| dc.description.abstract | Today, Escherichia coli (E.coli) is widely used as recombinant protein expression host. However, other species could be more suitable to express some recombinant proteins. In the present study, plasmids with XylS/Pm expression cassette were constructed to create shuttle vectors for E.coli and the Gram-positive bacterium Bacillus subtilis (B.subtilis). The purpose was to construct vectors suitable for recombinant protein expression in B.subtilis. First three vectors, pTH1xp-mCherry, pHCMC05-mCherry and pHT10-mCherry, were shown to be functional in E.coli and B.subtilis These vectors were fused to the DNA sequence encoding XylS/Pm expression cassette. The XylS/Pm controlled expression of the reporter gene mCherry The constructed vectors, named pSH1, pSH2 and pSH3, were successfully transferred to both E.coli and B.subtilis. Despite successful transformation, mCherry protein production from XylS/Pm was only observed in E.coli Consequently, the plasmid copy number and the expression level of mCherry and XylS mRNA were measured in E.coli and in B.subtilis to identify the cause of lack of recombinant protein expression in B.subtilis The copy number was much higher for all plasmids in E.coli than in B.subtilis Also, B.subtilis harboring the vectors expressed significantly less mCherry and XylS mRNA than \textsl{E.coli} harboring the same vectors. On the other hand, the results showed that mCherry and XylS mRNA level were to some degree elevated in B.subtilis Hence, some alterations on the XylS/Pm expression cassette, which improves XylS expression and/or increase the plasmid copy number, might lead to recombinant protein expression from B.subtilis harboring the vectors developed and tested.
Also, another Gram-positive bacteria, Lactobacillus plantarim (L.plantarum), were transformed with the vectors. Only pTH1xp-mCherry and pSH1 was successfully transferred. However, mCherry protein production was not detected. Due to this result, further work on L.plantarum was not performed. | en |
