NICE Leicester iGEM Team Nicotinamide Adenine Diphosphate (NAD + ) - PowerPoint PPT Presentation

NICE Leicester iGEM Team

Nicotinamide Adenine Diphosphate (NAD + ) Increased through Colonisation of Escherichia coli

The Team ● 7 Undergraduate Students - Biological Scientists ● 2 Supervisors - Professor Dalgleish and Dr Badge ● 1 Advisor - Emily Halsey (2012)

Our thought process Neurodegenerative disorder / Muscle fatigue Bacterial Organ Help treat a Human Genes in NAD + Disorder biosynthesis PncB NadD NadE

What we planned to do: ➔ Colonise the gut with E. coli K-12 ➔ Upregulate the enzymes: ◆ NadD- Nicotinamide mononucleotide adenylyltransferase (NMNAT) ◆ NadE - NAD synthetase ◆ PncB - Nicotinate phosphoribosyltransferase ➔ Increase availability of NAD + ➔ Export NAD + to be absorbed into the blood ➔ Build in kill switches to control colonisation

Colonisation http://2014.igem.org/Team:Imperial Aqualose CofA (A) Fibronectin BFP (E) http://www.nature.com/nrmicro/journal/v2/n2/fig_tab/nrm http://nptel.ac.in/courses/102103012/8 icro818_F2.html

Colonisation • BFP (Bundle Forming Pilus) gene about 14kb • Found in Enteropathogenic E. coli (EPEC) • Produces a type IV pilus o Grouped due to pilin subunit protein o Found in gram negative bacteria o Function varies  Generates motile forces  Adherence o Strong o Flexible

Increased NAD + Taken From: ZHOU, Y., WANG, L., YANG, F., LIN, X., ZHANG, S. and ZHAO, Z.K., 2011. Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD(+)-Auxotrophic Mutant. Applied and Environmental Microbiology, 77(17), pp. 6133-6140.

Why increase NAD + ? Can treat Neurodegenerative disorders Mitigates destruction of neurons Also Increased oxidative phosphorylation associated with ageing Can combat muscle fatigue Muscle fibre conversion Increased oxidative phosphorylation ....And we think this is better than supplements!

Why is NICE nicer than supplements? • NICE is an autonomous, one-off treatment • NICE does not need to be injected • NICE provides a continual supply of NAD +

Periplasmic Export ● NAD + cannot pass through the cell membrane ● So export the enzymes that synthesise NAD + ● The TAT(Twin Arginine Transport) system naturally occurs in E. coli Palmer, Tracy, and Ben C. Berks. 'The Twin-Arginine Translocation (Tat) Protein Export Pathway'. Nature Reviews Microbiology (2012)

BioBricks • Consisting of: o Promoter o Ribosomal binding site (RBS) o TAT Signal Peptide o The gene of interest • Also BioBrick the genes without the promoter, RBS and TATSP

Kill switch • A temperature dependant toxin/antitoxin kill switch using CCDB/CCDA • Kills bacteria that leave body Temperature SENSITIVE RBS Temperature INSENSITIVE RBS

What we actually did in the lab • Had a lot of fun! • Took part in the Measurement Study • Designed primers for PCR • Amplified NadD, NadE and PncB genes • Attempted to make them BioBrick compatible • Transformed gBlocks for the BioBricks from IDT into competent cells to make BioBricks • But...

Results Had a very low transformation efficiency or...

Future Considerations • Delivery? o Freeze dry the bacteria in an acid resistant capsule • Colonisation? o Insert BFP gene • Regulation? o Kill switches o Regulate NAD + level • Other disorders?

Human Practices Public Engagement / Dialogue- • Global survey with other teams • Survey for the Aix-Marseille team • Created our own survey

Survey

Human Practices Public Engagement / Dialogue - Online Surveys Ethics - Our survey asked the public what ethical problems they have with the project Education - Lesson plan, Talks in schools (Liam and Ross), GENIE Dynamic DNA (Liam)

Raising funds

Sponsored Walk (Walkathon) ~26 miles ● 26 miles ● Along Grand Union Canal The previous year have done the ● In the torrential rain!

Parkinson’s UK 400 feet!!

Interlab measurement study

We concluded from results that: ● Device 1 - very strong promoter ● Device 2 - fairly strong promoter ● Device 3 - very weak promoter

A NICE Summary • An addition to the native microbiome • Make E. coli export NAD + • Increased intracellular [NAD + ] • Treats neurodegenerative diseases, e.g. Parkinson's disease • The E. coli die on leaving the body https://commons.wikimedia.org/wiki/File:Digestive_system_diagram_edit.svg

Acknowledgements and Attributions Parkinson’s UK ● New England Biolabs ● Starlabs UK ● Heathrow sci ● Fisher sci ● IDT ● Promega ● S.Stibbards & Sons ● UoL Genetics Department ● And all our sponsors!! ●

Thank you for listening; Any Questions?