Development of an Absolute Standard for Quantitative PCR using
E. coli DH5α 16S rRNA Gene Cloned into a Plasmid
1
Moore ,
R.
1Oregon
H.
1
Poppen ,
F.
2
Colwell
State University Department of Microbiology, 2Oregon State University College of Oceanic and Atmospheric Sciences
Introduction
Summary
Methods
There is growing concern that the accumulation of greenhouse gases,
especially carbon dioxide (CO2), in the atmosphere is contributing to global
climate change. One solution for the removal of anthropogenic CO2
emissions is deep geologic carbon sequestration in basalt aquifers. A
project located at Wallula Gap in eastern Washington State (Figure 2) is
investigating the potential of the Columbia River Basalt Group (CRBG) to
act as a site for the deep geologic storage of supercritical CO2 (scCO2).
• DNA Extraction. DNA was extracted from overnight E. coli DH5α cultures using the MOBIO
UltracleanTM DNA Isolation kit according to the manufacturers’ instructions and the 16S rRNA
gene was amplified by PCR with primers 27F and 1492R.
• Linear regression R2 values and
amplification efficiencies (Table
1) suggest the standard curve
developed from entire genome
is the more accurate and
precise standard.
• Cloning and Transformation. Reactions were performed using the Invitrogen TOPO TA
Cloning® kit according to the manufacturer’s instructions.
• Quantitative PCR. For qPCR standards, plasmids were serially diluted to final concentrations
ranging from 1.0x107 – 1.0x102 plasmid/µL and were quantified with SYBR Green chemistry.
• qPCR is being used instead of the more traditional method of direct cell counts due to
difficulty visualizing environmental samples using DAPI stains.
Wallula Gap Pilot Well
• Using genomic DNA for a qPCR standard is the conventional method, however, using a
plasmid containing the gene of interest is considered more accurate because the number of
genes can be accurately determined.
Figure 4. Overview of the approach used to
develop the plasmid used in the construction of the
qPCR standard.
Results
Amplification Efficiency
From Plasmid
0.147
N/A
From Whole Genome
0.988
86.6%
Genomic Standard Curve
• The genomic standard (Figure 5) and the
plasmid standard (Figure 6) were constructed
and analyzed at the same time and under the
same conditions.
Storage in deep basalt formations is a viable long-term solution because of the
geochemical trapping properties of basalts. Geochemical trapping involves mineralization
reactions that occur when the CO2 reacts with the basalt. The injected CO2 dissolves in
water, producing carbonic acid, which creates the acidic conditions favoring the
dissolution of cations from mafic minerals. Once free, cations such as Ca2+ can interact
with carbonic acid, forming calcium carbonate (CaCO3). Thus, over time, the water-rock
interactions can help seal the formation with stable carbonate minerals and prevent
migration of the CO2.
3
3.5
4
4.5
5
5.5
6
Log (Copy Number)
Figure 5. Standard curve for a sample
developed from E. coli DH5α entire genome.
Standards were generated by 6 serial dilutions of
genomic DNA.
• Contribution from nonspecific amplification
during qPCR was minimal because of
experimentally determined optimal primer
concentrations and elevated data collection
temperatures (Figures 7 and 8).
L
Genomic Standard Melt Curve
–
+
1
2
3
4
5
6
7
30
28
26
24
22
20
14
2
2.5
3
3.5
4
4.5
5
5.5
6
Log (Copy Number)
Figure 6. Standard curve for a sample
developed from E. coli DH5α 16S rRNA gene
cloned into a plasmid. Standards were generated
by 6 serial dilutions of plasmid DNA
8
Plasmid Standard Melt Curve
0.35
0.35
Mineral Key
200 µm
A cut section of cap rock
at 2,525 feet. Migration of
scCO2 is prevented by
cap’s tight configuration.
(Courtesy of M. Fisk)
(O) Iron oxides
– black with straight edges
Fluorescence Derivative
Fluorescence Derivative
F
(P) Pyroxene
– darker, fractured
2.5
• Low R2 value for plasmid standard suggests high
variability in Ct measurement between replicates.
4kbp
0.25
900bp
0.15
0.05
0.25
0.15
References
0.05
F
O
(G) Groundmass
– black irregular
• Additional tasks may involve the
extraction and quantification of
archaeal DNA, as well as the
amplification and quantification
of bacterial and archaeal
functional genes involved in
single-carbon metabolism.
P
G
(F) Plagioclase feldspar
– white, rectangular
y = -0.104x + 32.67
R2 = 0.147
16
18
16
2
The Injection Site
• Future work will employ the
more accurate and precise
standard
when
quantifying
bacteria in filtered groundwater
samples from the Wallula Gap
site.
18
• Standard curve using plasmid DNA was
developed from a cloning reaction that had low
efficiency.
• The plasmid DNA used in each
experiment was taken from the
same low-efficiency cloning
reaction.
Thus,
continued
development of the plasmid
standard
from
a
more
successful cloning reaction is
necessary.
Plasmid Standard Curve
Threshold Cycle (Ct)
28
• Both curves are representative of the results
from multiple experiments for their respective
standards.
24
26
y = -3.69x + 36.6
R2 = 0.988
14
Develop an absolute standard for qPCR using a plasmid with an E.
coli DH5α 16S rRNA gene insert and compares it to a known absolute
standard developed from the entire genome of E. coli DH5α in
preparation for using qPCR to estimate microbial numbers in the
filtered groundwater from five depth intervals in the Wallula Gap pilot
well.
Threshold Cycle (Ct)
Objective
Linear Regression R2
30
One goal of the project is to survey the microbial population in water
samples taken from five different depths and investigate numerical changes
in the microbial population as it responds to the injection of scCO2.
Quantitative polymerase chain reaction (qPCR) will be used to estimate
copies of the bacterial 16S rRNA gene and by extension, the number of
cells present in the samples.
Standard
22
Figure 1. Different approaches to sequestering CO2. In the figure, 3b
best represents the method proposed for the onshore sequestration
of scCO2 at the Wallula Gap Site.
Table 1. Displays the linear regression correlation coefficient and the amplification efficiency of the standards. Amplification efficiency of the plasmid standard is not applicable because the large variation
in Ct values (inherent in the low R2 value) suggests a severe flaw exists in the standard.
20
Figure 2. Geographical location of the
Columbia River Basalt Group (CRBG). The
Wallula Gap Site where scCO2 will be
injected is indicated by the blue dot.
• Multiple experiments testing the
accuracy and precision of the
plasmid
standard
were
performed and the results
consistently suggest
the
plasmid standard is flawed.
P
-0.05
-0.05
60
65
70
75
80
85
90
95
Temperature (°C)
200 µm
A cut section of interflow
rock at 2,590 feet. This
section is representative of
interflow rock where scCO2
will be injected. (Courtesy
of M. Fisk)
Figure 3. Schematic of the pilot well
at Wallula Gap. The red bars show
the five microbiological sampling
depths.
Figure 7. Melt curve analysis performed on the
genomic standard during qPCR. The presence of
only one large peak is evidence of only one
amplicon, the desired 16S rRNA gene insert.
Figure 7. Invitrogen E-Gel produced after PCR
amplification of the plasmid extracted from competent
cells after low-efficiency cloning reaction. (L)
corresponds to a 1kb ladder, (-) and (+) are the
negative and positive PCR control respectively, and 18 correspond to eight different colonies collected from
the transformation plates. The bands at 900bp are the
desired bacterial 16S rRNA gene insert and the bands
at 4kbp are the vector.
60
65
70
75
80
85
90
95
Temperature (°C)
Figure 8. Melt curve analysis performed on the
plasmid standard during qPCR. The presence of
only one large peak is evidence of only one
amplicon, the desired 16S rRNA gene insert.
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research needs. Advances in Geophysics. Volume 43, 97-177, I-IX (2001).
2. McGrail, B.P. et al. Potential for carbon dioxide sequestration in flood
basalts. J. Geophys. Res. 111, 13 PP. (2006).
3. Dupraz, S., Parmentier, M., Ménez, B. & Guyot, F. Experimental and
numerical modeling of bacterially induced pH increase and calcite
precipitation in saline aquifers. Chemical Geology 265, 44-53 (2009).
4. Ehrlich, H. Geomicrobiology: its significance for geology. Earth-Science
Reviews 45, 45-60 (1998).
Acknowledgements
Research was made possible by funding from Oregon
State University's Subsurface Biosphere Initiative
(SBI) Fellowship program. Thanks to Rick Colwell and
Heather Poppen for their mentoring and support.