"
Team:BostonU/Data
From 2012.igem.org
Data Collected
We are also introducing a new promoter and gene pair to iGEM that forms a copper-dependent repressible system. Since the library of metal sensitive promoters was relatively small on the Registry of Standard Biological Parts, we decided to investigate a metal-dependent system. We chose mmoR and a σ54 promoter associated with it from a bacterium called Methylosinus trichosporium OB3b. mmoR is a σ54-dependent transcriptional activator.
It is unclear if the σ54 promoter will function in E. coli in the absence of mmoR. nBLAST results comparing the mmoR to the E. coli genomes available yielded very short hits, with the top hit showing matches for less than 200bp out of the possible 2070bp length of mmoR. However, the pBLAST hits showed stronger hits with other σ54-dependent transcriptional activators found in E. coli. The top nBLAST and pBLAST hits are shown below.
To determine if the σ54 promoter will function without mmoR present in E. coli, we PCR amplified 395bp of the σ54 promoter and cloned the product into the pSB1C3 BioBrick backbone. We then placed the promoter in front of GFP, RFP, and YFP using standard BioBrick cloning.
We also amplified the same region of the σ54 promoter using MoClo primers and cloned it into one of our Level 0 MoClo destination vector.
