Expert Tutors:
Prof Mike Young - UCW, UK
Dr Peter Mullany - UCL, UK

Introduction:
University of Nottingham scientists have recently adapted the L1.LtrB Group II intron to create a clostridial plasmid (pMTL007) which allows for the positive selection of gene knockouts in a variety of clostridial species. This is made possible through the use of an IPTG-inducible plasmid to regulate production of group II RNA, and the creation of a Retrotranspositional Activation Marker (RAM) based on an erythromycin resistance gene. Successful retargeting of the modified group II intron to the gene of interest may therefore be selected on the basis of acquisition of erythromycin resistance. The system has been tested in 3 different clostridial species (Clostridium difficile, Clostridium acetobutylicum and Clostridium sporogenes) with a 100% success rate. Typically, the number of mutants obtained per experiment number in the 100s, and from start to finish are generated within 8 days. These data suggest that this system may be universally applied to Clostridium sp and other Gram-positives, and open up the possibility of revolutionizing the assignment of function to clostridial genes identified by genome sequencing.

Aim:
The overall aim of the workshop is to undertake the key steps involved in the process of gene knock-out in three clostridial species, C. difficile, C. acetobutylicum and C. sporogenes. As part of this experimental process, you will be taught how to introduce plasmids into clostridia by electrotransformation and conjugative transfer from E. coli donors. You will also be taught how to design and construct the pMTL007 target vectors, and how to screen for the desired knock-out at the end of the process.
Interspersed within the practical modules, our expert tutors will deliver informal lectures on key aspects of the molecular genetics of clostridia. These will include, conjugative plasmid transfer, conjugative transposons and aspects of applied microbiology.

Schedule:
The precise details of the course modules and schedules will be given to attendees on arrival. A rough outline is given below:

DAY 1: Tuesday 12 September 2006

• Instruction in design of oligonucleotide pimers
• Generation of retargeting cassette by SOEing PCR
• Cloning of cassette into pMTL007

DAY 2: Wednesday 13 September 2006

• Conjugation of vector into Clostridium difficile
• Conjugation of vector into Clostridium sporogenes
• Transformation into Clostridium acetobutylicum

DAY 3: Thursday 14 September 2006

• Authentication of transconjugants/transformants
• Induction of pMTL007 retargeting

DAY 4: Friday 15 September 2006

• Isolation of retargeted clones
• Authentication by PCR screening
• Identification of gene knock-outs

All accommodation and meal costs will be met and paid for by the organisers.

Travel expenses will be reimbursed on production of original receipts*.
*- the average travel expenses are assumed to be 297 EURO

For details of how to get to Nottingham, please go HERE