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Team:BostonU/Data
From 2012.igem.org
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| - | <ul><h7>While we worked on making our MoClo parts, we began learning about how to characterize genetic devices that had fluorescent protein markers. One of those devices was an inverter under pBad (BBa_I13458-BBa_I13453) control. This inverter was built using BioBricks and was submitted with the rest of our parts this year (<a href="http://partsregistry.org/Part:BBa_K783067">BBa_K783067</a>). | + | <ul><h7><p dir="ltr">While we worked on making our MoClo parts, we began learning about how to characterize genetic devices that had fluorescent protein markers. One of those devices was an inverter under pBad (BBa_I13458-BBa_I13453) control. This inverter was built using BioBricks and was submitted with the rest of our parts this year (<a href="http://partsregistry.org/Part:BBa_K783067">BBa_K783067</a>). |
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| - | + | We began to work on our characterization workflow in September because we had originally planned to have MoClo Level 1 and Level 2 parts to test by then. Unfortunately, due to many technical challenges, we have not yet produced a Level 1 or Level 2 part to test. Instead, we decided to test devices that had been made in our lab using BioBricks so we could begin to learn how to characterize devices using flow cytometry. | |
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Revision as of 21:44, 3 October 2012
Data Collected
While we worked on making our MoClo parts, we began learning about how to characterize genetic devices that had fluorescent protein markers. One of those devices was an inverter under pBad (BBa_I13458-BBa_I13453) control. This inverter was built using BioBricks and was submitted with the rest of our parts this year (BBa_K783067).
We began to work on our characterization workflow in September because we had originally planned to have MoClo Level 1 and Level 2 parts to test by then. Unfortunately, due to many technical challenges, we have not yet produced a Level 1 or Level 2 part to test. Instead, we decided to test devices that had been made in our lab using BioBricks so we could begin to learn how to characterize devices using flow cytometry.
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.
