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From 2013.igem.org

Aer

 

Aer

Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.

 

EnvZ

Overview
EnvZ is the histidine kinase in the EnvZ-OmpR signal transduction system, located in the inner membrane. It contains two separate transmembrane regions. EnvZ autoposphorolates, then phosphorotales the transcription factor OmpR. EnvZ and OmpR are often seen as a single, polysistronic mRNA, although there is normally much less OmpR present in any given cell.

Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.
We chose Aer because it is a more recently studied gene, so little is known about it. According to Nishiyama , Aer also has temperature sensing potential, so this could be another interesting aspect our project.
Our construct consists of an Aer-EnvZ chimera protein created view overlapping extension PCR (OEPCR), and a transcription factor, OmpR. EnvZ is a histidine kinase that communicates with OmpR.

 

KanR

KanR provides kanamycin resistance to colonies with successful transformations.

 

pBR322

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

 

OmpR

OmpR is the transcription factor regulated by EnvZ. It is found in the cytosol. OmpR is phosphorolized by the kinase EnvZ, making the two dependent on each other. OmpR’s N-terminus must be phosphorolized by EnvZ in order to allow transcription of certain transmembrane genes; the C-terminus moderates multimerization of OmpR, which in turn controls transcription of outer membrane proteins..

 

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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