Difference between revisions of "Part:BBa K2213001"

Revision as of 18:00, 5 September 2017

Tet_EutMN

Ethanolamine Utilisation (Eut) bacterial micro-compartment (BMC) proteins EutM and EutN from E.coli , placed under the inducible Tetracycline promoter. Also contains RBS, terminators and all tetp components – thus this part alone can be used to synthesise EutM and EutN at varying concentrations, relevant to the experimental task. EutM is tagged with GFP and His6. EutN is tagged with FLAG. See Figure 1.

Tetracycline Promoter

The tetracycline expression system is based on two regulatory elements, the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO); both derived form the tetracycline-resistance operon of the E.coli Tn10 transposon. In our part, tetracycline (Tc) can bind rTetR, increasing its affinity for DNA binding. Thus upon Tc addition, rTetR can bind tetO, permitting transcription of any gene under control of the tet promoter. This is illustrated in Figure 2.

Several iGEM teams have previously carried out characterisation and worked with the Tet promoter system. For your interest and research we recommend looking at the HQ submitted part BBa_R0040 (https://parts.igem.org/Part:BBa_R0040) as a starting point.

EutM and EutN

The Ethanolamine Utilisation (Eut) bacterial micro-compartment (BMC) proteins EutM and EutN from E.coli are clustered together here, as they are found in nature. Coming soon...

Usage and Biology

Although it is possible to use this part for EutM and N expression without further assembly, we do not recommend doing this if the ultimate goal is to produce fully functional Eut BMCs. When forced to produce BMCs, E. coli are placed under a large amount of strain and begin to experience slowed and abnormal growth. Therefore, we suggest using a low copy number plasmid eg. pSB4A5 (https://parts.igem.org/Part:pSB4A5), as we have used in our project. By using a low copy number plasmid, cellular stress is minimised, but the experimenter still has the ability to induce BMC formation.

Sequence and Features