Part:BBa_K2992029

Acetone pathway: cb_PntnH-5-UTR+RBS-ca_thl-cb_ctfAB-cp_TFdx

Botulinum toxin-predicting acetone production pathway with C. botulinum PntnH driving expression of ctfAB and theC. acetobutylicum genes thl and adc.

Usage and Biology

This parts entry represents an acetone-production pathway for plasmid-borne expression in C. sporogenes for predicting botulinum neurotoxin production. The entry comprises the thiolase gene thl BBa_K2992008 and acetoacetate decarboxylase adc gene of C. acetobutylicum BBa_M36585 coupled with the two units of the ctfAB complex from C. botulinum BBa_K2992003 and BBa_K2992005 separated by their native intergenic region containing a partial RBS sequence for ctfB BBa_K2992007. This operon is regulated by the promoter BBa_K2992001 and associated 5’UTR+RBS BBa_K2992015 from the non-toxic non-haemagglutinin ntnH gene of C. botulinum.Transcriptional termination for this synthetic acetone-production operon occurs through the activity of Tfdx from C. pasteurianum K2284012 BBa_K2284012. In our project we used genome-scale modelling to predict the necessary genes required to produce acetone in our chosen surrogate strain C. sporogenes. We sought to link acetone production with C. botulinum neurotoxin production by the integration of the neurotoxin transcriptional regulator botR onto the chromosome of C. sporogenes and by using promoter regions from the regulon of botR to control the acetone-production operons. In doing so, we hoped to generate our surrogate host strain as a model for predicting neurotoxin production in foodstuffs following food manufacturing processes.

Characterisation

Data incoming.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 530
  Illegal PstI site found at 3425
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 3425
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 530
  Illegal PstI site found at 3425
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 530
  Illegal PstI site found at 3425
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Heap Modular Cornillo et al 1997