Environmental Engineering Reference
In-Depth Information
RHA1 16S rRNA gene sequence corresponding to E. coli positions 599 to 619
was chosen for designing the TaqMan-16S rDNA probe based on the presence
of only two other perfect matches for this primer, R. marinonascens and R.
globerulus . The probe contained FAM (6-carboxy-fluorescein) as a reporter flu-
orochrome on the 5′ end and TAMRA (N,N,N′,N′-tetramethyl-6-car-
boxy-rhodamine) as a quencher on the 3′ end of the nucleotide sequence. A
flask trial was performed to test the specificity of the designed probe, and
fluorescence during PCR amplification was observed for Rhodococcus strains
only. Similarly, primers and a suitable probe targeting the fcbB gene (chloroben-
zoyl-CoA hydrolase/dehalogenase) were designed and tested. In this case,
among some closely related Rhodococcus species, quantitative amplification of
the sequence was possible only for RHA1(pRHD34:: fcb ).
To test whether the recombinant organism could be detected within the
soil community, a soil microcosm experiment was conducted, similar to that
described in the above section, and the results of this study were summarized
in Rodrigues et al. (2002). After 30 days of incubation, the estimated numbers
of RHA1(pRHD34:: fcb ) cells per gram of soil measured by the two TaqMan
probes were similar to the values obtained for culturable rifampicin-resistant
RHA1( fcb ) cells (colony forming units , CFUs/g soil) throughout the growth
cycle. Growth was expected on the particular congeners in the PCB mixture,
and the absence of increased counts may have been due to protozoan graz-
ing, although this was not investigated. The method did allow measurement
of population dynamics in soil and an assessment of the stability of the
engineered gene (Figure 6.13).
Molecular Tracking of RHA 1 (fcbB) in
Picatinny Arsenal Soil Using Real Time PCR
40
R 2 = 0.9816
35
RHA 1 in Soil (CFUs)
Plasmid pRHD34 (copy #)
30
RHA1 Whole Cell DNA (copy #)
25
20
15
R 2 = 0.9984
10
R 2 = 0.9964
5
0
−4
1
6
11
16
21
26
31
ln CFUS or Copy Number
Figure 6.13 Molecular tracking GEMs in soil. (Redrawn from Rodrigues et al., J.
Microbiol. Meth. , 51, 181-189, 2002.)
 
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