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
LR1, LR2 and LR3
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.