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Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors

M Chawla1, S K Das Gupta

  • 1Department of Microbiology, Bose Institute (Centenary Building), P1/12 C.I.T. Scheme VIIM, Calcutta, 700054, India.

Plasmid
|March 24, 1999
PubMed

Insights

Transposition of IS1096 in E. coli-mycobacteria shuttle vectors caused deletions. Minimal vector derivatives, essential for replication and selection in mycobacteria, proved structurally stable for transformation.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Escherichia coli-mycobacteria shuttle vectors are crucial for genetic manipulation in mycobacteria.
  • Structural instability in these vectors can hinder their application.
  • The IS1096 transposable element from Mycobacterium smegmatis is implicated in vector alterations.

Purpose of the Study:

  • To investigate the structural alterations in E. coli-mycobacteria shuttle vectors.
  • To identify the cause of vector instability and its impact on replication.
  • To develop stable shuttle vectors for mycobacterial transformation.

Main Methods:

  • Construction of shuttle vectors using pAL5000 and pUC19.
  • Analysis of vector structural changes using molecular techniques.
  • Assessment of replication capability and stability in mycobacteria.

Main Results:

  • Transposition of IS1096 occurred at specific sites upstream of the kanamycin resistance gene.
  • Structural alterations resulted in large deletions, sometimes affecting ORF2 (RepB) coding sequences.
  • Replication capability was maintained despite deletions in ORF2, suggesting non-essentiality.
  • Deletion derivatives with minimal replication and selection sequences were structurally stable.

Conclusions:

  • The ORF2 coding sequences of the pAL5000 replication region are not entirely essential for vector replication.
  • Minimal, deletion-derived shuttle vectors are structurally stable.
  • These stable vectors hold potential for efficient mycobacterial transformation systems.

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