Lab rotation project description
The mini project will serve as an introduction to the available gene tools to be deployed in Clostridium pasteurianum. The student will:-
(i) receive a safety induction to cover safe laboratory practice within the confines of SBRC laboratories and equipment within the Centre;
(ii) be trained in the use of anaerobic cabinets and the cultivation of Clostridium pasteurianum;
(iii) undertake CRISPR/Cas9-mediated and ClosTron-based gene knock-out (www.clostron.com);
(iv) become trained in the application of ACE technology, by integrative correction of a mutant pyrE allele in the chromosome.
Through all these procedures, the student will learn the basics techniques required to undertake the project. This will include, how to electro-transform and conjugate from E.coli into Clostridium, how to screen putative mutants for intron/ transposon integration by colony PCR and agarose gel electrophoresis, and how to derive nucleotide sequence data covering the intron/transposon insertion site. They will also be training in the use of the required DNA analysis software, including DNASTAR, CLC Bio workbench and the Intron Design Tool at www.clostron.com.
LR1, LR2 and LR3
One of the greatest challenges facing industry and society are the future sustainable production of chemicals and fuels from non-food feedstocks while at the same time reducing Green House Gas (GHG) emissions. To compete with existing petrochemical-based chemical manufacturing processes, low cost feedstocks for biological fermentation processes are essential, since the feedstock typically equates to over 60% of the overall production cost. To date, the focus has been to use lignocellulosic feedstocks. Their use is reliant on an energy intensive pre-treatment step, and thereafter, the addition of costly exogenous hydrolytic enzymes needed to convert the partially deconstructed biomass into the sugars needed by the fermentative process organisms. The costs involved are making the development of economic processes extremely challenging.
AIM: Glycerol is a versatile carbon and energy source and presently it is produced in large scale as the principle by-product (10% w/w) of the biodiesel industry where the rapidly expanding market for biodiesel has dramatically altered the cost (for crude glycerol, prices have decreased from $0.25 per pound to $0.05 per pound) and availability of glycerol. It is now essentially a waste product of biodiesel industry and it has been estimated that the production of crude glycerol from biodiesel industry will reach 37 billion gallons by 2016. Clostridium species, and in particular C. pasteurianum, can grow particularly well on glycerol as a sole carbon and energy source and transform it into a number of useful chemicals such as 1,3-propanediol (PDO), acetone, butanol, ethanol etc. Butanol has many superior properties over ethanol as an alternative fuel. It has a higher energy content (110,000 Btu’s per gallon) than ethanol (84,000 Btu per gallon for ethanol), and its low vapour pressure and its tolerance to water contamination in petrol blends facilitate its use in existing petrol supply and distribution channels. Moreover, butanol can be blended with petrol at higher concentrations than existing biofuels, without the need to retrofit vehicles and offers better fuel economy than petrol-ethanol blends.
STRATEGY: Until now, the opportunity to use Clostridium strains for the high level production of butanol from glycerol has been limited due to the lack of genetic engineering tools. However, the requisite tool box needed for the optimisation of the metabolic pathways required for butanol production by Clostridium pasteurianum has been developed within the Clostridia Research Group of SBRC Nottingham. It is the purpose of this PhD project to use synthetic biology approaches and the University of Nottingham tool box to generate an engineered strain of C. pasteurianum able to over-produce butanol using glycerol as a feedstock. This studentship will focus on the identification of the bottlenecks in butanol production through a novel approach recently devised at Nottingham, and thereafter implement strategies to circumvent them, through the use of proprietary methods for gene knock-out and knock-in, orthogonal expression and a novel method for directly selecting compensatory SNPs. Strategies that result in the overproduction of PDO will also be implemented.
TRAINING: This translational project will be carried out within the BBSRC/EPSRC Synthetic Biology Research Centre (SBRC) at Nottingham which comprises 70+ graduate and postdoctoral researchers (www.clostron.com/people.php) and a current budget of £27M. The study will allow for training in a unique multidisciplinary environment, incorporating anaerobic gas fermentation, Synthetic Biology, microbial physiology, metabolic engineering and computer modelling. The project forms part of a Colombia-UK collaboration providing the student with the opportunity to visit collaborators in Cali.