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SPS ingapore Controlled missiles: Targeted treatment of tumors with - - PowerPoint PPT Presentation

Aug 25, 2022 156 likes •579 views

SPS ingapore Controlled missiles: Targeted treatment of tumors with engineered E. coli Team SPSingapore In a Nutshell Biological Programming Simplifying Circuits In a Nutshell Biological Programming HP: Simplifying Circuits In a Nutshell

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SPSingapore

Controlled missiles: Targeted treatment of tumors with engineered E. coli

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Team SPSingapore

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In a Nutshell Biological Programming HP: Simplifying Circuits

In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

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Quorum Sensing

In a Nutshell Biological Programming Simplifying Circuits

O2

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Quorum Sensing

Prevents bacterial activity unless sufficient bacteria are present

In a Nutshell Biological Programming Simplifying Circuits

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O2 O2 O2

Anaerobic switch

Prevents bacterial activity outside of the hypoxic tumour region

O2 O2 O2

In a Nutshell Biological Programming Simplifying Circuits

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O2 O2 O2

QS OFF Anaerobic ON

No expression

QS ON Anaerobic OFF

No expression

In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

Anaerobic switch Quorum sensing

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In a Nutshell Biological Programming Simplifying Circuits

In a Nutshell Biological Programming HP: Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

Anaerobic switch Quorum sensing

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In a Nutshell Biological Programming Simplifying Circuits

Anaerobic switch Quorum sensing

aaaaaaaaaa

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nirB/FNR anaerobic system

  • nirB promoter is anaerobic-responsive

In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

Anaerobic switch Quorum sensing

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EsaR/EsaI quorum sensing system

In a Nutshell Biological Programming Simplifying Circuits

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Anaerobic switch Quorum sensing

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Invasin + Listeriolysin effector proteins

  • Updated documentation of

BBa_K299812

  • Sequence differs significantly

from parts registry

  • BioBricks suffix is missing in

construct (Pstl only)

Invasin Listeriolysin In a Nutshell Biological Programming Simplifying Circuits

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Invasin + Listeriolysin effector proteins

  • Updated documentation of BBa_K299812
  • Listeriolysin is ‘codon-optimised’
  • Invasin is 300bp longer
  • Invasin matches Yersinia enterocolitica

Listeriolysin Invasin

4146bp in parts registry Documented

Listeriolysin

4460bp from sequencing Sequenced

Invasin +300bp

In a Nutshell Biological Programming Simplifying Circuits

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Invasin + Listeriolysin

  • Constitutive expression of Invasin and Listerolysin

(From BBa_K299812)

Listeriolysin Invasin Evaluate ability of part to confer invasion phenotype and escape from endosome

Mammalian cell

Positively-regulated by FNR protein in Anaerobic conditions

Invasin

PnirB

Plac

In a Nutshell Biological Programming Simplifying Circuits

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Testing our tools: PnirB-Invasin vector

  • There was increased invasion under anaerobic conditions

In a Nutshell Biological Programming Simplifying Circuits

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Testing our tools: Plac-GFP

  • Plac promoter shows high basal expression

Green fluorescence detectable at 488nm

Plac GFP

In a Nutshell Biological Programming Simplifying Circuits

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Testing our tools: Plac-GFP

Untransformed E. coli pSB1C3-PlacGFP

In a Nutshell Biological Programming Simplifying Circuits

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Submissions to parts registry

Registry ID Name of Construct Purpose BBa_K299812 pSB1A2-inv-hly Sequence confirmed sample BBa_K1804001 pSB1C3-plac-gfp Constitutively expressing GFP BBa_K1804002 pSB1C3-pnirB PnirB Anaerobic promoter In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

In a Nutshell Biological Programming HP: Simplifying Circuits

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Anaerobic switch Quorum sensing

Explaining our system

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Genetic Circuits

  • Informative, but unappealing

In a Nutshell Biological Programming Simplifying Circuits

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Brute-force representation

In a Nutshell Biological Programming Simplifying Circuits

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Simplified genetic circuits

  • A teaching tool to introduce genetic circuit

designing

  • Simplified abstraction of genetic circuit elements

In a Nutshell Biological Programming Simplifying Circuits

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In a Nutshell Biological Programming Simplifying Circuits

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  • To test the tool, we integrated it into a workshop.
  • Presented it as a set of puzzles
  • Varying levels of difficulty
  • Multiple ways of obtaining the solution

Workshop use

In a Nutshell Biological Programming Simplifying Circuits

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Workshop

  • One-day workshop
  • Mostly first-year biology

students, and some participants from other disciplines

  • Theoretical and experimental

components

In a Nutshell Biological Programming Simplifying Circuits

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Introduction & Theory

Quick introduction to genetics Genetic circuit puzzles

In a Nutshell Biological Programming Simplifying Circuits

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Wet lab: Fusion PCR

Basic Laboratory Training Introduction to gel electrophoresis

In a Nutshell Biological Programming Simplifying Circuits

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Wet lab: Bacteria with GFP

Viewing GFP-transformed bacteria Microscope view

In a Nutshell Biological Programming Simplifying Circuits

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After the workshop

  • Most enjoyed the workshop, with puzzles being

the most popular.

  • Non-biology majors picked up on the puzzles fairly

quickly.

  • Participants displayed good conceptual

understanding.

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After the workshop

  • We also sought further feedback from educators.
  • Received interest in developing it for science outreach
  • Ideally we would like to develop the tool further,

and make it open-access.

In a Nutshell Biological Programming Simplifying Circuits

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What we’ve done this summer

  • Designed a dual-switch system
  • Submitted three parts to the BioBricks registry
  • Developed an educational tool on genetic circuits
  • Conducted a workshop testing the tool

In a Nutshell Biological Programming Simplifying Circuits

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THE END

We would like to thank:

Special Programme in Science Faculty of Science, National University of Singapore Mentors: Dr. Leslie Gapter, Prof. Linda Kenney, Dr. Stuti Desai Integrated DNA Technologies