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Volume 13, Issue 4
  • ISSN: 2211-5501
  • E-ISSN: 2211-551X

Abstract

Introduction

Enterococci are natural inhabitants of the human gastrointestinal tract and contain plasmids, which are often used to generate plasmid vectors. RGK7, a strain of isolated from neonatal faeces, natively contains a 1935 bp plasmid pRGK7d.

Objective

This study focused on the molecular characterization of the plasmid pRGK7d and its exploitation for the generation of shuttle cloning vectors.

Methods and Results

The isolate RGK7 was identified as by phenotypic and genotypic methods, including 16S rRNA sequencing. Phylogenetic analysis also confirmed that the strain belongs to the group of . BLAST analysis of the pRGK7d plasmid sequence revealed the highest homology with genes for replication protein. ORF Finder analysis of the 1.9 kb plasmid pRGK7d showed a total of 17 ORFs in multiple overlapping frames, and the biggest ORF sequence showed 97% homology with the Rep protein of In order to make it an shuttle vector, the plasmid was first cloned in pUC18 and then recloned in T vector by incorporating antibiotic selection markers ampicillin resistance ( and erythromycin resistance ( for its selectivity in Gram-negative and Gram-positive . The shuttle vector was reduced in size and made more effective by cloning only the Rep protein instead of the whole plasmid along with the gene.

Conclusion

An shuttle vector was generated utilizing the plasmid pRGK7d, which, on transformation, conferred ampicillin and erythromycin resistance to the and respectively, that was otherwise sensitive. The shuttle vector was found to be stable in both organisms.

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2025-01-31

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/content/journals/cbiot/10.2174/0122115501352974241106100118
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Isolation of Plasmid from a New Strain of Enterococcus faecium and its Construction for Use as Shuttle Cloning Vector
Curr. Biotechnol. 13, 237 (2024); https://doi.org/10.2174/0122115501352974241106100118
/content/journals/cbiot/10.2174/0122115501352974241106100118
/content/journals/cbiot/10.2174/0122115501352974241106100118
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  • Article Type:     Research Article
Keyword(s): 16S rRNA sequencing; erythromycin resistance; lactic acid bacteria; ORF; phylogenetic analysis; Shuttle vector

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