THE EXPERIMENTAL PRACTICE OF GAPDH GENE CLONING AND SEQUENCING IN SPECIES OF BASIL AND CILANTRO

THE EXPERIMENTAL PRACTICE OF GAPDH GENE CLONING AND SEQUENCING IN SPECIES OF BASIL AND CILANTRO Experiment Completed: November 29, 2011 Experiment Submitted: December 9, 2011 Genetics Lab Tuesday 11AM Matt Smith

The purpose of this experiment was to clone and sequence the GAPDH gene apparent in Basil and Cilantro plant species. GAPDH (Glyceraldehyde -3-phosphate dehydrogenase) is a primary enzyme utilized in the process of glycolysis. This gene has been shown to appear in the majority of plants, based on the fact that glycolysis is a primary function of life. In order to amplify this particular sequence, two methods of a polymerase chain reaction was used, including initial and nested, followed by purification. Negative results in the initial PCR suggested no amplification, however the nested and purified PCR results showed results. Pipetting errors were thought to be a potential cause of error. A blunt-end ligation was used for isolation and transformation was used to create a live culture of plasmid-inserted bacteria. Sufficient colonies were transformed and colonized using AMP/IPTG plates. Miniprep techniques facilitated the isolation of only the plasmid insert. This included the use of a BglII digest using particular restriction cut sites had found electrophoresis bands of 1000bp and 3000bp in the Cilantro plant, while three bands were found in the Basil plant. Overall success was determined by the successful amplification isolation of the gene, expressed through these methods. While not enough time was allotted for sequence analysis, an automation technique was the intended protocol for this sequence.

I. Introduction Cloning has provided immense advances in the world of genetics with the introduction of genetic engineering. It has been acknowledged in centuries past that one can manipulate and eventually reproduce particular plants simply through the process of replanting spawns (Seidel 2001). However, it has been found in recent decades that such genetic processes can apply to somatic cells, and in turn, animals. This, however, is a multi step process that involves much more than the simple seed. Since the first mammalian clone produced in 1990, the entire process has been observed on a more finite, molecular level. This can occur as it portrays itself on a bigger stage, such as a sheep (Seidel 2001). With this information comes an array of applications for the process. Uses range anywhere from cloning valuable animals/plants to commercialization of synthesized foods, and even transplantation of embryonic cells. Still, a large percentage of gene manipulation remains in research, and hopefully finding novel insights into cell function. According to Mulligan (2008), basic techniques of gene manipulation are used to grow a course of identical organisms, observing the efficiency of recombination. In bacteria, the plasmid is the central form of DNA, containing all genes. Due to its circular shape and ease of manipulation in the cell, the bacterial cell remains a popular medium for gene cloning. With this bacterial cell, one is able to insert fragments or genes of DNA onto this plasmid through DNA, creating subsequent recombinants (Mulligan 2008). This experiment will utilize bacterial cells in order to manipulate certain genes. Glyceraldehyde -3-phosphate dehydrogenase, otherwise known as GAPDH, is an enzyme used in the process of glycolysis (Figge and Cerff 2001). According to Figge and Cerff (2001), this gene has the ability to catalyze the two-sided inter-conversion between step 6 and 10 of glycolysis. GAPDH is seen within eukaryotes, eubacteria, and archaebacteria. However, most of these genes belong to one of two Gaps. It is believed that both gaps were originated from endosymbiotic gene transfers of certain bacterial descendants (Figge and Cerff 2001). This idea of gene transfer suggests the GAPDH gene can be manipulated and eventually recombined. Due to the importance of the gene in the role of glycolysis, GAPDH resides on the genome of all plants (Figge and Cerff 2001). While individual nucleotide sequences may vary, the end result remains the

same. GAPDH stands as an accessible gene that can be utilized for certain gene transfer processes. This is especially true when considering plants. All plants contain the GAPDH gene within the genome, thus giving them the drive to undergo glycolysis. Therefore, when considering gene manipulation and cloning, plant provide an abundant source of this particular gene across an array of species. The following experiment will seek to amplify a certain region of DNA encoded with this gene in order to observe the transfer of DNA fragments through cloned cells. Since plants will be used in the experiment, the plant sources used will optimistically possess the highest yield of DNA in order to conduct the experiment with success. Although without research, one cannot determine the amount of contained DNA from a simple phenotype. The focus will shift to the individual sample from each species taken instead of the species chosen. Since plant cells divide and grow most evenly soon after sprouting, the DNA contained in young leaf samples will be just as abundant, and remain far from plant death. As plants maturate and begin to die, the DNA lyses itself with the help of endonucleases. This can be seen on larger, discolored leaves that possess more of a brittle texture. Therefore, young, healthy plant samples would be beneficial for improving the yield of DNA for the experiment. In this experiment, two plant species will undergo a multistep process utilizing a number of different methods. One of the main techniques used is a polymerase chain reaction (PCR). This is a scientific technique used to amplify a particular piece of DNA, generating millions of copies of a DNA sequence (Joshi, 2010). This reaction has become a powerful technique used internationally in molecular biology due to its quick and inexpensive nature. Efficacy of a PCR reaction is measured by specificity, yield, and reliability of final results (Cha, 1993). Technique can go through one of two methods with separate purposes. Initial PCR carries out initial replication of target DNA, while nested PCR is a more specific modification of the prior technique, focusing on reducing product contamination seen from binding sites of unanticipated primers (Joshi, 2010). Utilization of the PCR reaction ultimately increases efficacy of target sequence. This experiment will begin by extracting plant sample DNA using spin columns and centrifugation. The obtained DNA will undergo two steps of PCR for amplification and purification and eventually tested on an electrophoresis gel. Using a blunted ligation, the

PCR products will be ligated and transformed with bacterial cells on an agar plate. This plasmid DNA will meet a restriction digest analysis and second purification. Once combined with sequencing primers and mailed to a sequencing facility, the purified plasmid sequence will be analyzed using Geospiza web portal. My hypothesis states that Basil and Arabidopsis will exhibit very similar DNA sequences based on the presence of the GAPDH gene in both plant species. Overall success of the experiment will be determined by the effective amplification of target GAPDH sequence using a number of described techniques, primarily, polymerase chain reaction. II. Methods DNA Preparation This study was conducted using two chosen plant species of Basil and Cilantro. Young, healthy leaf samples were obtained from each plant and ground with added lysis buffer and micropestle. Centrifugation pelleted material to the bottom of the microcentrifuge tube, leaving the supernatant for collection and dilution with 70% ethanol. Eluate was spun through capless collection tubes and columns. DNA was washed with wash buffer multiple times, and spun again to dry material. Addition of sterile water to the column bed was followed up by quantification of sample DNA with the NanoDrop program. Initial PCR 5 PCR tubes were labeled with appropriate solutions: 1-negative control, 2- Arabidopsis gdna, 3-positive control, 4-Basil, and 5-Cilantro. Blue Master-Mix containing initial primers was added to each tube, along with sterile water. A small sample of the DNA assigned was added to the appropriate PCR tubes and mixed with the pipette. These were placed into a thermal cycler, and eventually into a -20 C storage area. A 1% agarose gel was prepared with included GelStar. Small samples of each PCR tubes was placed into the wells of the gel, along with obtained ladder. The electrophoresis gel was run at 250V for 20 minutes.

Nested PCR Using previous prepared samples of DNA, exonuclease was added to fresh PCR tubes with each PCR samples. Once mixed with the micropipette, the exonuclease mixture was incubated at 37 C for 15 minutes. This was transferred to an 80 C water bath for another 15 minutes. After incubation, sterile water was placed into each tube for dilution. A small sample of this diluted mixture was placed into fresh tubes and centrifuged. Prepared samples were combined with Yellow Master-Mix, and samples taken from the assigned template. Once again, samples were placed into a thermal cycler and stored in 20 C. Purification of PCR Products Utilizing a process known as Size-Exclusion Chromatography, a PCR Kleen Spin column was used to purify obtained nested PCR products. This column contains beads with specific size pores to separate larger and smaller molecules of a mixture. Beads in the column were suspended and forcibly flicked them back down with a downward flick. Nested PCR products were placed on top of the beads in two columns, and centrifuged, disposing of everything but the column. Using a 1% agarose gel, nester and purified PCR products were checked for apparent bands using electrophoresis. Samples were set to run at 250V for 20 minutes. Ligation/Transformation Ligation has the ability to take the form of sticky-ended or blunt-ended methods. A blunt-end ligation was chosen for this experiment due to ease of cleavage, and high binding efficiency due to a lowered cost of primer synthesis. A blunting reaction was prepared with the following reagents: 2XLB (buffer), distilled water, purified PCR products, and proofreading polymerase. This solution mixture was incubated at 70 C and transferred to ice for cooling. A ligation reaction was prepared with reagents of T4 DNA Ligase and pjet1.2 blunted vector, which was combined with the blunting reaction. E.coli cells were chosen for this transformation and placed together with the ligation reaction. Cells were incubated on ice, then heat shocked at 42 C, and put back

on ice. SOC was placed into each tube and spread onto the corresponding AMP/IPTG plate and incubated at 37 C for 24 hours. Miniprep/Restriction Digest Utilization of the miniprep process allows isolation of the plasmid of interest. This was conducted by first creating a live culture of the (hopefully) transformed bacteria. Centrifugation pelleted bacteria, which allowed the supernatant to be removed, containing the plasmid. Lysis solution was added and mixed by inversion. A neutralization solution was consequently added after a period of 3 minutes to combat the added lysis, and then centrifuged, collecting a pellet. The supernatant was added to an appropriate column and spun, eventually washed with wash buffer. After a dry centrifuge run, elution solution was added on top of the column. Columns were spun, discarded, and capped only the tubes with the minprep plasmid DNA, which was stored at 4 C short term. Restriction digest was used to confirm insertion of the fragment into the host plasmid. BglII Master-Mix was added to the digestion tubes, along with miniprep DNA. Centrifugation collected materials and was then incubated at 37 C for 1 hour. A 1% agarose gel was used to check the digest. Sequencing Small samples of the obtained gene plasmids were combined with the appropriate primer needed for sequencing. The template is as follows: Basil-pJET Forward Cilantro-pJET Forward Basil-pJET Reverse Cilantro-pJET Reverse Basil-GAP Forward Cilantro-GAP Forward Basil-GAP Reverse Cilantro-GAP Reverse Upon combination of the samples with intended sequencing primers, samples were sent off to a sequencing facility, which used a technique of automation sequencing.

III. Results The GAPDH gene used in this experiment was ultimately cloned in order to analyze sequences of the particular gene fragment. Through sequential steps leading to the end sequence, success was determined numerically and physically at each step. gdna Extraction Upon retrieval of the plant material, the genomic DNA of both the Basil and Cilantro was extracted through the use of silica spin columns, collecting the purified DNA. Pelleted DNA was tested using the Nano-Drop system, recording results shown in Table 1. Positive results of notable DNA indicate the successful extraction of DNA from the plants. Initial mass of each plant sample varied slightly, thus wavering the difference in DNA concentrations for each plant. Initial PCR The initial PCR set-up utilized a 2x blue master mix along with each of the 5 sample lanes. Electrophoresis was the test of choice to test success of the procedure, indicating any presence of the correct bands at the correct base pair levels. Using a 500 base pair ladder, the gel image in Figure 1 confirms a bright band in lane 3 and even brighter band in lane 4. Lane 3, containing the positive control gdna, generated a band size of 1000 base pairs. This control acted as a check within the experiment to ensure proper plasmid activity. Lane 4, containing the pgap GAPDH gene, also generated a 1000 base pair band, at an even brighter and more concentrated brightness. Experimental samples of Basil (5) and Cilantro (6) generated no apparent band, of any size. Nested PCR/PCR Purification The nested PCR procedure hoped to increase the amount of target sequence, as well as ensure specificity of the target. Following a similar procedure, but instead used a 2x yellow master mix with nested primers. Along with these set-up nested PCR products, were the purified nested PCR products. Electrophoresis was once again the test of choice, portraying bands shown in Figure 2. Using a 500 base pair ladder. Bands were generated in lanes 2-7. Lane 1 (negative control) generated no band, as expected. Bold bands were

expressed in lanes 3-5, and 7-8. Both nested PCR products (P1, P2) and purified nested PCR products (PP1, PP2) generated bold bands at the 1000 base pair level. Dark bands were apparent in these described lands. Lane 6 generated two faint bands at the same base pair level. Ligation/Transformation The insertion of the purified PCR product into a sufficient vector (plasmid) was chosen to utilize the pjet plasmid. The purified fragment was inserted to the pjet plasmid through blunt-end ligation in which the ligase added two end phosphodiester bonds to link the product to the plasmid. Eco471R, coded for a restriction enzyme, provided a layer of selection in the ligation process (Figure 3). Using AMP/IPTG selection plates, and competent E.coli cells, the ligated product was plated and transformed overnight, producing total colonies of 16 for Basil and 11 for Cilantro (Table 2). Transformation efficiency was calculated for each of the samples, resulting in efficiencies seen in Table 2. Hoping to see numerical data of similar value, the efficiencies varied by about 10,000 colonies/µg DNA. Miniprep/Bgl II Digest The plasmid of interest was isolated through a miniprep process and inserted into a new host. Success of the procedure was tested on an agarose gel through electrophoresis. Figure 4 expresses both bands of 1000 bp and 3000 bp sizes. Using a 500 base pair ladder, Lane 2 (P1) expressed a band at 3000 bp, but generated two more bands, one showing a size of 500 base pair. Lane 3 (P2) exhibited expected results with bands at the 1000bp and 3000 bp size. Sequencing Directing protocol from Table 3, GAPDH gene plasmids were combined with sequencing primers before sending off to the sequencing facility.

IV. Discussion Success of this experiment was contingent on a number of factors and techniques utilized in the described protocol. Although several methods were used, overall success was thought to be determined primarily by the successful amplification of the target DNA sequence of GAPDH using necessary PCR reactions. With the outcome of the experiment determined by early processes, the techniques used were done so in a careful manner. Plant samples of Basil and Cilantro were collected based on overall age and health. Younger leaves were preferred since their cells would still be in the process of growing, ensuring lively cells full of DNA needed for growth. The initial PCR process tested the quality of DNA. An electrophoresis gel, however, did not show any bands for each of the plant samples. This suggested that no DNA was amplified, however a possible source of error may have been regarding the loading of the gels. The micropipette could have released a portion of the sample into the buffer; possibly creating a band that was too faint to see without adequate amount of sample solution. The nested PCR technique generated a gel image with expected solid bands of the positive/negative control and Arabidopsis sample. Interestingly enough, both plants had generated bands of the nested and purified PCR technique. This ensured amplification had in fact been conducted on the target sequence. Ligation and transformation using E.coli cells had provided positive results with sufficient colonies collected. Miniprep procedure had been completed successfully, with the isolation of the plasmid. The BglII digest results were apparent on an electrophoresis gel. The sample of Cilantro DNA sequence had produced two expected bands. Basil had produced three bands, one of which was faint in brightness. This was most likely due to the presence of another BglII restriction site. This created an additional cut site, which generated three pieces of DNA instead of the intended, two. With the presence of purified bands on this gel, proper DNA sequences had been prepared well enough to be sent off to a sequencing facility. There was not enough time, however, to receive and analyze the proper sequences. If more time was allotted, future studies could observe the sequence of each plant DNA as compared to the sequence of Arabidopsis. Expected results would show these plant species are very similar in terms of nucleotide sequences.

References: Cha, R.S., and Thilly, W.G. 1993. Specificity, efficiency, and fidelity of PCR. Journal of Genome Research. 3: 18-29. Figge, R., Cerff, R. 2001. GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity. Journal of Molecular Biology and Evolution. 28(12): 2240-2249. Joshi, M., and Deshpande, J.D. 2010. Polymerase chain reaction: methods, principles, and application. International Journal of Biomedical Research. 1(5): 81-97. Mulligan, P. 2008. Genetic engineering. AccessScience [serial online] 1-12. Seidel, G. 2001. Cloning (genetics). AccessScience [serial online] 1-9.

P1 P2 1 2 3 4 5 6 7 8 1000bp 1000bp 500bp Figure 1. Electrophoresis Gel exhibiting amplification of particular genomic regions through an initial PCR reaction of both Basil and Cilantro Plant Samples. A 500 base pair ladder was used in lane 1. P1 portrays Basil, while P2 represents Cilantro. Lane 2 represents a negative control, Lane 3 represents Arabidopsis gdna, Lane 4 portrays a positive control, Lane 5 represents Basil plant sample, and Lane 6 represents Cilantro plants sample. DNA samples were combined with blue master mix. Agarose gel was run at 250 V for 20 minutes.

L A + P1 P2 PP1 PP2 1 3 5 7 9 11 13 15 1000bp 500bp Figure 2. Electrophoresis gel expressing amplification of nested PCR products and purified nested PCR products of both Basil and Cilantro plant samples. A 500 base pair ladder was used. P1 represents Basil, while P2 represents Cilantro. PP1 and PP2 represent purified PCR products of the two samples. Negative (-) and positive (+) controls are portrayed in lanes 3 and 7 respectively. Arabidopsis (A) is portrayed in lane 5.

Not I Eco52 I Bgl II Kpn2 I PsoX I Eco88 I Xho I Xba I Bgl II Btg I Eco130 I Nco I Bsu15 I bla(amp R ) pjet 1.2 2.974 bp Eco471R Rep(pMB1) Figure 3. pjet 1.2 blunted vector with incorporated Eco471R coding for restriction enzymes. Several restriction enzymes are listed in green section including BglII, the enzyme of interest. Bla(amp r ) and Rep(pMB1) are incorporated as transcriptional regulators of the plasmid in the cell.

P1 P2 1 2 3 4 5 6 7 8 3000bp 1000bp 500bp Figure 4. Electrophoresis gel of restriction digestion protocol for both Basil and Cilantro plant samples. A 500 base pair ladder was used. P1 portrays Basil, while P2 portrays Cilantro. Agarose gel was run at 250 V for 20 minutes.

Table 1. Concentration of genomic DNA samples taken from plant species using Nanodrop. Plant Name Part of Plant Weight 1 Concentration of DNA 2 Basil Leaf 0.096 82.7 Cilantro Leaf 0.105 99.2 1 =milligrams (mg) 2 = ng/µg

Table 2. Transformation and ligation of a purified PCR product containing GAPDH using the pjet plasmid. Plant Name Colonies Observed Transformation Efficiency 2 Basil 1 16 33,927 Cilantro 1 11 23,325 1 =AMP/IPTG selection plates 2 =colonies/µg DNA