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  • Fusion of two stable gene confers plasmid instability to the vector containing the hybrid gene
  • Ali Behboodian,1,* Saman Hosseinkhani,2 Farangis Ataei,3
    1. Tarbiat Modares University
    2. Tarbiat Modares University
    3. Tarbiat Modares University


  • Introduction: Cloning DNA fragments in a plasmid is usually uncomplicated, but bacterial cells attack the plasmid from time to time. Plasmid loss and elimination of a part of the plasmid during bacterial cell duplication threatens subsequent utilization of a plasmid. Plasmid instability may originate from expression of toxic gene products, metabolic burden of the plasmid, plasmid copy number, the genotype of the host strain, or recombinogenic potential of sequence present in the plasmid. For instance, repetition of a particular sequence and formation of secondary structures can confer instability to a plasmid. Overall, lengthy sequences and sequences that are from different genes increases the risk of plasmid instability. Sometimes it is hard to distinguish plasmid instability from common errors in cloning, and it is even called ‘unclonability’. While tackling plasmid instability can be challenging, there are some reports that claim lowering the cultivation temperature is advantageous. If this strategy fails, the alternative approach is to examine bacterial hosts that allow faithful maintenance of the plasmid. E.coli strain Stbl4 has been shown to be effective in cloning unstable DNA inserts. E.coli strain Stbl4 has endA1 mutation which enhances the plasmid stability. E.coli strain Stbl4 has the following genotype: mcrA, Δ(mcrBC-hsdRMS-mrr), recA1, endA1, gyrA96, gal-thi-1, supE44, λ-relA1, Δ(lac-proAB)/F', proAB+lacIqZΔM15, and Tn10 (TetR).
  • Methods: In this study, We fused the sequence of a membrane protein with the sequence of a polymerase from two vectors. Both of these vectors showed no plasmid instability during propagation, and we were able to extract high yields of plasmid. We fused the two sequences with overlap-extension PCR. Then the PCR product was double-digested and purified. Consequently, the purified PCR products were ligated with double-digested pet21 vectors.
  • Results: After transformation of E.coli DH5-Alpha with ligated products, the colony-PCR showed that the transformed bacterial cells possessed a shorter gene than the expected size. Interestingly, these completely rearranged the hybrid gene after several rounds of duplication. Since lowering temperature during transformation was not effective, we employed E.coli strain Stbl4 strain as the host. Around 10 percent of screened transformed Stbl4 colonies showed correct size, which indicates the plasmid stability.
  • Conclusion: Therefore, we observed that fusion of two genes undermined the stability of the plasmid harboring the hybrid gene. Fortunately, this plasmid instability can be overcome by employing Stbl4 strain.
  • Keywords: Plasmid Instability- Colony PCR- Hybrid Gene- Transformation- Recombination