Biotechnology and Biological Sciences Doctoral Training Programme
   
   
  

Regulation of clostridial butanol production by cell-cell communication

 

Lab rotation project description

This   mini project will serve as an introduction to anaerobic microbiology and the   biology of the genus Clostridium. Its scientific objective will be the   phenotypic characterisation of a C. acetobutylicum quorum sensing mutant. 

The student will: 

1. Be trained in anaerobic   microbiological methods and techniques such as the use of anaerobic cabinets   
2. Be given a C. acetobutylicum mutant   defective in a quorum sensing gene; this will be grown in batch culture,   together with a wild-type control and in the presence and absence of   synthetic signal molecules
3. Learn to record the growth of these   cultures by taking optical density readings, colony-forming-unit counts, and   determination of total protein
4. Microscopically examine these   cultures and learn to recognise vegetative cells, clostridial forms, and   endospores
5. Determine the concentration of   fermentation products in culture supernatants by gas chromatography
6. Learn how to determine granulose   content and to perform endospore counts
7. Learn how to set up plate assays to   identify bacterial signal molecules
8. Learn   how to exploit previously obtained RNA seq data for experimental design and   hypothesis testing
9. Learn to critically analyse data,   qualitatively and quantitatively, and in comparison to the literature

Fact file

Research theme

IBB

Location

Life Sciences

Rotation

LR1, LR2 and LR3

Contact

2nd supervisor


BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

Several anaerobic Clostridium species are well known for their ability to convert sugars and   starches into organic acids and solvents. During the first half of the last   century, these bacteria were used for the industrial production of acetone   and butanol, but today the classical AB (acetone-butanol) fermentation   process is no longer economically viable. Thus, considerable efforts have   been devoted to improving the organisms’ performance through metabolic   engineering. However, decisive breakthroughs are yet to be made. A major   reason for this is our limited understanding of the organisms' physiology and   metabolism, in particular the mechanisms that govern timing and extent of   solvent formation.
  

In a previous PhD project, we discovered a total of ten quorum sensing   systems in C. acetobutylicum which enable individual cells of a population to communicate with one another via diffusible signal molecules.  At least seven of these systems were shown   to strongly influence the production acetone and butanol, as well as sporulation, but the underlying molecular mechanisms remain unknown. However, a thorough understanding of the physiological factors and regulatory mechanisms constraining solvent formation is a prerequisite for successful metabolic engineering.
    
 The aims of the proposed PhD study are therefore to
    (i) obtain a detailed understanding of the transcriptional, translational,   and physiological changes occurring in quorum sensing-deficient mutants;  
    (ii)  identify the genes directly   regulated by quorum sensing;
    (iii) exploit this knowledge to generate strains in which butanol formation   can be maximised.
    
Selected quorum sensing systems will be characterised in detail, using targeted mutagenesis, fluorescent reporter systems, and state-of-the-art ‘omics’ techniques including RNA-seq, ChIP-seq and iTRAQ-based proteomics.
 Next generation sequencing (RNAseq) will be carried out to compare the transcriptomes of selected quorum sensing mutants with that of the wild type. Genes found to be differentially expressed will be confirmed by RT-PCR and   phenotypic analyses (based on the genes’ predicted functions). Chip-seq will  be used to identify those directly controlled by quorum sensing. This subset will be investigated further through inactivation and overexpression to   establish its role in butanol formation and sporulation. Based on the   outcome, strains will be constructed that combine several of these modifications and tested for butanol production. If appropriate a high throughput robotics platform will be utilised to facilitate these analyses.
    
In a parallel approach, signal molecules produced by C. acetobutylicum will   be isolated from culture supernatants and identified using liquid chromatography combined with mass spectrometry. The influence of synthetic   quorum sensing signals on butanol formation, when added back to cultures, will be investigated.
    
Training: The project offers training in anaerobic microbiology, advanced microbial   genetics, next generation sequencing (RNAseq.ChIP-seq), high-throughput   robotics, molecular biology, continuous culture systems, gas/liquid   chromatography, mass spectrometry, and metabolic engineering. We are part of the BBSRC/EPSRC Synthetic Biology Centre Nottingham and have strong   links  with groups in the   Bioenergy/Renewables sector in Europe, the US, China and India, providing ample opportunity to take part in international conferences, workshops, and exchange programmes.
    
 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk