Download The development of restriction analysis and PCR

THE DEVELOPMENT OF RESTRICTION ANALYSIS AND PCR-BASED
SCREENING METHODS FOR THE IDENTIFICATION OF TRANSFORMED CELLS
CONTAINING RECOMBINANT PGEX-GFPS65T PLASMIDS IN E. COLI.
CHRISTOPHER HEARD
KEY WORDS: RESTRICTION ANALYSIS, PLASMID SCREENING, POLYMERASE CHAIN REACTION,
PGEX-2T, PGEX-GFPS65T
Abstract
The identification of transformed bacterial cells and recombinant plasmids is important in
biochemical research and has ramifications for industry. Accurate screening methods based
on restriction analysis techniques and PCR technology were developed for the commonly
used pGEX-2T plasmid with a view to identifying cells containing recombinant pGEXGFPS65T. An experimental trial was conducted to determine the efficacy of the proposed
methods, using agarose gel electrophoresis to approximate DNA fragment lengths. Standard
curves were constructed to allow approximation of fragment lengths (and thus plasmid
identity) using the proposed methods. The efficacy of the restriction analysis and PCR-based
techniques was confirmed, with the PCR-based method determined to be the most accurate.
Introduction
The bacteria Escherichia coli is of substantial importance as a model organism for the study
of genetics, microbiology, and biochemistry (see, for example Natarajan et al. 2011). Its
genetic material is well understood, and the use of specialised plasmids to facilitate the
expression of novel gene products has expanded the usefulness of this organism (Madigan et
al. 2009). The incorporation of genetic material, particularly in the form of plasmids, into E.
coli cells poses challenges. The uptake of genetic material is commonly induced by
transformation (Snustad & Simmons 2009), however this method is imprecise and
successfully transformed cells must be identified before further analysis can occur.
The identification of transformed bacterial cells and recombinant plasmids is thus an
important task in biochemical research and has ramifications for the biotechnology and
pathology industries. Successfully transformed bacterial cells can be identified using a variety
of methods including visual inspection (for species with aesthetic phenotypes) and antibiotic
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selection (Nelson & Cox 2008). Restriction analysis and polymerase chain reaction (PCR)
provide effective methods for screening bacterial cells to identified transformed organisms.
pGEX-2T is a commonly used plasmid, so the development of effective screening methods
which can be used to identify transformed cells containing recombinant pGEX-2T plasmids
will have broad applications.
To address this requirement, accurate screening methods based on restriction analysis
techniques and PCR technology were developed for identifying transformed cells containing
recombinant pGEX-2T plasmids. The efficacy of these methods was then confirmed via
experimental methods.
The aim of this series of experiments is to use molecular biology techniques to clone GFP
into pGEX-2T, transform the recombinant plasmid into E. coli and use screening techniques
to identify the cells which have been transformed with the desired plasmid, with a view to
analysing and characterising GFP.
Method
The pGEX-2T and pGEX-GFPS65T plasmids
The pGEX-2T plasmid was selected for its broad application and relevance in the biological
sciences. The standard structure of the pGEX-2T plasmid is provided at Figure 1. Figure 1
also provides detail of the expected structure of the pGEX-GFPS65T recombinant plasmid.
The GFPS65T gene is a simple gene encoding a variant of green fluorescent protein (GFP)
which is widely used as a molecular marker. The recombinant plasmid was produced by
exposing pGEX-2T plasmids to the restriction enzymes BamHI and EcoRI in the presence of
GFPS65T genes with sutiable ‘sticky ends’. Ligase was used to seal the gene into the
plasmid.
The E. coli cells were exposed to a mixture of both plasmid types, reflecting the imprecision
associated with simple in vitro recombination methods. The cells were then plated on agar
containing ampicillin (to ensure that only transformed cells survived) and allowed to form
colonies.
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Figure 1: Design of Plasmids
The pGEX-2T plasmid system consists of a GST gene upstream of a multiple cloning site (detail magnified) and an ampicillinresistance gene. The pGEX-GFPS65T recombinant plasmid is distinguished by the insertion of a GFPS65T gene in the
multiple cloning site. The locations of the BamHI and PstI restriction sites are marked and the expected fragments from
restriction analysis are indicated.
Restriction Analysis
An accurate restriction analysis method requires suitable restriction enzymes and knowledge
of the expected products of their action. The BamHI and PstI restriction enzymes were
selected. The locations of the restriction sites on which these enzymes act are marked in
Figure 1. The restriction sites flank the multiple cloning site, which means that the presence
of the GFPS65T gene in the recombinant plasmid will result in fragments of different sizes.
Fragments 3977bp and 971bp in length were anticipated from transformed cells containing
the a non-recombinant pGEX-2T plasmid. Fragments 3977bp and 1687bp in length were
anticipated from transformed cells containing recombinant pGEX-GFPS65T plasmids.
A colony was selected from the agar plate for analysis and the cells of the bacteria were lysed.
A plasmid digestion was then prepared according to the volumes in Table 1. A positive
control was also prepared by using a known sample of pGEX-GFPS65T (prepared according
to Table 1).
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The products of the digestion were then run on a gel, alongside a 1KB DNA ladder to allow
the sizes of fragments to be determined.
Component
Volume (μL)
10 x NEB Buffer 3
2
Water (to 20μL)
13
10 x BSA
2
DNA (3μg)
2
BamH1
0.5
Pst1
0.5
Total
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Table 1: Restriction Digest Components
PCR Analysis
The selection of PCR primers was dictated by similar considerations as the selection of
enzymes for the restriction analysis.
Primer 1 is complementary to the sense (+) strand such that the 3’ end is towards (but short of)
the BamH1 and EcoR1 restriction sites. Thus, it is the forward primer. Primer 6 is the reverse
primer as it is complementary to the antisense (-) strand with the 3’ end towards (but short of)
the restriction sites. These primers also have chemical characteristics consistent with those
required for PCR (source?).
The binding sites of the selected primers are indicated on Figure 2. Figure 2 also contains
predictions of the sizes of the PCR fragments obtained. A 152bp long fragment is expected
for the pGEX-2T plasmids, while a 868bp long fragment is expected for the recombinant
pGEX-GFPS65T plasmid.
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Figure 2: PCR Primer Binding Sites
The selected primers bind to positions flanking the multiple cloning site. The reverse primer binds upstream of the
ampicillin-resistance gene while the forward primer binds in the GST gene. The direction of transcription if indicated by the
arrows. The expected PCR products are noted in purple, along with their predicted lengths.
Table 2 illustrates the composition of the PCR reactions prepared to test the effectiveness of
the primers. A negative control (known pGEX-2T), positive control (known pGEX-GFTS65T)
and template free control were prepared. An unknown sample was also collected from the
culture and tested.
Component
Negative Control
Positive Control
Unknown
Template-Free
Template
2 μL
2 μL
2 μL
0 μL
Forward Primer
2 μL
2 μL
2 μL
2 μL
Reverse Primer
2 μL
2 μL
2 μL
2 μL
Taq
0.08 μL
0.08 μL
0.08 μL
0.08 μL
dNTPs
0.4 μL
0.4 μL
0.4 μL
0.4 μL
Buffer
2 μL
2 μL
2 μL
2 μL
Water
11.52 μL
11.52 μL
11.52 μL
13.52 μL
Total
20 μL
20 μL
20 μL
20 μL
Table 2: PCR Reaction Components
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Results
Section 5: Gel Electrophoresis Result
To assess the efficiency of the two proposed screening methods, the DNA products of the
restriction analysis and PCR were investigated through the use of gel electrophoresis. Figure
3 shows the gel obtained from the restriction analysis.
The positive control (lane 3) was unsuccessful, as the presence of a nuclease in the restriction
analysis preparation which was activated by a component in the buffer caused loss of the
DNA.
Figure 3: Restriction Analysis Gel
Lanes numbered from left to right.
Lane 1: 1KB DNA Ladder
Lane 2: ‘Unknown’
Lane 3: pGEX-GFPS65T Digest (unsuccessful)
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Figure 4 shows the gel obtained from the PCR products.
Figure 4: PCR Gel
Lanes numbered from left to right.
Lane 1: DNA Ladder
Lane 2: Negative Control (pGEX-2T)
Lane 3: Positive Control (pGEX-GFPS65T)
Lane 4: ‘Unknown’
Lane 5: Template-Free Control
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Discussion
A logarithmic standard curve was produced from the 1KB DNA ladder to allow the sizes of
the fragments in lane 2 to be approximated. The resultant standard curve is at Figure 5. The
equation of the curve is noted on Figure 4 and was:
Where y is fragment length (bp) and x is distance migrated (mm).
Figure 5: Restriction Analysis Standard Curve
A semi-logarithmic grid is used. The model is noted beside the curve as y=31430e-0.086x.
For example, for the furthest migrating band in the second well:
So the fragment in this band is approximately 1304bp long. The other band in the second well
was approximated via the same method to be 4738bp in length.
The expected fragment lengths were 3977bp for both plasmids, 971bp for pGEX-2T and
1687bp for pGEX-GFPS65T. Since the band identified as containing 4758bp fragments is
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probably the band of 3977bp fragments (corresponding to the plasmids), the standard curve
appears to overestimate the fragment length. Thus, the 1304bp band is likely the 971bp
fragments from pGEX-2T, indicating that pGEX-2T was the ‘unknown’ in lane 2.
A similar standard curve was developed from the ladder used in the PCR electrophoresis. The
formula describing the curve was determined to be
. The band in lane one
was estimated to be 183bp in length, the band in lane two 1001bp in length and the band in
lane three 936bp.
The band lengths for the ‘negative’ control (pGEX-2T) and ‘positive’ control (pGEXGFPS65Y) were reasonably close to the predicted lengths of 152bp and 868bp respectively
(compared to approximated/calculated values of 183bp and 1001bp). The absence of bands in
lane 4 (the template free control) accords with expectations.
The ‘unknown’ sample was pGEX-GFPS65T. This is indicated by the approximated band
length of 936bp (which is significantly closer to 868bp than 152bp) and is also apparent upon
visual inspection of the gel. The unknown band migrated a very similar distance to the
positive control, which contained pGEX-GFPS65T.
Both methods produced the expected results. The PCR method appears to have been more
accurate. The failure of the positive control in the restriction analysis to produce correct
results is a significant shortcoming of this experimental process. Additional analysis would
be necessary to ensure that this method is suitable for screening.
Conclusion
The purpose of this investigation was to develop and assess potential restriction analysis and
PCR-based screening methods for identifying transformed E. coli cells containing
recombinant pGEX-GFPS65T. Both the proposed restriction analysis and PCR designs
performed satisfactorily, producing unambiguous results which allowed plasmid
identification.
The PCR method appeared to be the most accurate and was more thoroughly validated by the
evidence, owing to the failure of the positive control in the restriction analysis. As the
proposed methods have shown potential, further research may be able to refine the screening
methods to improve precision and ensure their effectiveness.
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References
Madigan, M & Martinko, J & Stahl, D & Clark, D 2009, Brock: Biology of Microorganisms,
13th edn, Pearson, New York.
Natarajan, C et. al. 2011, ‘Expression and Purification of Recombinant Hemoglobin in
Escherichia coli’, PLOS One, vol. 6, no. 5, viewed 06/08/2011,
<http://apps.webofknowledge.com>.
Nelson, D & Cox, M 2008, Lehninger: Principles of Biochemistry, 5th edn, W. H. Freeman
and Company, New York.
Snustad, DP & Simmons, MJ 2009, Principles of Genetics, 5th edn, John Wiley & Sons,
Hoboken.
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