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From 2013.igem.org

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Revision as of 03:22, 6 July 2013

HemAT

 

HemAT

Oxygen gas, sensor FixL or HemAT (also need HemC). list of Direct Oxygen sensors . FixL, was fused with 5 different kinases : “it was likely that the autophosphorylation activity of the histidine kinase domain could not be regulated by the O2 association/dissociation in the sensor domain in the chimeric proteins” Intracellular & redox sensor.  O2 sensor, would be interesting.  & may also be able to sense  CO in the absence of oxygen  It seems like FixL is an O2 sensor. 

Sensing domain is from 1-186, its cytoplasmic, so it might not work on EnvZ.  It also does not HAMP domain so we do not think it would work on a transmembrane kinase domain.

Chimeric sensory kinases containing O2 sensor domain of FixL and histidine kinase domain from thermophile.

Oxygen‐Sensing Histidine‐Protein Kinases: Assays of Ligand Binding and Turnover of Response‐Regulator Substrates.

Heme-based oxygen sensor protein FixL: its structure and function.

Direct oxygen sensor protein.

Amino Acid Sequence:
MLFKKDRKQETAYFSDSNGQQKNRIQLTNKHADVKKQLKMVRLGDAELYVLEQLQPLIQENIVNIVDAFYKNLDHESSL

MDIINDHSSVDRLKQTLKRHIQEMFAGVIDDEFIEKRNRIASIHLRIGLLPKWYMGAFQELLLSMIDIYEASITNQQELLKA

IKATTKILNLEQQLVLE

 

KanR

KanR provides kanamycin resistance to colonies with successful transformations.

 

pBR322

The pBR322 ori is a low copy origin of replication derived from

 

 

NtrB

Details for NtrB coming soon.Details for NtrB coming soon.Details for NtrB coming soon.

 

NtrC

Details for NtrC coming soon.Details for NtrC coming soon.Details for NtrC coming soon.

 

T7 Promoter

The T7 promoter is constitutively expressed by the phage T7 RNA polymerase. The polymerase is orthogonal to the wildtype E. coli promoters.

 

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Gel samples

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Flow sample

 

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