Team:Sydney Australia/Parts

Part Number Name Type Function Description Medal
BBa_K1996000 pSB1C3-EtnP Basic Putative promoter region for ethylene oxidation genes in Mycobacterium NBB4 Native sequence from Mycobacterium NBB4 , contained within the ethylene metabolism operon Bronze
BBa_K1996001 pSB1C3-EtnR1 Basic DNA-binding protein for EtnP in Mycobacterium NBB4 Coding sequence of EtnR1 from Mycobacterium NBB4 , codon-optimised for expression in E. coli K12 Silver
BBa_K1996002 pSB1C3-EtnR2 Basic Putative ethylene receptor in Mycobacterium NBB4 Coding sequence of EtnR1 from Mycobacterium NBB4, codon-optimised for expression in E. coli K12 Bronze,Silver
BBa_K1996003 pSB1C3-amilCPpink Basic Pink mutated version of the chromoprotein amilCP from Acropora millepora Coding region of the pink mutant version of amilCP Gold
BBa_K1996004 pSB1C3-amilCPpurple Basic Purple mutated version of the chromoprotein amilCP from Acropora millepora Coding region of the purple mutant version of amilCP Gold
BBa_K1996005 pSB1C3-amilCPgreen Basic Green mutated version of the chromoprotein amilCP from Acropora millepora Coding region of the green mutant version of amilCP Gold

EtnP

EtnR1

EtnR2

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.

amilCP Pink

amilCP Purple

amilCP Green

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

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School of Life and Environmental Sciences
The University of Sydney
City Road, Darlington
2006, New South Wales, Sydney, Australia