Part:BBa_K3265026

EutSMN bacterial microcompartment

This is an improved version of part BBa_K2213012 from team Manchester 2017. We down-regulated the base expression by exchanging the RBS to a the very weak RBS J61100. EDIT

Characterization

To test if our part could now produce the EutM and EutN proteins at levels low enough so that it would not be toxic to the cells anymore, we redid the OD measurement with the original EutSMN part.

Procedure

100mL LB cultures were inculcated with a single colony from an overnight transformation of pSEVA441 vector carrying our improved EutSMN part. E. coli strain DH5alpha was used.

Cultures were grown for 4 hours at 37° 300 rpm and then induced with 4.5 ng/mL Anhydrotetracylcine (ATC) or 45 ng/mL Tetracycline (Tetc) and 1mM Arabinose ((+)araB) each. One culture was not induced (control).
All concentrations are to be interpreted as final concentrations in the liquid culture. 45 ng/mL Tetc/ATC = 0.1 uM

Measurements were taken each hour for 9 hours and a final measurement was taken after 20 hours.
ATC was used at 10x less concentration since its a much stronger inducer than Tetc and has lower toxicity.

Results

It seems that our very weak RBS J61001 is doing a good job at keeping the base expression levels low enough so that not a lot of the protein is produced.

Imaging

We confirmed with imaging that the protein is produced at high enough levels to form compartment:

This picture was taken 10 hours past induction, the signal does not seem very strong but there were a moderate number showing micrompartments.

The control was also imaged:

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1293
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1232
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1067
  Illegal AgeI site found at 3824
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3212