Cloning and Sequencing of a portion of a Capsicum chinense x Capsicum frutescens hybrid ghost pepper’s GAPDH gene (GAP C)

Introduction

Background Information

The primary project of my Molecular Biology Techniques class, also known as BITC-2441, was to clone and sequence a plant chosen by student pairs to learn the genetic sequence of the glyceraldehyde-3-phophate cytosolase (GAPC) gene. This paper contains the results from ghost pepper, also known as ‘Bhut Jolokia’. Fresh ghost pepper was crushed, measured and a lysate generated following resuspension in buffers. 2 sequential PCR reactions, the first using degenerate primers and the second using primers directed against a highly conserved region of the GAPC gene, were done to amplify regions of GAPC. The expected DNA band was detected with gel electrophoresis. This PCR product was purified using size exclusion chromatography and spliced into E.coli HB101 plasmid. This plasmid was isolated from the bacteria and analyzed with gel electrophoresis. DNA samples were then Sanger sequenced using pJET (plasmid-specific) and pGAP (gene-specific) primers. The sequence was analyzed with Geneious Prime bioinformatics software.

Ghost pepper originates from northeastern India, a region known for its wide variety of extremely hot peppers and spicy cuisine. Other varieties include ‘Naga Jolokia’ and ‘Bih Jolokia’ (Bosland et al., 2007. 1). It is a cross between Capsicum chinense x Capsicum frutescens and has a Scoville Heat Unit (SHU) of 1,001,304. The cultivar is relatively well understood owing to a great deal of attention paid to it (Bosland et al., 2007, 1) by hot pepper aficianados.

The lab is trying to compare the Ghost pepper GAPC gene with that of other plants. There are many steps to cloning and sequencing genes of a plant. The primary reason the lab is important is due to contributing to the knowledge about GAPC within plants, in this case in ghost pepper. GAPC is a well-known housekeeping gene sequence within the Glyceraldehyde-3-phophate dehydrogenase (GAPDH) family that is conserved across many plant species, making it ideal for research (Bio Rad.com). Moreover, the portion of GAPC to be amplified encodes for two-thirds of the gene, including the enzyme’s active site. GAPC encodes for an NAD⁺-dependent cytostolic GAPDH (Bio Rad, 18). Furthermore, the GAPC gene of ghost pepper has never been sequenced before in BITC 2441 at Lone Star College.

Purpose

The purpose of this experiment is to clone and sequence the GAPC gene of ghost pepper using primers directed highly conserved GAPC sequences.

Hypothesis

If the instructions of the protocol are followed, the GAPC gene from ghost peppers will have more sequence similarities to the gene from closely related plants compared to more distantly related species.

Methods and Materials

Extracting gDNA

Prior to extraction, a ghost pepper plant was kept in the dark for 24-48 hours to maximize the cellular DNA. 200 ul of lysis buffer was added to a 1.5 ml microcentrifuge tube. Ghost pepper leaves were added to the lysis buffer and micro pestles were used to crush and lyse the cells. After a homogenous mixture was obtained, an additional 500 ul of lysis buffer was added. The tube was then centrifuged at full speed for 5 minutes. Afterwards, 400 ul of supernatant was removed and added to 500 ul 70% ethanol to create plant lysate. 800 ul of the resulting plant lysate supernatant was then added to a mini-column and centrifuged again. 700 ul of wash buffer was added and centrifuged and the resulting flowthrough was discarded. The wash process was repeated two more times. The column was transferred to a new microcentrifuge tube, 80 ul of 70C of sterile water was added and held for 1 minute to and then centrifuged. The eluate containing DNA was then collected and stored.

Amplifying GAPC region and assessing results with Gel Electrophoresis

To carry out the PCR with degenerate primers, the PCR tubes were put on ice and 20 ul of 2x Master Mix with Initial Primers (MMIP) was pipetted into each tube. Afterwards 15 ul of sterile water was added to each tube. Using a fresh tip each time, 5 ul each of negative control, pGAP, ghost pepper, Celosia, and Arabidopsis DNA templates were added to the tubes. The tubes were placed into a thermal cycler for 1 round of initial PCR.

This amplified gDNA was prepped for nested PCR by treating with 1 ul of exonuclease I to destroy the leftover degenerate primers from the initial reaction. The template DNA was incubated at 37C for 15 minutes and then 80C for another 15 minutes to heat-inactivate the exonuclease I enzyme. 98 ul of sterile water was added and an additional 2 ul of the appropriate initial PCR into the tubes was added. The tube was then vortexed and placed on ice to keep cool. 20 ul of yellow master mix (2x MMNP) was pipetted into each PCR tube. The tubes were placed into the thermal cycler.

The results of both rounds of PCR were separated through agarose gel electrophoresis in a 1% gel. The gel lanes were run according to Figure 2: Lane 1 was loaded with the 500 base pair molecular ladder. 5 ul of gDNA and 1 ul of loading were loaded onto lanes 2 and 3. Lanes 4 and 5 were loaded with 20 ul of initial PCR reaction was transferred into a new tube along with 2 ul of loading dye. 5 ul of nested PCR and 1 ul of loading dye were added to lanes 6 and 7. Lane 8 was loaded with 20 ul of Arabidopsis gDNA and 2 ul loading dye were added to act as a control.

Purifying and ligating PCR product into a plasmid vector

The products of the nested PCR reaction were used for cloning of the amplified GAPC sequence into a sequence vector. Beads in the PCR spin column were resuspended. The cap of the spin column was snapped off and the column centrifuged for 2 minutes. 30 ul of yellow nested PCR reaction was added onto the column bed. The column was centrifuged for 2 minutes and supernatant collected.

The resulting supernatant with the amplified DNA were combined with 2x ligation reaction buffer, proofreading polymerase, 2 ul of blunting reaction and 1.5 ul of sterile water. The tubes within were centrifuged to force contents to the bottom and incubated in the thermocycler for 5 minutes at 70C, then cooled on ice for an additional 5 minutes. Centrifugation was applied to force contents to collect at the bottom. 9.0 ul of blunt reaction was added, followed by 0.5 ul of T4 DNA ligase and 0.5 ul pJet 1.2 blunted vector. The reaction was centrifuged to collect contents at the bottom of tube. The tube was incubated at room temperature for 5-10 minutes. The reaction tube was then stored at -20C until further use.

Transformation

10 ul of E. coli HB 101 was streaked via the quadrant method onto a LB plate. This was then incubated at 37C overnight and then stored at 4C until the day of the lab.

1.5 ml of C-growth medium was pipetted into a 15 ml culture tube and warmed for 10 minutes at 37C. 150 ul of fresh starter culture prepared from the starter plate was then pipetted into the C-growth medium. Afterwards, 250 ul each of transformation reagents A and B from BioRad were combined into a separate microcentrifuge tube, kept on ice. After a 20-40 minute incubation, the C-growth culture was transferred to a new microcentrifuge tube. This was centrifuged at 17,000 rpm for 1 minute and put on ice. The resulting supernatant was removed without disturbing the bacterial pellet. This pellet was resuspended with 300 ul of cold transformation buffer. It was then incubated on ice for 5 minutes and immediately centrifuged for 1 minute. The supernatant was removed. 120 ul of cold transformation buffer was added and kept on ice for 5 minutes. The cells were now competent for transformation.

5 ul of ligated plasmid were added to the 50 ul competent cells and mixed by pipetting twice. The tube was incubated on ice for 10 minutes. The entire volume of transformation was pipetted onto an LB Amp IPTG plate warmed at 37C. An inoculation loop was used to gently spread the bacteria around the plate. It was immediately placed upside down in an incubator at 37C until the bacterial colonies were apparent. This plate was used to inoculate four tubes of LB/AMP broth (20 ml LB broth and 100 ul ampicillin).

Isolating Plasmid from Bacteria

1.5 ml of miniprep culture of transformed E. coli was transferred into a microcentrifuge tube and centrifuged. The supernatant was removed without disturbing the pellet. This process was repeated two more times. 250 ul of resuspension solution was added and the mixture vortexed. Another 250 ul of lysis buffer was added and the tube inverted 6-8 times. 350 ul of neutralization solution was added within 5 minutes and then gently inverted 6-8 times. The tube was centrifuged for 5 minutes. The resulting supernatant was added onto a spin column spun for 1 minute at top speed. The flowthrough was discarded. Subsequently, 750 ul of wash solution was added to the column and centrifuged for 1 minute. Flowthrough was discarded and the column was centrifuged for 1 minute to dry. The column was transferred to a new tube and 100 ul of elution solution was added to the column. The solution was allowed to settle for 1-2 minutes. The column was centrifuged for 2 minutes. The column was discarded, and the sample capped. The miniprep DNA was stored at -20C until further use.

Miniprep DNA was analyzed by restriction digestion with Bgl II. 10 ul of plasmid DNA was combined with 10 ul 2x Bgl ll master mix (2 ul 10x Bgl II reaction buffer, 7 ul sterile water and 1 ul of Bgl II enzyme). Reactions were incubated at 37C for 1 hour. The samples were analyzed by 1% agarose gel electrophoresis: 10 ul of 500 bp molecular ladder was added to Lane 1. 5 ul of undigested DNA was combined with 15 ul of sterile water in a separate tube to compare digested DNA.1 ul of 6x loading dye was added to each of the digested and undigested samples. Immediately afterwards, 20 ul of each DNA sample was added to each well. The gel was run at 100 Mv and the results are shown in Figure 5.

Sequencing DNA and performing bioinformatics

10 ul of miniprep DNA were combined with 1 ul of each sequencing primer. The mixture was added to assigned wells on the 96-well plate. The plate was sent to BioRad for Sanger sequencing. The resulting sequence data were analyzed with Geneious Prime bioinformatics software.

Results

DNA Preparation

Figure 1.a shows the yield of crushed ghost pepper plant leaf material was prepared from ghost pepper leaves. Figure 1.b shows the 60-80 ul of genomic DNA solution extracted from the DNA material.

GAPDH PCR

The results are shown in Figure 2 from left to right. PCR was done at an early stage to determine the presence of GAPDH genes in the various products and Arabidopsis was utilized to further measure the presence and intensity of PCR products since it has been thoroughly sequenced in many laboratories.

The LB AMP IPTG plate below was inoculated with competent cells that had the PCR products inserted into them. Colonies were selected that could grow in Ampicillin-indicating uptake of plasmid ampicillin gene and GAPC gene. However, to maximize the chances for colonies to grow, Tube 1 and 3 were inoculated with colonies from my own plate while 2 and 4 were inoculated with a different plate’s E. coli. 1 and 3 were the ones showing signs of heavy and cloudy growth. Tubes 2 and 4 were clear and showed no signs of growth. Inoculated 2 and 4 again with plate this time. Nonetheless, the plates that were inoculated again showed no growth, indicating that the previous cells used up all nutrients and the new inoculation was unable to grow and take hold.

PCR for Bgl II enzyme

Restriction digestion of GAPC construct was performed, with results shown in Figure 5. Digestion with Bgl II enzyme excised the PCR fragment. A single band shows that the enzyme was successfully digested. The presence of two bands in Lane 4 indicates the size of the vector and size of inserted DNA.

Bioinformatics sequence analysis

The sequences were sent away to be sequenced. Afterward, Geneious Prime bioinformatics software on Windows 10 was used to . The current results are shown in Figure 6 below and how far into sequencing and what needs to be done for a more complete picture.

One way to gain a more complete understanding is to compare the sequences that are most similar to the ghost pepper GAPC sequence with those lower in the list. As can be seen above, there were 5 results with an E value of 0. All of those were from the Solanum family.

Discussion

Conclusions

The top 3 E-value results, which all have values of 0 indicating a perfect match, are Capsicum frutescens glyceraldehyde-3-phosphate dehydrogenase (GAPC), Solanum pinnatisectum, and Solanum melongena glyceraldehyde-3-phosphate dehydrogenase. The genus that the GAP C sequence shares the highest identity with is that of the Solanum family. Members of this family include tomatoes, potatoes, and eggplants.

An interesting thing to note is that no other pepper species were returned, indicating that the GAPC of this pepper species differed significantly from other previously sequenced pepper species.

Explanation of Conclusions

Several factors may explain these experimental results. The first two rounds of PCR, initial and nested, focused on amplifying portions of GAPC gene from gDNA of ghost pepper. With initial PCR a set of blue primers using multiple degenerate (less specific) sequences were utilized to amplify the GAPC gene from the gDNA. Exonuclease I was used to remove the primers from the initial PCR so that they do not amplify the target DNA. The enzyme was then inactivated. Then in the second round of PCR (nested) a more specific set of yellow primers were used to amplify the GAPC from the initial products. The PCR products from the initial round now contain a high proportion of GAPC-like sequences relative to the amount of gDNA. The results are shown in Figure 2. A 500 bp molecular ladder was used to measure target DNA. The gDNA show the brightest bands, which indicate the amount of DNA extracted from the first step. The initial PCR shows the denaturing of the DNA while the nested bands show that the amplification from the specific set of primers were successful. Arabidopsis was added because the plant also contains a GAPC region and yielded a single bright band as expected.

Bgl II sites were cut to separate insert DNA from plasmid DNA. Figure 5 demonstrates the results for Bgl II PCR. If the isolated plasma contains the insert of interest then there would be two bands, one containing excised PCR fragment and another with the vector DNA. If there is only one band in an unintended lane, then the digest was unsuccessful.

Works Cited

Bosland, P., and Baral, J. (2007) ‘Bhut Jolokia’-The World’s Hottest Pepper Known Chile Pepper is a Putative Naturally Occurring Hybrid. HORTSCIENCE 42(2):222–224.

Bio Rad. (n.d.). Cloning and sequencing explorer series. BioRad.com. Retrieved March 23, 2022, from https://www.bio-rad.com/en-us/product/cloning-sequencing-explorer-series?ID=43739f03-3540-4719-b42f-1809e2199b9f

Bio Rad. (n.d.). Bioinformatics supplement – bio-rad.com. Bio Rad. Retrieved March 24, 2022, from https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lse/literature/10040011.pdf