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December 03-04, 2018 | Chicago, USA
American Pathology and Oncology Research
&
15
th
International Congress on
Microbial Genetics and Molecular Microbiology
International Conference on
Journal of Clinical & Experimental Pathology | ISSN: 2161-0681 | Volume 8
Dual labeling of
Pseudomonas Putida
ND6 with fluorescence proteins for exploring the conjugal
transfer of pND6-1 and pND6-2 plasmid
Shan Wang, Shanshan Li, Keke Qian, Dan Wang, Dan Du
and
Wei Yan
Xi'an Jiaotong University, China
D
ual labeling of
Pseudomonas putida
ND6 with fluorescence proteins for exploring conjugal transfer of pND6-1 and pND6-
2 Plasmid: Gram-negative
Pseudomonas putida
ND6 possess two large plasmids pND6-1 and pND6-2. The former one
which carries the genes encoded for naphthalene degradation in the catechol-meta-cleavage pathway belongs to the IncP-
7 conjugative plasmid. Several genes involved in the Type IVB Secretion System are located in the later plasmid. In order
to well-understand the characteristics of these two plasmids during conjugation, pND6-1 and pND6-2 were labeled with
red fluorescent protein gene (dsred) and green fluorescent protein gene (gfp) respectively by homologous recombination via
biparental mating. In view of the narrow host range of the IncP-7 plasmid, Poprl promoter (located before the oprl gene) from
Pseudomonas putida
ND6 was attached to dsred and gfp and inserted into the non-functional region of plasmid together to
avoid affecting the expression of functional genes on the plasmid. Both red and green fluorescent proteins were co-expressed
in the isolated conjugon GROND6 (pND6-1::dsred, pND6-2::gfp). Furthermore, the results suggested that Poprl promoter
could better improve the red fluorescent expression when compared with the green fluorescent protein in
P. putida
ND6. The
dual-labeled GROND6 with red and green fluorescent proteins was subsequently tested its conjugation transfer by mating
experiment with
P. putida
KT2440 as the recipient. The screened transconjugant KT2440RG exhibited both red and green
fluorescence under fluorescence microscopy, indicating that the constructed dual-fluorescent-labeled strain GROND6 (pND6-
1::dsred, pND6-2::gfp) can be used to in situ detect the transfer of two mobile plasmids in ND6 in the various environment.
Biography
Shan Wang is pursuing her Doctor’s degree in Power Engineering and Engineering Thermophysics at Xi’an Jiaotong University. Her study focuses on the functional
mechanism of the conjugative transfer system in
Pseudomonas putida
ND6 and the monitoring of conjugation in distinct environments in situ.
1582354536@qq.comShan Wang et al., J Clin Exp Pathol 2018, Volume 8
DOI: 10.4172/2161-0681-C5-058