Part:BBa_K2213002

araB_eutLK

The ethanolamine utilisation bacterial microcompartment (BMC) proteins, EutLK (from E. coli) under control of the arabinose inducible promoter. Contains RBS, terminators and all tetp components. EutL is tagged with His6. EutK is tagged with FLAG (see figure 1).

Fig 1: Schematic of the araBAD_EutLK part (BBa_K2213002)

araBad Promoter

The ara expression system is advantageous as in addition to being induced by arabinose, gene expression can be rapidly downregulated as repression can occur due to catabolite repression in the presence of glucose or by competitive binding of the anti-inducer fucose. Plasmids containing the ara expression system have minimal levels of background expression (Siegele and Hu, 1997).

EutLK

Characterisation

Understanding growth defects upon EutLK protein expression
Manchester iGEM 2017 recorded optical density measurements at 600nM for EutS, EutMN, EutSMN and EutLK. OD measurements were taken at 0 hours, 4 hours and at 20 hours (see figure 2). It was observed that between 4 and 20 hours, the OD of cultures containing the constructs EutMN, EutSMN and EutLK were reduced by 75.53%, 81.77% and 67.93% respectively. In contrast to this, the OD of the EutS culture continued to rise and had increased by 45.28% when the final reading was taken at 20 hours. This suggests that the production of microcompartment subunits EutM, EutN, EutL and EutK are toxic to the cell, however, the production of EutS may be less toxic. This may be due to less strain being put on the cell due to the expression of a single microcompartment subunit, rather than multiple subunits being expressed simultaneously. Overall this data indicates that the expression of complete microcompartments is likely to be toxic to the cell and should be highly regulated.


Figure 2. Average optical density at 600 nM of EutS, EutMN, EutSMN constructs induced and non-induced. Measurements were taken at 0 hours, 4 hours and 20 hours.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1300
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1239
  Illegal BamHI site found at 2164
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1074
  Illegal AgeI site found at 1775
  Illegal AgeI site found at 1952
  Illegal AgeI site found at 2252
  Illegal AgeI site found at 2315
  Illegal AgeI site found at 2362
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1056
  

References Siegele, D.A. and Hu, J.C., 1997. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences, 94(15), pp.8168-8172.