Medieval Blue Genes: Reducing Industrial Indigo Dye Pollution of the Environment

BACKGROUND:
The current industrial process for the dyeing of cotton yarn for denim jeans consumes about 2 kg of sodium dithionite for every 3 kg of indigo reduced. Disposal of the by-products of dithionite oxidation is a significant environmental burden. In the Middle Ages, cloth was dyed in a fermenting woad vat, which dissolved the added indigo by reducing it to a soluble form. Recently, when mediaeval techniques of woad preparation and fermentation were reproduced, the bacterium responsible for the reduction of indigo was identified as Clostridium isatidis [Padden et al, Nature 1998; 396:225]. Proof that this was indeed the bacterium responsible for the medieval process was subsequently obtained when spores of this Clostridium were revived from a 1000-year-old Viking woad vat at an archaeological site in York [John, Biologist 2006; 53:29-33].

The environmental damaged caused by the currently employed synthetic indigo process are of increasing concern to society. A return to the natural woad-based process could have real benefit, particularly if the effectiveness and reproducibility of the process could be improved. Rational changes are, however, not possible as the mechanism by which the bacteria reduce indigo remain unknown. The recent development of advanced gene tools for forward and reverse genetics in clostridia now provide the means by which insight into the process may be gleaned.

AIM:
The student will undertake a random transposon mutagenesis of an isolate of C. isatidis recently identified in a cellulolytic anaerobic consortium by the Clostridia Research Group (CRG). By screening for colonies that fail to form blue colonies on agar media containing indigo, mutants affected in its reduction will be identified. Their role and function of essential genes will thereafter be determined using a combination of genetic and biochemical assays and whole genome sequencing

STRATEGY:
The student will work with Clostridium isatidis isolated by the CRG. Initially its entire genome sequence will be determined using Next Generation Sequencing. Thereafter, through the introduction of an orthogonal expression system into the genome using ACE technology [Heap JT et al 2012; Nucl Acids Res 40:e59], mariner transposon mutagenesis will be implemented [Zhang et al. 2015, PLoS One; 10(4):e0122411]. Libraries will be screened for organisms unable to reduce indigo using a simple agar plate assay (colourless colonies as opposed to blue). The identity of inactivated genes will be confirmed through nucleotide sequencing of inverse PCR products, and the functionality of the gene confirmed by deliberate knock-out using ClosTron technology [www.clostron.com]. Thereafter, the role of the identified genes/proteins in indigo reduction will be determined using a combination of genetic and biochemical assays.

TRAINING:
Nottingham’s Synthetic Biology Research Centre (SBRC) are renowned experts in Clostridium genetics, and with 100+ postdoctoral/ graduate researchers represent the largest clostridial molecular biology group in the world. The project offers training in anaerobic microbiology, advanced genetics, Next Generation Sequencing, bioinformatics, fermentation and microbial physiology.