Phenol:chloroform:isoamyl
alcohol and then centrifuging the mixture for 5 minutes at maximum speed in order to separate
the thick interphase layer from the lysate. We transferred the lysate into a new microcentrifuge
tube and added 150 µL of 5M NaCl. Next we precipitated the DNA by adding 680 µL of
ispopropyl alcohol and allowing the mix to stand for 10 minutes. We collected the precipitate by
centrifuging the mixture at maximum speed for 10 minutes. Then we decanted the supernatant
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and added 500 µL of 70% ethanol to the cell pellet. In order to collect the final precipitate we
centrifuged for another 3 minutes at maximum speed and allowed the pellet to air dry. Next we
re-suspended the pellet in 50 µL of TE buffer (10mM Tris-Cl (pH 8), 1mM Na2EDTA) and
finally we added 1 µL of RNase A(10 mg/mL in 50 mM potassium acetate (pH 5.5)) and
incubated at 37 °C for 15 minutes.
Quantification of Genomic DNA
We prepared a 1:100 and 1:200 dilution of our DNA in two separate microcentrifuge
tubes. The spectrophotometer was allowed to warm for at least 15 minutes and we blanked the
spectrophotometer with the water used to make our dilutions. Finally we filled the quartz cuvette
with 0.1 mL of the 1:100 DNA dilution and then took the reading. We repeated this process with
the 1:200 dilution.
Lab 5-Polymerase Chain Reaction
(Reference lab 5 handout) The oligonucleotide primers that we used for the PCR were
designed using the bioinformatics lab data, and the template was derived from the genomic DNA
we isolated in the previous lab. The primers were given to us in lyophilized form so we resuspended them in sterile ddH2O to a final concentration of 200 µM for the stock and then
diluted it in a separate tube to have 200 µL of a 20 µM working stock. Next we prepared our 6
polymerase chain reaction mixes. Three solutions will have 1 µL of 1.0 µg genomic DNA added
and the other three will have 1 µL of wild type cDNA (1:10 Dilution WT) added. In addition to
this we added 25.5 µL of sterile distilled water, 1 µL of 0.2 mM dNTPs, 10 µL of 1.0 M Betaine,
10 µL of 1X GC buffer (1.5 mM MgCl2), 0.5 µL of 1.0 unit (U) Phusion polymerase, 1.0 µL of
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0.2 µM TF primer, 1.0 µL of 0.2 µM TF primer. Thermocycler was programmed to heat the
reactions for 1 minute at 98°C to denature genomic DNA, and then run 34 cycles of 20 seconds
at 98 °C, 25 seconds at primer annealing temperature (53°C, 55°C, 57°C; one of each gDNA at
each temp. along with one of each cDNA at each temp.), and then 1.5 minute polymerase
extenstion at 72°C. After the cycles 10 minutes at 72°C and then they were held at 4°C.
Lab 6-Agarose Gel Electrophoresis
(Reference lab 6 handout) First we weighed out 0.75 grams of agarose gel in order to
obtain a 1.5% mixture. Then added 50 ml of 1X TAE, mixed the solution, and covered it in
plastic wrap to place in microwave for 1 minute. Then it took another 20 second and then
another 10 sec cycle in the microwave for the agarose to completely dissolve. Placed comb into
the electrophoresis casting tray using the 1.5 mm side. Once the agarose solution cooled down
we added 0.5 µL of 10 mg/ml Ethidium Bromide. Then we poured the agarose into the casting
tray and allowed it to solidify for 30 minutes. Once this was complete we filled the
electrophoresis chamber with 1X TAE until it just covered the gel. Finally we loaded 5 µL of kb
ladder into the first lane. We mixed each of our 10 µL samples of gDNA or cDNA with 1 µL of
10x dye (xylene cyanol and bromphenol blue) and then ran them through the agarose gel at a
constant 90-120 volts. This took approximately 30 minutes.
Lab 7-Cloning PCR Product
Removal of Primer Dimers
(Reference lab 7 handout) First we added 305 µL of distilled water into the sample resevior
of a montage PCR device. Next we added 95 µL of cDNA and spun the montage PCR unit at
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1000 X g for 15 minutes. Then we added 20 µL distilled water and inverted the reservoir into a
clean vial and spun it at 1000 X g for 2 minutes.
Quantification of PCR Product
We made 100 µL of a 1:50 dilution of our cleaned PCR product ran it through the
spectrophotometer to read the absorbance ratings at A260 and A280.
TOPO Cloning
First we mixed the TOPO cloning reaction; 2.26 µL of distilled water, 1.74 µL of the
1:10 dilution coding DNA PCR product, 1 µL of salt solution, and then 1 µL of TOPO vector.
Once the solution was mixed, it was incubated for 10 minutes at room temperature. Then we
allowed a vial of chemically competent E. coli to thaw on ice for 5 minutes. Once this was
complete we added 2 µL of the TOPO cloning reaction to the vial of E. coli. This was then
incubated on ice for 10 minutes and heat shocked at 42°C for 30 seconds. Next we added 250
µL of room temperature SOC Medium and shook the tube horizontally at 200 rpm and 37°C for
40 minutes in a shaking incubator. Finally we spread 200 µL of the solution to a pre-warmed
plate containing 50 µg/mL kanamycin using sterile glass beads and incubated these overnight at
37°C. After the incubation they were placed in a 4°C cold room.
Lab 8-Plasmid Mapping
(Reference lab 8 handout) We mapped our plasmid by using a gene construction
program. This consisted of opening up a pENTR/TOPO-D circular plasmid map and pasting our
gene sequence over a highlighted section of CACC. This created a new plasmid map with our
gene sequence.
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On the new plasmid map that we created we marked the sites for commonly used
restrictive enzymes. Once this was done we picked 2 enzymes to use in order to cut the circular
plasmid at 3 locations, one section of which is mainly our gene. Finally we printed a picture of
the predicted digest bands that would result from running the broken plasmid through gel
electrophoresis. The two digestive enzymes picked were NheI and AvrII.
Lab 10- Restriction Enzyme Digest of Plasmid
(Reference lab 10 handout) The day before the lab we inoculated six 2 mL sultures of LB
liquid media tubes (containing 50 µL/mL Kanamycin) with six transformant colonies. We did
this by using six different 6 inch wooden sticks to pick up the different colonies to place in 2 mL
LB-Kan media. They were then placed in a shaking incubator at 37°C.
On the day of the lab we transferred 1.5 mL of each culture into microcentrifuge tubes
using a plastic transfer pipet. This was centrifuged on maximum speed for 2 minutes. Next we
decanted the supernatant and added 350 µL of Sucrose Lysis Buffer. The pellet was resuspended
by pipeting up and down. Then we added 25 µL of lysozyme solution (10 mg/mL in TE) and
mixed by inverting several times. This was incubated at room temperature for 5 minutes before
being heated at 99°C for 1 minute in a boiling water bath. Next we centrifuged the tubes for 15
minutes at maximum speed before removing the pellet. In order to precipitate the supernatant we
added 40 µL of 3M NaOAc and 220 µL of isopropanol to the supernatant. This mixture was
incubated at room temperature for 5 minutes and centrifuged for 10 minutes at maximum speed.
Next we poured off the supernatant and washed the plasmid pellet in 1000 µL of 70% ethanol
and proceeded to centrifuge this for 2 minutes at maximum speed. Finally we removed the
supernatant and dried the pellet before resuspending the pellet in 50 µL Tris-EDTA (TE) buffer.
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Now we were ready to make the enzyme digest cocktail. This consisted of 14 µL of 1X
Buffer from 10X buffer stock, 2.5 µL Nhe1, 3.5 µL of Avr11, 1.4 µL of 1X BSA from 100X
BSA stock, and 96.6 µL of distilled water. This cocktail was a 7X mixture, 1 for each plasmid
isolated and 1 extra for pipet errors. We then put 17 µL of the cocktail in new microcentrifuge
tubes along with 3 µL of isolated plasma from the 1-6 cultured colonies into the 6 different
tubes. These tubes were then incubated at 37°C for an hour before adding 10X sample dye and
running them on a gel (see Lab 6 for this procedure).
Results
Lab 3-Bioinformatics
A)
SIRT6- Homo sapiens (Protein sequence)
MEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECA
KCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIV
NLQPTKHDRYADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRING
SIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS
B)
Number one:
2.7 e-32
1|ENSEMBL:ENSP00000337332|REF
3.0001718090399e- SEQ:NP_057623|H- INV:HIT000032
IPI(HUMAN) IPI:IPI00383640.3
49
167|VEGA:OTTHUMP00000078069
Ta
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x_Id=9606 Splice Isoform 1 of
Mono-ADP-ribosyltransferase si
rtuin-6
SGD
YPL015C
HST2 SGDID:S000005936, Chr XVI
from 526880-525807, reverse c
omplement, Verified ORF, "Cyto
plasmic member of the silencin
g information regulator 2 (Sir
1.000095655533e-07
2) family of NAD(+)-dependent
protein deacetylases; modulate
s nucleolar (rDNA) and telomer
ic silencing; possesses NAD(+)
-dependent histone deacetylase
activity in vitro"
C) Number two:
2.8e-20
1|ENSEMBL:ENSP00000337332|REF
SEQ:NP_057623|H- INV:HIT000032
1.9991517704233e- 167|VEGA:OTTHUMP00000078069
IPI(HUMAN) IPI:IPI00383640.3
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Tax_Id=9606 Splice Isoform 1 of
Mono-ADP-ribosyltransferase si
rtuin-6
SIR2 SGDID:S000002200, Chr IV from 378442376754, reverse complement, Verified ORF, "Conse
rved NAD+ dependent histone deacetylase of the Sirtuin
SGD YDL042C 6.0015700855979e-12 family involved in regulation of lifespan; plays roles in
silencing at HML, HMR, telomeres, andthe rDNA
locus; negatively regulates initiation of DNA repl
ication"
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D) Number three:
5.5e-16
HST2 SGDID:S000005936, Chr XVI
from 526880-525807, reverse c
omplement, Verified ORF, "Cyto
plasmic member of the silencin
g information regulator 2 (Sir
SGD YPL015C 3.9998859049333e-09 2) family of NAD(+)-dependent
protein deacetylases; modulate
s nucleolar (rDNA) and telomer
ic silencing; possesses NAD(+)
-dependent histone deacetylase
activity in vitro"
1|ENSEMBL:ENSP00000337332|REF
SEQ:NP_057623|H- INV:HIT000032
167|VEGA:OTTHUMP00000078069
1.0000099287337eTa
IPI(HUMAN) IPI:IPI00383640.3
53
x_Id=9606 Splice Isoform 1 of
Mono-ADP-ribosyltransferase si
rtuin-6
E) SIRT6- Tetrahymena thermophila 00313730 (protein sequence)
MDTAHKTVNEKKEYFDSPELLEAKVTQLADMIKQSNHFVCFTGAGISTSAGIADFRSGVN
TVLKTGPGLWEKMAQKVGNQPKKHKVIMSRAVPTKSHMALVKLNQEGILKYLISQNIDGL
HRRSGFNPNSLSELHGNTNLEKCLKCGKSYMRDYRVRKALDVHDHLTGRICDNQKCGGEL
VDTIVNFGENLPKKDMEQGFFNSKQADLHLVLGSSLRVTPAADMPLATAQNGNKLVVVNL
QKTPLDSLCALRIYALIDDVMVLLMKKLGLEIPEFILQRTIVIKKTNQNTINVFSEDKDG
CPYDIFKQIVLDQGKKQPFEIQVKAPYIFNITNPQFAIKLGFFEHYKEGPFRLDLNLQNL
PLGQKTKYLIQFSPKLQKWISCEKIQ
F) SIRT6 Tetrahymena thermophila 00313730(Coding sequence)
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ATGGATACTGCTCATAAAACAGTAAACGAAAAGAAAGAATACTTTGATTCTCCGGAATTA
TTAGAAGCAAAAGTCACTTAGTTGGCAGATATGATTAAATAGTCAAATCACTTTGTTTGC
TTTACAGGTGCTGGAATATCTACTTCAGCAGGTATAGCTGATTTTAGAAGTGGAGTTAAC
ACAGTCTTGAAAACTGGACCTGGTTTGTGGGAAAAGATGGCTTAAAAAGTAGGAAATCAA
CCTAAAAAACACAAAGTTATAATGTCTAGAGCTGTTCCAACTAAAAGCCATATGGCACTA
GTTAAGTTGAATCAAGAAGGAATTCTTAAGTATTTAATCAGTTAAAATATAGACGGCTTA
CATAGAAGAAGTGGATTCAACCCTAATAGCCTATCTGAACTACATGGAAACACTAATTTA
GAGAAATGTTTAAAATGTGGAAAGTCTTATATGAGAGATTATAGAGTGAGAAAAGCTTTA
GATGTTCATGACCACTTAACAGGAAGGATTTGCGACAATTAGAAATGTGGTGGCGAATTA
GTAGATACAATTGTTAATTTTGGAGAGAATTTACCCAAAAAAGATATGGAATAAGGTTTT
TTTAACTCAAAATAAGCAGATTTACACTTAGTTTTAGGAAGTAGTTTAAGAGTTACTCCA
GCAGCTGATATGCCTCTAGCAACTGCTTAAAATGGAAATAAATTAGTTGTGGTTAATTTA
CAAAAAACCCCTCTAGATAGCTTGTGTGCCTTAAGAATATATGCTTTGATTGATGATGTC
ATGGTTCTACTTATGAAAAAGCTAGGTTTAGAGATACCAGAATTTATTCTGCAGAGAACT
ATTGTGATTAAAAAGACAAACTAAAATACAATAAACGTTTTTAGTGAAGACAAAGATGGT
TGTCCATATGACATTTTTAAGTAAATTGTTTTGGATTAAGGCAAAAAGTAACCATTTGAG
ATTTAGGTTAAAGCACCTTACATATTCAATATCACAAATCCATAATTTGCCATAAAGCTA
GGATTTTTTGAACATTATAAAGAAGGACCATTCAGATTAGATTTAAATTTATAAAATCTA
CCTCTTGGATAAAAGACTAAGTATTTAATATAATTCTCACCAAAATTACAAAAGTGGATT
AGTTGTGAAAAAATATAATGA
G) Homolog protein comparison
Homo sapiens
USERSEQ1
(256 aa)
Tetrahymena thermophila
USERSEQ1
(386
)
Figure 1: Bioinformatics of SIRT6 homolog in Tetrahymena- A) Protein sequence for the
Homo sapiens gene SIRT6. B), C) and D) are the IPI (Human) and SGD protein homologs and
their e-values for each of the top three tTHERM numbers. E) Is the SIRT6 T. thermophila
protein sequence for tTHERM 00313730. F) Is the SIRT6 T. thermophila coding sequence for
tTHERM 00313730. G) is the homolog protein comparison between the Homo sapiens and the
T. thermophila homolog for SIRT6. There are no introns on this gene.
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Lab 4-Quantification of Isolated Genomic DNA
Table 1-Spectrophotometer readings for isolated DNA
Dilution
A260 Reading
A280 Reading
A260/ A280
µg/ µL of DNA
1:100
1:200
Average
1.957
1.556
1.757
.863
.669
.766
2.267
2.33
2.299
9.785 µg/ µL
15.56 µg/ µL
12.67 µg/ µL
Lab 5-Polymerase Chain Reaction
THD11 TF
5’-CACCCTCGAGGATACTGCTCATAAAACAGTAAAC
THD11 TR
5’-AGAGCCTAGGTCATTATATTTTTTCACAACTAATC
Figure 2- These are the nucleotide primer sequences used to clone the T. thermophila gene THD11 by
PCR. TF is the forward primer (yellow highlighted region added for cloning) and TR is the reverse primer
(blue highlighted region added for cloning).
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Lab 6-Agarose Gel Electrophoresis
1
2
3
4
5
6
7
8
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Figure 3: Agarose Gel Electrophoresis of THD11 PCR- This shows the
results of the Agarose gel electrophoresis. Lane 1 is the 1KB ladder. Lanes 2, 3, 4 are the gDNA
at 53°C, 55°C, and 57°C respectively. Lanes 5 and 6 are empty. Lanes 7, 8, and 9 are cDNA at
53°C, 55°C, and 57°C respectively. The 1161 bases was predicted in lab3, bioinformatics
Lab 7-Cloning PCR Product
Table 2-Spectrophotometer reading after purification process
cDNA
A260
A280
A260/ A280
.023
.017
1.35
Table 3-Plates of cultured E. coli with 200 µL of TOPO solution
Plate
Number of cultured dots
First plate
Second plate
2
103
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Lab 8-Plasmid Mapping
Figure 4- Shows what the E. coli plasmid looks like with the T. thermophila gene (shown in
green). Also shows the locations of commercially available digestive enzymes. The two
digestive enzymes used to cut the plasmid at three different locations, making 3 bands, are
AvrII and NheI (highlighted in black). The pink is the kanamycin resistant band and the yellow
band is the pUC origin of replication.
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Figure 5- Digest chart showing sizes of bands after circular plasmid is digested and where the
digestive enzymes are located on the plasmid. The figure on the right is what the gene
construction program predicts the gel will look like after running the digested plasma through.
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Lab 10- Restriction Enzyme Digest of Plasmid
1
2
3
4
5
6
7
8
9
10
Figure 6: Restriction Digest of the pENTR, THD11 Clones- Lane 1 is the
1KB ladder. Lanes 2, 5, and 8 are empty. Lanes 3, 4, 6, 7, 9, and 10 were each ran with the
plasmids digested by Avr11 and Nhe1, from the 6 different picked transformant colonies. To the
right of the gel are the predicted base sizes for the 3 bands, obtained from lab 8.
Written Results
Through lab 3, “Bioinformatics,” we found 3 homolog genes to SIRT6 and selected
tTHERM 00313730, letter D) in Figure 1 to clone. We also obtained the protein sequence,
coding sequence, along with the genomic sequence for the SIRT6 homolog from these online
databases. There were zero introns. These sequences were used later to create the necessary
forward and reverse primers for PCR. We then isolated the DNA and ran it through a
spectrophotometer. The A260/ A280 result (Table 1) for this was 2.29 which is higher than the
necessary amount to determine if it’s pure enough to use (1.8). Now that the isolated DNA had
been found pure enough we were able to proceed in running it through PCR, using the forward
and reverse primers found in lab 3. We found the results for our PCR lab after lab 6, “Agarose
Gel Electrophoresis.” The picture of our gel in Figure 3 shows that our cDNA was successfully
duplicated through PCR but that the gDNA wasn’t. Due to these results we used the cDNA
through out the rest of the experiment. Our cDNA had primer dimers that would have to be
cleaned out in the next lab.
After running the cDNA through the cleaning process we checked its purity with the
spectrophotometer and got an A260/ A280 reading of 1.35 (Table 2) which we determined was an
appropriate reading to continue on with the lab. We made the TOPO cloning cocktail and spread
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200 µL of the solution on a plate. Results for the first plate were 2 transformant colonies.
Results for second run were more successful with 103 cultured transformant colonies of E. coli
(Table 3). These colonies were used in lab 10, “Restriction Enzyme Digest of Plasmid”. It was
then necessary to map the plasmid and find effective digestive enzymes to use, found in Figure
4. We chose enzymes Avr11 and Nhe1. These would cut the plasmid into three pieces, one of
which would be mostly our gene; the predicted band sizes were 2140, 1348, and 266 bases;
shown in Figure 5. This predicted what our results should look like after running the digested
plasmid through a gel, in lab 10.
Next we made the restriction enzyme digest cocktail to cut the plasmid and ran this
solution through agarose gel electrophoresis. The gel (Figure 6) showed us bands at the correct
lengths of 2140, 1348, and 266 bases indicating that our gene was successfully cloned into the E.
coli plasmid.
Discussion/Conclusion
The first step in this experiment was to find a proper homolog for the mammalian gene
SIRT6, which we did using online Tetrahymena databases. Lab 3, Bioinformatics, gave us the
necessary sequences in order to run our picked homolog of SIRT6 through PCR. It also indicated that our
gene had zero introns. We then isolated the DNA in lab 4, where we got an A260/ A280 that was too
high. We attributed the spectrophotometer reading, of 2.29, being a little high to having a
slightly too concentrated solution of DNA. This would not affect future labs. The isolated DNA
was used as a template to make millions of copies through polymerase chain reaction, using the
forward and reverse primers found in lab 3.
In lab 5, “PCR,” we found the primer annealing temperatures for the genomic DNA and
the cDNA. We used 3 different temperatures, figured using the forward and reverse primers.
These 3 temperatures were 53°C, 55°C, and 57°C. The cDNA was expressed and produced the
highest amount of duplicated cDNA when the primer annealing temperature in the thermo cycler
was set at 55°C. The gDNA did not run through PCR successfully which we attribute to human
error in making the gDNA PCR cocktail, for we did not mix up the master solution before
separating into 3 different tubes. Lanes 2, 3, and 4 in Figure 3 were samples from these 3 tubes.
We noticed that the Phusion polymerase sank straight to the bottom of this master mix so when it
was separated into the three different tubes the concentrations varied immensely. Our gel
showed primer dimers present, indicating a need to purify our DNA before continuing.
After the purification process in lab 7, TOPO Cloning, we ran the product from the 55°C
PCR through a spectrophotometer and obtained an A260/ A280 reading of 1.35 which is low. This
was due to not having a high enough concentration of PCR product. Also because of the weak
concentration we combined our 53°C, 55°C, and 57°C product. Next we made a 1:10 dilution of
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the PCR product for the TOPO reaction. Since our PCR product was already a weak
concentration I believe the extra dilution, combined with having to run the shortest allotted times
in the incubator/shaking incubator, caused our first run Agar plates to produce a low number of 2
transformant colonies. The second run with a higher concentration and appropriate times
produced 103 cultured transformant colonies, so we made the first run our negative control.
Next we mapped the E. coli plasmid, with the SIRT6 homolog inserted, using a plasmid
construction program. This allowed us to find the best commercially available enzymes to use
for digesting the plasmid so that we could determine if our gene was in fact properly inserted and
cloned. We picked Avr11 and Nhe1. The plasmid construction program predicted the digestive
bands to be 2140, 1348, and 266 bases long. Next, we made restriction enzyme digest solution
and ran it on an agarose gel. The gel confirmed the presence of our inserted gene for there were
bands at the appropriate predicted lengths. The band length of 1348 included mainly our gene
and since our gene was 1161 bases long the presence of this band on the gel indicated the
successful insertion of the SIRT6 homolog, THD11, into the E. coli plasmid. Now that this gene
has been cloned into the plasmid it can easily be cultured and stored for future use and research.
This research can include: studying the gene function, tracking the gene with green fluorescing
proteins, knocking out or promoting the gene, and eventually discovering the T. thermophila
proteome.
References
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Aging-like Pheynotype in the Absance of Mammilian SIRT6." Cell 124 (2006): 315-29.
2. Rodgers, Joseph T., and Pere Puigserver. "Certainly Can't live With Out this: SIRT6."
Cell Metabolism 3 (2006): 77-82.
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