Part:BBa_K909010

YcgZ promoter with multiple BluR (YcgE) operator sites

The BLUF-EAL protein BluF (YcgF) acts as a blue light sensor in E. coli. The degenerate EAL domain of BluF does not have cyclic-di-GMP phosphodiesterase activity, but BluF directly antagonizes the MerR-like repressor BluR (YcgE). BluR acts as a repressor on the ycgZ-ymgABC operon by binding to Operator sequences at the YcgZ promoter.

Without blue light activation BluR is free to perform its repressing activity (Fig 1). Blue light however activates BluF homodimerization and further heterotrimerization with BluR. The heterotrimer is no more able to repress expression and the promoter is active (Fig 2).

The YcgZ promoter can therefore be used to engineer a blue light switch.

Fig 1: Without activation by blue light BluF (YcgF) is not active and thus BluR (YcgE) is free to bind DNA and perform its repressing function.

Fig 2: Blue light activates the dimerization of BluF (YcgF) and further the heterotrimerization with BluR (YcgE). This inhibits DNA binding and therefore induces expression.

The previous constructs found in the parts registry (BBa_K238013 and BBa_K360041) were designed before the BluR binding regions / inverted repeats were scientifically determined. However these were determined recently by DNA footprinting by a [http://www.ncbi.nlm.nih.gov/pubmed/22783906 research team of FU Berlin] (Fig 3).

We used this information to improve the previously submitted parts by doubling the amount of BluR binding sites.

Fig 3: A) The DNA footprint done in the research of [http://www.ncbi.nlm.nih.gov/pubmed/22783906 Natalia Tschowri 2012] that shows the binding regions / inverted repeats for BluR (YcgE) indicated with arrows in the sequence below. B) Shift Assay: Point mutations in the determined binding regions drastically decrease DNA binding of BluR (YcgE).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]