Projects

Systems Biology of Clostridium acetobutylicum - a possible answer to dwindling crude oil reserves (COSMIC-SysMO)

Clostridium are bacteria which evolved before the earth had an oxygen atmosphere. To them the air we breathe is a poison. To survive they produce a spore resting stage, resistant to physical and chemical agents. Some species cause devastating diseases, such as the superbug Clostridium difficile. On the other hand, most are totally harmless, and make a wide range of chemicals useful to man. The best example is Clostridium acetobutylicum which makes butanol. Butanol is an alcohol, which may be used as a replacement for petrol. Despite their importance, our understanding of their biology has lagged behind that of the more recently evolved bacteria which ‘breathe’ oxygen. The aim of COSMIC is to use a systems biology approach to allow more accurate predictions of the behaviour of clostridia at the cellular level.

 

ClosTron Gene Knock-out System

  • Changing 15 bp (EBS1 & EBS1d) retargets group II intron + ErmRAM to any gene sequence
  • Retargeting region (350bp) is generated by SOEing PCR and cloned between HindIII – BsrGI sites
  • Retargeted plasmid introduced into Clostridia by either electroporation or conjugation
  • Retargeting of the intron induced by addition of IPTG
  • Retargeted clones selected as EmR colonies - 100s obtained!!
  • Insertion in correct gene checked by simple PCR
  • Process takes 8 -10 days

Experimental Setup

  • Common strain ATCC 824 used by all partners
  • Common experimental setup:
    • Chemostat (phosphate limited)
    • pH 6.0: Acids; pH 4.5: Solvents
    • Cell density (via phosphate concentration)
    • Sporulation (via batch culture)
  • Common standards and protocols for Omics analysis

Modelling and Simulation

The Workpackages