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

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

Sep 03, 2022 351 likes •645 views

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

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SLIDE 1

NICE

Leicester iGEM Team

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

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The Team

  • 7 Undergraduate Students -

Biological Scientists

  • 2 Supervisors - Professor

Dalgleish and Dr Badge

  • 1 Advisor - Emily Halsey (2012)
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Our thought process

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

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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

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Colonisation

http://2014.igem.org/Team:Imperial

Aqualose

http://www.nature.com/nrmicro/journal/v2/n2/fig_tab/nrm icro818_F2.html

BFP (E) CofA (A) Fibronectin

http://nptel.ac.in/courses/102103012/8

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SLIDE 7
  • BFP (Bundle Forming Pilus) gene about 14kb
  • Found in Enteropathogenic E. coli (EPEC)
  • Produces a type IV pilus
  • Grouped due to pilin subunit protein
  • Found in gram negative bacteria
  • Function varies
  • Generates motile forces
  • Adherence
  • Strong
  • Flexible

Colonisation

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SLIDE 8

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.
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Why increase NAD+?

Can treat Neurodegenerative disorders Mitigates destruction of neurons Increased oxidative phosphorylation Can combat muscle fatigue Muscle fibre conversion Increased oxidative phosphorylation

Also associated with ageing

....And we think this is better than supplements!

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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+
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Periplasmic Export

  • NAD+ cannot pass through the cell membrane
  • So export the enzymes that synthesise NAD+
  • The TAT(Twin Arginine Transport) system naturally
  • ccurs in E. coli

Palmer, Tracy, and Ben C. Berks. 'The Twin-Arginine Translocation (Tat) Protein Export Pathway'. Nature Reviews Microbiology (2012)

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BioBricks

  • Consisting of:
  • Promoter
  • Ribosomal binding site

(RBS)

  • TAT Signal Peptide
  • The gene of interest
  • Also BioBrick the genes

without the promoter, RBS and TATSP

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Kill switch

  • A temperature

dependant toxin/antitoxin kill switch using CCDB/CCDA

  • Kills bacteria that

leave body

Temperature SENSITIVE RBS Temperature INSENSITIVE RBS

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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...
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SLIDE 15

Results

Had a very low transformation efficiency or...

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Future Considerations

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

Public Engagement / Dialogue-

  • Global survey with other teams
  • Survey for the Aix-Marseille team
  • Created our own survey
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SLIDE 18

Survey

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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)

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Raising funds

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Sponsored Walk (Walkathon)

~26 miles The previous year have done the

  • 26 miles
  • Along Grand Union Canal
  • In the torrential rain!
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Parkinson’s UK

400 feet!!

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Interlab measurement study

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We concluded from results that:

  • Device 1 - very strong promoter
  • Device 2 - fairly strong promoter
  • Device 3 - very weak promoter
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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

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SLIDE 26

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!!
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Thank you for listening;

Any Questions?