A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis

Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.

Effects of Sr2 + on the preparation of Escherchia coli DH5α competent cells and plasmid transformation

Bacterial gene transformation used with Escherichia coli as a desired microorganism is one of the important techniques in genetic engineering. In this study, the preparation of E. coli DH5α competent cells treated with SrCl2 and transformation by heat-shock with pUC19 plasmid was optimized by Response Surface Methodology (RSM). Other five E. coli strains including BL21 (DE3), HB-101, JM109, TOP10 and TG1, three different sizes plasmids (pUC19, pET32a, pPIC9k) were used to verify the protocol, respectively. The transformation mechanism was explored by scanning electron microscope combined with energy dispersive spectrometer (SEM-EDS), atomic absorption spectroscopy (AAS) and Fourier-transform infrared spectroscopy (FT-IR). An equation of regression model was obtained, and the ideal parameters were Sr2 + ions of 90 mM, heat-shock time of 90 s and 9 ng of plasmid. Under this conditions, the transformation efficiency could almost reach to 106 CFU/µg DNA. A small change of the cell surface structure has been observed between E. coli DH5α strain and competent cells by abovementioned spectrum technologies, which implied that a strict regulation mechanism involved in the formation of competent cells and transformation of plasmids. An equation of regression model for the competent cells preparation and plasmid transformation could be applied in gene cloning technology

TRANSFORMASI PLASMID YANG MENGANDUNG GEN merB PADA Escherichia coli BL21(DE3)

ABSTRACTDNA transformation is one of the methods for inserting DNA into bacterial cells. The current transformation method is widely used to transfer plasmids containing genetic material. This study aims to evaluate the results of plasmid transformation containing merB gene in Escherichia coli BL21(DE3) bacteria. The stages of the research carried out were preceded by the microbiological identification of the E. coli BL21(DE3) bacteria used as hosts. Then the plasmid transformation containing merB gene into the E. coli BL21(DE3) host cell using the heat shock method was carried out. The transformation results were evaluated by observing at the presence of E. coli BL21(DE3) colonies on agar Luria Bertani (LB) media containing ampicillin antibiotics. Plasmids in E. coli BL21(DE3) were isolated and analyzed by 1% agarose gel electrophoresis. The results showed the success of the transformation indicated by the growth of E. coli BL21(DE3) bacteria in agar LB media containing ampicillin and the visualization on agarose gel resulted that the plasmid which carried the merB gene could be transformed in to the E. coli BL21(DE3) bacteria.Keywords : Plasmids, merB genes, heat shock, Escherichia coli BL21(DE3)ABSTRAKTransformasi DNA merupakan salah satu metode untuk memasukkan DNA ke dalam sel bakteri. Metode transformasi saat ini dipakai secara luas untuk mentransfer plasmid yang mengandung bahan genetika. Penelitian ini bertujuan untuk mengevaluasi hasil transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli BL21(DE3). Tahapan penelitian didahului dengan identifikasi secara mikrobiologi bakteri E. coli BL21(DE3) yang digunakan sebagai inang. Selanjutnya dilakukan transformasi plasmid yang mengandung gen merB kedalam sel inang E. coli BL21(DE3) menggunakan metode heat shock. Hasil transformasi dievaluasi dengan melihat adanya koloni E. coli BL21(DE3) pada media agar Luria Bertani (LB) yang mengandung antibiotik ampisilin. Plasmid pada E. coli BL21(DE3) diisolasi dan dianalisis dengan elektroforesis gel agarose 1%. Hasil penelitian menunjukkan keberhasilan transformasi dengan adanya pertumbuhan bakteri E. coli BL21(DE3) pada media LB yang mengandung ampisillin dan hasil visualisasi pada agarose gel terlihat bahwa plasmid yang membawa gen merB dapat ditransformasikan ke dalam bakteri E. coli BL21(DE3).Kata Kunci : Plasmid, gen merB, heat shock, Escherichia coli BL21(DE3)