BBa_K1996000 to BBa_K1996002
These three parts were obtained from the ethylene metabolism operon in Mycobacterium NBB4, as they were suspected to function together to regulate the expression of genes responsible for ethylene oxidation. EtnR2 (BBa_K1996002) is thought to bind to ethylene and phosphorylate EtnR1 (BBa_K1996001), which binds to EtnP (BBa_K1996000) and activates bidirectional transcription, resulting in the expression of ethylene degradation genes.
EtnR1 has been shown to bind to EtnP through an electrophoretic mobility shift assay , demonstrating its function as a DNA-binding protein. The characterisation of EtnR2 as an ethylene receptor, and the binding of EtnR1 and EtnR2 is still underway.
BBa_K1996003 to BBa_K1996005
Each part is an improvement on the basic amilCP part submitted by Team Uppsala Sweden 2011 (BBa_K592009). Each part contains the coding regions of different coloured mutant amilCP chromoproteins, created by error-prone PCR. BBa_K1996003 is the pink mutant chromoprotein, BBa_K1996004 is the purple mutant chromoprotein and BBa_K1996005 is the green mutant chromoprotein. Colour can be readily observed by the naked eye following growth in liquid media or on agar plates for 1-3 days, rendering the parts useful as reporter genes.
Figure 1: Purple, pink and green mutants compared to the wild type. Protein was extracted from 50mL of culture by bead beating and resuspended in 0.5mL TE buffer. Maximum absorbance for the purple mutant was at 579nm (Figure 2), indicating that it had been shifted slightly compared to the wild type’s maximum at 589nm. The absorbance maximums for the pink and green mutants are also shifted, to 576nm and 609nm respectively (Figure 2).
Figure 2. Spectrum scan of the extracted protein from the different coloured amilCP mutants, with the absorbances adjusted mathematically to be equal, to show each peak. Pink = BBa_K1996003, Purple = BBa_K1996004 and Mint green = BBa_K1996005