Part:BBa_K4370010:Experience

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Applications of BBa_K4370010

BBa_K4370010 was introduced between NotI sites into the pOSV805 plasmid, replacing the initial cloning module that contains the blue chromoprotein encoding gene amylCP (BBa_K1155003) under the control of E. coli promoter (BBa_K4370009, not functional in Streptomyces).

Since BBa_K4370010 contains two promoters (one active in Streptomyces, one active in E. coli), we had to check first that there was no promoter interference and that the mRFP1 gene could be properly expressed from this module at a level sufficient to allow a visual screening for further applications.

Therefore, after cloning BBa_K4370010 into pOSV805, we observed the color of the colonies on plate. We could clearly distinguish red and white colonies, harboring or not the BBa_K4370010 part, respectively (Figure 1). Thus, whereas bacteria pOV805 were blue, the bacteria transformed by pOSV805- BBa_K4370010 are now red. This result indicates that pOSV805-BBa_K4370010 can now be used as a chassis vector for further cloning of gene of interest for a constitutive expression in Streptomyces, the red/white screening being a convenient means of screening the clones containing the new construction (Figure 2).

Figure 1: E. coli colony phenotype after transformation with a ligation mix harboring pOSV805-BBa_K4370010 or pOSV805 devoid of cloning module

Figure 2: E. coli colony phenotype after transformation with pOSV805 parental plasmid (blue) or its derivative harboring the pOSV805-BBa_K4370010 cloning module (pink)

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