Part:BBa_K1893012:Experience

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Applications of BBa_K1893012

This construct should be able to control transcription and thus regulate gene expression of a coding sequence as part of a two-plasmid system. For experimental characterisation, the first plasmid should contain this construct. The second plasmid should be the reporter plasmid containing the superfolder GFP (SFGFP) gene with a ribosome binding site immediately downstream of the STAR-target (pAD1 plasmid attenuator) sequence. The SFGFP coding sequence is under the control of a constitutive Anderson promoter and also has its own TrrnB terminator. The relative levels of transcription of either STAR and Anti-STAR when regulated by two constitutive promoters of differing strengths should regulate the amount of gene expression of the SFGFP.

User Reviews

Even though they are very small sequences, STAR and Anti-STAR with their associated terminator were both easy to amplify by PCR. However, when we attempted to ligate STAR and Anti-STAR, both under the control of the J23119 promoter, into the same plasmid, using the BioBrick assembly method, we were unsuccessful even though we tried a number of times to optimise various steps in the protocol. We think that perhaps a recombination event was occurring in the cells as the sequence of the promoter is duplicated. We did not experience any major difficulties when assembling STAR and Anti-STAR under the control of the quorum-regulated promoters separately (BBa_K1893020-BBa_K1893023).

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