Cloning the Saccharomyces RAD23 Gene in Tetrahymena

Cloning the Saccharomyces RAD23 Gene in Tetrahymena

David Brannan

Lab Partner-Shannon Smith

10/10/08-BMS 110/999

Lab Report

Abstract:

This lab report contains the steps and results of a project that seeks to clone a gene of Tetrahymena thermophila into a plasmid. If successful, the cloned gene of the Tetrahymena plasmid could possibly be used for future research. For this experiment, we are seeking to clone the corresponding gene of Saccharomyces RAD23 in Tetrahymena. The protein Saccharomyces RAD23 is used for DNA repair through Nucleotide Excision Repair (NER) which removes DNA lesions. However, Saccharomyces RAD23 is an accessory protein that helps maintain peak function, but isn’t essential to NER simply taking place. These functions of RAD23 suggest that the corresponding gene in Tetrahymena may have similar abilities; and that successfully cloning this gene into a plasmid may lead to results that could be applicable to genetic research. The processes that are included in this lab report include Bioinformatics, isolating DNA, amplifying DNA, testing it with electrophoresis, cloning DNA, inserting it into E. coli, creating a plasmid map, and purifying the plasmid with restriction enzyme digest. The results from the majority of the labs were fairly successful, though some failures were encountered. Overall, the results of these experiments were satisfactory until the TOPO cloning reaction failed to form colonies. Due to this, we used colonies for the final lab from another lab group that successfully formed colonies with RAD23. When we carried out the plasmid purification and restriction enzyme digest, the results failed to yield plasmids that could be

Abstract: (Continued)

used for future research. Therefore, in order to achieve a product, we would have to start back at the TOPO cloning reaction and make another attempt to have it work. If that fails, the most promising place to restart would probably be to once again isolate the gDNA of Tetrahymena.

Introduction:

In this project, we are seeking to clone the corresponding gene of the protein SaccharomycesRAD23 in Tetrahymeana to create a plasmid that could potentially be used for research. Tetrahymena are free living ciliate protozoa that are common in fresh water, used for research, and have a variety of complex and specialized cell structures. The protein Saccharomyces RAD23 is involved in Nucleotide Excision Repair (NER);2,3 which serves as a DNA repair mechanism for possible mutations. The RAD23 gene was originally found in yeast, but has since been found to perform a variety of functions including binding to DNA during replication, initiating the targeting of proteolytic substrates, and being partially responsible for ultraviolet induced DNA repair. The importance of RAD23 can be easily seen in DNA replication. When RAD23 doesn’t function correctly or is absent in DNA replication, the phosphate base thymine binds with another thymine, as opposed to adenine (the proper binding pair). In humans, this abnormality causes Xeroderma pigmentosum, which makes human skin unable to repair itself, and therefore precludes an individual from getting UV exposure8. This information displays the importance that RAD23 plays in NER and though not completely essential to it, it fulfills a crucial role.

Introduction: (Continued)

Given the importance of the protein Saccharomyces RAD23 in NER, successfully cloning the corresponding gene in Tetrahymena could be used to research certain genetic abnormalities. In this case, cloning refers to isolating a defined DNA sequence and obtaining multiple copies of it1. Cloning has essentially four processes: fragmentation, ligation, transfection, and screening/selection. Fragmentation is where the DNA is essentially separated into fragments and amplified, usually through Polymerase Chain Reaction. Ligation is a procedure where the amplified fragment is incubated with the enzyme DNA ligase under appropriate conditions. Transfection is the process where the DNA is transfected into cells, which can be done chemically or through electroporation. Finally, the transfected cells are cultured. Ultimately, the goal of this project is to clone a gene of Tetrahymena into a plasmid that could be used by other researchers.

Materials and Methods

Bioinformatics

The amino acid sequence of Saccharomyces RAD23 was found through the database and the corresponding Tetrahymena homolog was found through the webpage The top three results can be found in the results section. The IPI (Human) and SGD protein homologs for the TTHERM_# were also found and recorded. The protein sequence of the Tetrahymena homolog was found with a ORF Translation coding sequence and labeled Tt RAD23 amino acid using the webpage The Tetrahymena homolog with the highest e-value was used to find the nucleotide sequence with the resource and labeled as Tt RAD23 CDS. The genomic sequence of Tt RAD23 was found with the highest TTHERM_# at With the same resource, the Tt RAD23 sequence was found with NTS and underlined EST’s. Finally, the amino acid sequence of Saccharomyces RAD23 was compared with the sequence Tt Saccharomyces RAD23; a second comparison was made between the amino acid sequence of Saccharomyces RAD23 and the results of the first comparison using the website

Tetrahymena Genomic DNA Isolation

First, 1.4mL of Tetrahymena culture was pipeted into a microcentrifuge tube. The tube was then placed in the centrifuge for one minute at 10,000 revolutions per minute; afterwards the supernatant was removed. The pellet that was formed from the centrifuge cycle was then flicked to loosen residual supernatant, and 700µL of Urea Lysis Buffer was added to the tube to protect the pellet from a possible change in pH. Next, 600µL of phenol-chloroform was added to the tube; the tube was then mixed. The tube was placed into the centrifuge for 5 minutes; the top layer was then transferred to a new tube. The process from when the phenol chloroform was added to now was repeated. 150µL of 5M NaCl was added to the removed lysate. To precipitate the DNA, 700µL of isopropyl alcohol was added to the lysate; the tube was then inverted 10 times, stood for 10 minutes, and was centrifuged for 10 minutes. After centrifuging the supernatant was removed and 500µL of 70% ethanol was added. The tube was then centrifuged for 3 minutes, supernatant was removed, and the pellet was air dried. 50µL of Tris-EDTA was then added to the tube and mixed; 1µL of RNase A was added and the tube was incubated at 37 °C for 10 minutes. Finally, the tube was labeled accordingly.

Polymerase Chain Reaction

First, the primers that were designed for my reaction were distributed by the instructor and were resuspended with the appropriate amounts of sterile ddH2O. The stock solution was determined to have a final concentration of 200uM; it was later diluted in a different tube to have 200µL of a 20uM, which will be called the working stock. The amount of sterile ddH2O to add to each was then calculated to be: Gene TF=136.5µL, and Gene TR-163.0µL.

Figure 1.

The coding sequences below represent the coding sequence for the primers that were ordered for both the RAD23-TF and RAD23-TR genes to isolate the genomic DNA of both through PCR.

RAD23-TF (34-mer; Tm=54°C)

5’-caC CCt cga gaa gat caa cat taa gac ttt aaa g-3’

RAD23-TR (30-mer; Tm=54°C)

5’-cct agg tca tta ata cat aaa atc atc gtc-3’

The water was added after briefly centrifuging the tube. The working stock solutions were then created. The PCR reactions were now set up according to the calculations made in the Pre Lab5. Four reactions were set up; that is two genomic DNA with primers for each person. Our solutions were divided as follows: 1-DB gDNA, 2-DB gDNA, 3-SS gDNA, and 4-SS gDNA. The two letters before each reaction represent the initials of me and my lab partner. The master mix was then created, and as was the working stock; the components can be found in Figure 2.

Polymerase Chain Reaction (Continued)

Figure 2.

The following table shows the amount of each ingredient that was used to make the mixes for the four genomic primers as well as the master mix.

Final concentrationStock concentration Master Mix

1.0 µg (total) genomic DNA0.43 µL 2.32_ (calculated) 1.29 µL

0.2 μM sense primer1.25 μL 20 μMx 3 3.75uL

0.2 μM antisense primer 1.25 μL 20 μMx 3 3.75uL

1 unit (U) Phusion polymerase0.5 μL 2 U/μL x 3 1.5µL

1X HF or GC buffer (1.5mM MgCl2)10_ μL 5X concentrated x 3 30 µL

0.2 mM dNTPs 1_ μL 10 mM x 3 3 µL

Sterile distilled water 35.57μL x 3106.7 µL

______

FINAL VOLUME: 50 μL 150 μL

The correct amount of master mix was then pipeted into each tube; to make a total volume of 50µL in each tube. When this was complete roughly 48µL of master mix remained. The four tubes of primer were then placed in a thermocycler at the closest temperatures available to those calculated in Pre-Lab 5(can also be found in results). The thermocycler will then take the primer tubes through an incubation process that will heat up and cool the primers to amplify the DNA.

Agarose Gel Electrophoresis

Agarose gels in electrophoresis chambers were already in place for the beginning steps of this experiment. First, the comb and side walls of the gel covering were removed and 1X TAE was poured into the electrophoresis chamber until the top had only a thin layer over it. Sample dye (1µL dots) had been previously made by the laboratory instructor. The first slot that the comb made in the gel was filled with 5µL of 1 kb ladder. Next, 10µL of each primer was mixed with the dyes and placed in the gel slots. With the primers in place, 90 volts of electricity were then added to the electrophoresis chamber for 75 minutes. The sample dyed primers that were placed into the gel slots eventually came close to the middle of the gel. The gel apparatus was then visualized to determine the quality of the primer.

TOPO Cloning and E. coli Transformation

We began setting up the TOPO cloning reactions by adding the necessary components. The following represents the quantities that were calculated to be necessary for the TOPO cloning reaction. The amount of PCR to be used was determined by visually comparing the ladder key of base pairs (bp) with the SS-4 gDNA image that was taken in lab 7(Result 4-Figure 2). It was visually determined to be 250ng, which went through the following calculation: (250) x (10) x (0.1)=250ng. The amount of PCR product was determinedby 10µL250ng=0.04µL/ng. It was determined that 10 250=0.04ng; therefore 0.04ul/ng x 10ng=0.4µL; 0.4µL of PCR product will be added to the solution. The other solutes that were added to the TOPO mix are as follows: Salt Solution: 1µL and TOPO Vector: 1µL. Since the final volume of the solution must be 6µL, the amount of sterile water to was calculated as follows: 6µL-1µL-1µL-0.4µL=3.6µL of sterile water. After calculating and combining the solutes, the solution was mixed by pipeting up and down. The tube was then allowed to sit for 10 minutes at room temperature. Next, we thawed our 5mL tubes of E.coli for 5 minutes, after doing so we added 2µL of the TOPO solution made previously to the E. coli tube and gently mixed them with a pipet tip. The E. coli tube was then incubated on ice for 10 minutes. After incubating, the E. coli tube was heat shocked for 30 seconds in a 42 °Ctub of water and was immediently transferred to ice. After transfering to ice, 250µL of SOC Medium was added at room temperature. The tube was then placed in a horizontal shacking incubator for 50 minutes. Once the incubation was complete, roughly 20 sterile glass beads were added to two different plates. Now, 200µL and approximately 50µL of the TOPO mix were added to each plate. The plates were swirled with the beads to ensure that the solution was adequately spread.

TOPO Cloning and E. coli Transformation (Continued)

The beads were then removed from the two plates and incubated overnight at 37 °C.

Constructing a Plasmid Map and Restriction Enzyme Digestion Design

Plasma Maps are graphical representations of plasmids that show the major sites such as genes, plasmid names, and restriction enzymes. The first step was to retrieve the RAD23Tetrahymena Sequence and the plasmid sequence used to clone the PCR product from the online Blackboard resource. After acquiring these sequences, a gene construction program was opened to create the plasmid map. The program then opened a circular plasmid that had the RAD23 Tetrahymena sequence inserted into it. We next found the region where the gene will be inserted between the brown colored sequences where the sequence was blue and read CACC. This sequence matched part of the primer sequence made for our primers. The CACC was then highlighted and pasted into the gene sequence and the junction marker was highlighted and deleted. We next changed the gene sequence to green; and highlighted the intron sequences and copied them. The RAD23 sequence was then pasted in the gene construction program; all the introns were highlighted and changed to the color black. We then went back to the plasmid and highlighted the green and black regions. The gene

Constructing a Plasmid Map and Restriction Enzyme Digestion Design

(Continued)

construction kit was used to change the green and black regions to the ‘widest line’ setting on the plasmid map. The plasmid map was then saved as pENTR, TtRAD23. The previously saved plasmid map was used to mark the restriction enzyme sites found in the handout from Lab 7(see lab notebook for handout). A toolbar was opened and the program was set to ‘Commercial’ and each constriction enzyme was added. The program then displayed all the sites found in the plasmid that correspond to a certain palindromic sequence. The plasmid map was then evaluated and it was determined that the NHE1 restriction enzyme was the best for determining that the RAD23 gene was in the plasma. The NHE1 sequence was then highlighted and copied. The document type was then changed to Gel; the gel bands from the digest were pasted in. The size of the DNA was then changed by setting the threshold to 500 base pairs. The gel picture was saved as pENTR, TtRAD23, NHE1 digest. The gel was changed to a table and saved as pENTR, TtRAD23, NHE1 Table. All of the documents were then saved for future use and reference.

Plasmid Purification and Restriction Enzyme Digest

The first step in this lab was to grow the bacteria that would be used later in the lab. We began by taking 6ml of LB with 50µg/ml Kanamyacin in three different glass test tubes(six total, three per person). A LB/KAN plate was labeled pENTR, TtRAD23, DBSS; a grid was drawn on the plate that contained six numbered squares. We then used sterile six inch wooden sticks to take colonies from the colonies transformation plate and mark them on the grided plate. It must be noted for this experiment that we used colonies from lab students that were also assigned the RAD23 gene. The remaining material on each wooden stickwas mixed with the liquid in the LB/KAN test tube. The test tubes were then placed in the shacking incubator at 37 C overnight. Next, a total of eighteen 1.5ml tubes were labeled as 1A, 2A, 3A, 2A, 2B, 2C, etc. Culture was then pipeted into each tube with roughly 1ml left in the test tube. The tubes were centrifuged at maximum speed for three minutes. The supernatant was removed from each tube. We then added 250uL of buffer P1 to each A tube of the six sets. The pellet was then resuspended by pipeting up and down; after pipeting, the contents of each A tube were transferred to the B tube and resuspended. Finally, the contents of the B tube was transferred to the C tube and resuspended. We added 250µL of Buffer P2 to the C tube and inverted it to create a through mix. The solution then turned blue, but after gaining a through mix we turned the tube right side up and added 350uL of Buffer N3 to ensure the plasmid DNA wasn’t damaged. The tubes were then inverted until the solution turned clear and was homogeneous; and the tubes were centrifuged at maximum speed for 10 minutes. Pipets were then set to 900µL and used remove the supernatant (roughly 850µL) from the tubes and pipeted into QIA prep spin

Plasmid Purification and Restriction Enzyme Digest

(Continued)

columns. The QIA prep spin columns were then centrifuged for 1 minute and the flow through top of the spin column was removed and discarded. We next centrifuged the tubesfor 1 additional minute to remove any residual buffer. The column was placed in a new 1.5ml tube with 50µL of Elution buffer. The tube stood for 2 minutes, and was centrifuged for 1 minute; the column was then discarded. The tubes were placed on ice with the labels 1Rad23DB and 2Rad23SS. We now confirm the plasmid by inserting the plasmid PCR with restriction enzyme digest. The restriction enzyme was determined to be NHE1 from the previous lab and the restriction buffer to be used is NHE1 Buffer 2. It was also determined with the handout that the reaction will require a BSA. We then created a cocktail that included the following: 14µL of Buffer2, 1.4µL of BSA, 3.5µL of NHE1 Buffer 2, and 107.1µL of sterile water. We then pipeted 18ul of the cocktail in six tubes and labeled the tubes with DBSS, NHE1, and the plasmid number(1-6). We next added 2µL from the plasmid tube to the corresponding cocktail tube; which was incubated at 37 °C overnight. Finally, we determined the quality of the primers through Agarose Gel Electrophoresis (steps can be referenced in the Agarose Gel Electrophoresis Part of Methods) to determine if the samples will have the necessary qualities to be used in the future.