Part:BBa_K2992035

Acetone pathway: csp_Pfdx-5-UTR+RBS-ca_thl-ca_ctfAB-cp_TFdx

Botulinum toxin-predicting acetone production pathway with C. sporogenes Pfdx driving expression of C. acetobutylicum-derived ctfAB, 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 BBa_M36585 coupled with the two units of the ctfAB complex M36581 BBa_ M36581 and M36582 BBa_ M36582 which are all derived from C. acetobutylicum. This operon is regulated by the promoter BBa_K2992016 and associated 5’UTR+RBS BBa_K2992017 from the ferredoxin fdx gene of C. sporogenes.Transcriptional termination for this synthetic acetone-production operon occurs through the activity of Tfdx from C. pasteurianum 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 569
  Illegal PstI site found at 2136
  Illegal PstI site found at 3401
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2136
  Illegal PstI site found at 3401
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 569
  Illegal PstI site found at 2136
  Illegal PstI site found at 3401
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 569
  Illegal PstI site found at 2136
  Illegal PstI site found at 3401
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Heap Modular Cornillo et al 1997