Abstract:

Objective To investigate the expression of tryptophan synthase α subunit-encoding gene (trpA) of Escherichia coli (E. coli), fusion fragment of trpA, yellow fluorescent protein gene (yfp) and chloramphenicol-encoding gene (cat) was knocked into E.coli strain MG1655 genome using λ-Red system. Methods Target genes trpA, yfp and cat were amplified by PCR, and the recombinant fragment was obtained by fusion PCR. The recombinant fragment was transformed into E.coli strain MG1655 harboring genes of λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation, the fusion protein expression was observed by fluorescence microscopy. Results The recombinant fragment trpA-yfp-cat was successfully obtained and transformed into MG1655, and the positive clones were screened out and confirmed by sequencing. The yellow fluorescence was observed throughout the bacterial cytoplasm, indicating that trpA-yfp fusion protein was successfully expressed in MG1655. Conclusion The trpA gene could be replaced by trpA-yfp fusion fragment in situ using λ-Red system, and its expression could be reflected indirectly by observing the expression of yellow fluorescent protein. The function of trpA and bacterial growth have not been changed, which lays foundation for the further study of E.coli tryptophan synthesis.

Key words: λ-Red system, fusion PCR, yellow fluorescent protein, tryptophan synthase &alpha, subunit