The experiment extracted, cloned, and sequenced the GAPC gene from thyme plant DNA inserted into E. coli plasmid vectors. Nested PCR and gel electrophoresis showed the thyme DNA was suitable for cloning. Restriction digest confirmed the E. coli plasmids accepted the GAPC insert. Sanger sequencing determined the plasmid sequences, which BLAST analysis found were nearly identical to the known GAPC gene sequence, indicating the experiment successfully cloned and sequenced the thyme GAPC gene.

1. The Extraction, Cloning, and Sequencing of the GAPC gene from Thyme DNA using E. Coli cells as a Plasmid Vector
Lance Rhyne, Hailey Medder, Madison Rost
Biology Department
130 Natural Science Building, WCU
Cullowhee, NC 28723
Abstract
Introduction
Results Conclusion
Acknowledgements
References
The goal of this experiment was to extract and clone the
GAPC DNA segment from Thyme DNA and sequence this
segment to determine whether or not the experiment was
successful. Based on the Nested PCR gel electrophoresis,
Thyme was chosen as the DNA genome to be further tested for
cloning. The restriction digest gel showed that the E. Coli
plasmid did accept the GAPC insert and cloned the DNA
sequence multiple times. The first BLAST was completed to
clean the DNA sequence by trimming the ends of the
chromosomes and vectors as well as eliminate the primers. The
final BLAST ran on the contig of the sequenced DNA
removed discrepancies between the known and verified bases
of the chromosomes to improve the quality values. Overall,
this experiment was a success based on the results as the
GAPC gene was extracted and cloned from Thyme to the E.
Coli plasmid as the gene is present in the DNA results from the
sequenced products. Based on the E-value score of 0 and grade
of 97.7%, it is safe to assume that the sequence obtained from
the Thyme is nearly identical to the original GAPC sequence.
The GAPC sequence from the plant Thyme was cloned and sequenced as a
plasmid inserted into E. Coli cells using DNA extraction, PCR, gel
electrophoresis, and Sanger Sequencing. DNA extraction separates DNA
from cell components and cell contamination but every cell type has
different obstacles extraction must work around. Extracted DNA should be
purified after removed before PCR or other experiments occur to remove
any additional molecular contamination. Silica-based purification methods
were used to purify the extracted gDNA from the Thyme using beads with
different pore sizes to prevent breakage of the large molecules. PCR or
Polymerase Chain Reaction was then used to create multiple copies of the
specific section of DNA (2) through a repeated process of DNA
denaturation, elongation, and annealing utilizing heat and Taq Polymerase.
Nested PCR is a type of PCR, also utilized, that completes two rounds of
PCR with different primers to increase the specificity of the desired genes.
These PCR products were then used to form plasmids. Plasmids are
separate DNA segments that replicate independently, away from
chromosomal DNA (2) that act as agents for the transfer of genetic material.
In order to complete the plasmid transformation process, selection of the
transformed colonies (Fig. 2) and further purification of the DNA was
conducted. The transformation process required growing E. Coli cells that
could accept the transformed plasmid containing the pJET 1.2 plasmid
vector by pelleting, chilling, washing, and, finally, resuspension. (2) The
cells that contained and accepted the transformed plasmid survived and
grew on media plates which provided colonies of ideal E. Coli samples. To
ensure the plasmid’s acceptance and reproduction, gel electrophoresis was
run to allow the PCR products to be checked for additional DNA segments
or bands in the sequence. Finally, Sanger sequencing was used to determine
the plasmid sequence via chain termination of single stranded DNA
templates mixed with dideoxynucleotides to determine the sequence of the
segment. Overall, the purpose of this experiment was to sequence the
GAPC Segment of plant DNA from Thyme leaves and stems while
providing a better understanding of how DNA cloning and sequencing
functions. Final results for this experiment should show the sequence from
the bacteria plasmid that contain the Thyme GAPC sequence with high
quality base pairs which will be present in the BLAST sequence generated
via Geneious 8.1.9.
Based on the results, this experiment was a success as the GAPC gene
was extracted from the Thyme plant, cloned, and inserted into the E. Coli host
cells. When looking over the Nested PCR electrophoresis gel, one can easily
identify the smudged band. The misshapen band was due to the well either being
pierced while pipetting the sample into the well or the well was made
extraordinarily thin. As always, possible contamination of the sample could have
occurred as well from surrounding particles or improperly cleaned equipment.
The culture samples that grew also proved that the plasmid was correctly
transferred into the E. Coli cells because the inserted plasmid sequence enabled
the cells to live in the conditions made by the plates. The plates contain
ampicillin that normally kills E. Coli cells; so, only cells that up took the plasmid
would survive as these cells would have an ampicillin resistant gene in their
genome form the plasmid they accepted. The plasmid also disrupted the eco471R
gene in the E. Coli that normally acts as a toxin to the cell also allowing the cell
to grow and multiply with the plasmid influence. The restriction digest
electrophoresis gel indicated that the primer added to the plasmid insert sequence
was present via the presence of only two bands. The two bands present meant
that the primers worked efficiently due to them only being able to cut at specific
nucleotide bases along the plasmid insert sequence. The BgI II site may also
cause a band to occur if the restriction digest runs incorrectly but as there are
only two bands on this gel, the restriction digest of the E. Coli cells and plasmids
ran properly and proved that the insert and plasmid were successfully inserted
into the bacteria cells.
Table 1 shows the top three hits for the pJET SEQ F, pJET SEQ R,
GAP SEQ F, and GAP SEQ R primer coverage of the GAPC gene. These genes
have been cleaned and trimmed so all that remains is high quality base pairs for
later modification. Based on the very first top hit for each primer the gene
samples are of high quality and all contain similar sequences from the
Arabidopsis thaliana chromosome 3 sequence. All of the E-values for the
Arabidopsis thaliana chromosome 3 sequence are zero and all the pairwise
identity percent's are above 97% meaning that the suspected GAPC gene in these
sequences is close to being identical to the literature value of GAPC. Table 2
shows the top five hits for the second BLAST run on the combined gene
sequence contig. This sequence was further modified by fixing any discrepancies'
between base pairs to ensure the highest quality available was achieved. Based
on the top hit Arabidopsis thaliana chromosome 3 sequence which actually
contains a GAPC sequence.
Figure 1. Nested PCR
The negative control generated no band and the pGAP plasmid
did generate a band indicating the successful preparation and
lack of contamination in these samples. The gDNA formed a
long column of dye suggesting there was an error in
preparation of this sample. Based on this gel, the Thyme was
chosen for further experimentation with cloning and
sequencing. Estimated band lengths: Thyme-1,000 bp,
Maleberry-1,000 bp, gDNA-unidentifiable bp, pGAp-1,000 bp,
and Sterile Water- 0 bp.
Figure 2. E. Coli Cultures
Both plates contain E. Coli cells that received the pJET 1.2 plasmid. Each plate
did have bacterial growth meaning the plasmid was accepted by the cells.
Figure 3. Restriction Digest
This gel provides evidence that the pJET 1.2 plasmid and insert were
accepted by the E. Coli cells, I,e, successfully transformed E. Coli cells.
There are only two bands present on this gel, one for the plasmid at 3,000
bp and one for the insert at 1,000 bp meaning that there was no
contamination on the gel from the BgI II digest or other molecules.
1. Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts,
Walter (2014) DNA Cloning by PCR, Manipulating and
Analyzing DNA Molecules, Essential Cell Biology Ed. 4,
2014, pgs. 327-330, 335-338. Garland Science, Taylor &
Francis Group, LLC, New York, New York.
2. Western Carolina University (2016) Cloning and
Sequencing. Cellular and Molecular Biology 333 Lab
Manual. 2016, Week 1-5 manuals, pgs. 1-10.
We would like to thank Western Carolina University and
Instructor Beyer for providing the materials, instruction, and
opportunity to complete this cell cloning experiment. As well
as to all our prior biology and chemistry professors for
providing us with the necessary knowledge to understand and
grow as a scientist from this experience.
Primer Description E-Value Bit-Score % Pairwise Identity Query Coverage Grade
pJET SEQ F Arabidopsis thaliana
chromosome 3 sequence
0 1,716.29 99.1% 20-993 98.6%
Arabidopsis lyrata subsp. lyrata
unplaced genomic scaffold
ARALYscaffold_3, whole
genome shotgun sequence 0 1337.26 91.7% 20-993 94.9%
Camelina sativa cultivar DH55
chromosome 19 genomic
scaffold, Cs Chr19, whole
genome shotgun sequence 0 1130.2 86.6% 22-993 92.2%
pJET SEQ R Arabidopsis thaliana
chromosome 3 sequence
0 1,757.77 99.3% 13-1005 97.7%
Camelina sativa cultivar DH55
chromosome 19 genomic
scaffold, Cs Chr19, whole
genome shotgun sequence 0 1162.66 86.8% 13-1003 91.4%
Capsella rubella cultivar Monte
Gargano unplaced genomic
scaffold scaffold_3, whole
genome shotgun sequence 0 1129.59 85.6% 13-1003 90.8%
GAP SEQ F Arabidopsis thaliana
chromosome 3 sequence
0 924.611 99.1% 6-530 99.1%
Arabidopsis lyrata subsp. lyrata
unplaced genomic scaffold
ARALYscaffold_3, whole
genome shotgun sequence 0 744.274 91.8% 6-530 95.4%
Camelina sativa cultivar DH55
chromosome 19 genomic
scaffold, Cs Chr19, whole
genome shotgun sequence 1.78e-176 632.465 88.2% 24-259 91.8%
GAP SEQ R Arabidopsis thaliana
chromosome 3 sequence
0 1045.44 99.3% 1-589 99.7%
Arabidopsis lyrata subsp. lyrata
unplaced genomic scaffold
ARALYscaffold_3, whole
genome shotgun sequence 0 787.555 90.1% 1-589 95.1%
Camelina sativa cultivar DH55
chromosome 19 genomic
scaffold, Cs Chr19, whole
genome shotgun sequence 5.36e-171 614.432 84.2% 1-589 92.1%
Table 1. BLAST Results, Run #1
These results are from the cleaned and trimmed chromosomes obtained from the E. Coli
cells that took up the plasmid. The vectors and chromosomes ends were automatically
trimmed by the Geneious 8.1.9 program and the primers removed. The E-values being close
to zero and the percentages being near 100% indicate that these chromosomes are nearly
identical to the actual GAPC gene and of high quality.
Description E-Value Bit-Score % Pairwise Identity Query Coverage Grade
Arabidopsis thaliana
chromosome 3 sequence 0 1757.77 99.3% 30-1022 97.7%
Arabidopsis lyrata subsp.
lyrata unplaced genomic
scaffold ARALYscaffold_3,
whole genome shotgun
sequence
0 1413.32 91.9% 30-1022 94.0%
Camelina sativa cultivar
DH55 chromosome 19
genomic scaffold, Cs
Chr19, whole genome
shotgun sequence
0 1162.66 86.8% 32-1022 91.3%
Capsella rubella cultivar
Monte Gargano unplaced
genomic scaffold
scaffold_3, whole genome
shotgun sequence
0 1126.59 85.6% 32-1022 90.7%
Camelina sativa cultivar
DH55 chromosome 1
genomic scaffold, Cs Chr1,
whole genome shotgun
sequence
0 1106.75 85.0% 32-1022 90.4%
Tabel 2. BLAST Results from Contig 2nd Run
These results show that the genome sequences are closely related to the GAPC gene
sequence found in the Arabidopsis thaliana; however they are not identical. Also, as the E-
value is zero, the grade high at 97.7%, and the pairwise identity high at 99.3%, the
sequenced gene is nearly identical to the literature value of the GAPC gene. The four
sequences listed below Arabidopsis thaliana are the other top hits for this BLAST results
with low E-values and high grade and pairwise identities meaning these genes are also very
similar to the genes sequenced in this genome other than the GAPC.
Sterile Water (Negative Control)
pGAP
gDNA
Maleberry
Thyme
5,000
4,500
3,500
3,000
2,500
2,000
1,500
1,000
500
Transformed E.
Coli cells
5,000
1,000
1,500
2,000
2,500
3,500
500
E. Coli post-transformation
Figure 4. Contig
The final contig sequence for the transformed plasmids focusing on the
GAPC1 gene sequence found within this specific DNA segment.