Part:BBa_K2213001

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.

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 1.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2190
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1516