Synthetic Biology and Rational Design Keith Shearwin University of - - PowerPoint PPT Presentation

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Synthetic Biology and Rational Design Keith Shearwin University of - - PowerPoint PPT Presentation

Feb 21, 2023 12 likes •627 views

Synthetic Biology and Rational Design Keith Shearwin University of Adelaide Synthetic biology what is it? Analogy with engineering Learning by building: natural and synthetic gene circuits 1 (2) (1) 2 (1) Understand natural

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Synthetic Biology and Rational Design Keith Shearwin University of Adelaide

  • Synthetic biology – what is it?
  • Analogy with engineering
  • Learning by building: natural and synthetic gene circuits
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(1) (2)

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“The complex genetic circuits found in cells are ordinarily studied by analysis of genetic and biochemical perturbations. The inherent modularity of biological components like genes and proteins enables a complementary approach: one can construct and analyse synthetic genetic circuits based on their natural counterparts. Such synthetic circuits can be used as simple in vivo models to explore the relation between the structure and function of a genetic circuit.”

(1) Understand natural circuits

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(2) Build useful stuff – rational design Examples of applications of synthetic biology

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Malaria ~ one million deaths annually, mostly children Artemisinin: Anti-malarial compound found in plant

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Produce artemisinin in microorganisms

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Enzymes from 3 species, plus extensive modification of individual steps Initial attempts yielded ~0.1 g/L 2012: yield ~40g/L. Over express all enzymes, in yeast Some metabolic engineering, some high throughput screening.

Produce artemisinin in microorganisms

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Early 2013

http://www.zagaya.org/2012/12/culmination-of-the-artemisinin-project/

http://www.nature.com/news/malaria-drug-made-in-yeast-causes-market-ferment-1.12417

  • Sugar cane
  • 200 000 litre

fermentors ~ tens of tonnes Sell at cost price Profit from other pathway intermediates

  • Farnesene (long chain hydrocarbon)
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Building new circuits – analogy with engineering

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Synthetic Biology: analogy with engineering

  • modularity
  • standardisation
  • mathematical description
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Modularity

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Standardisation - engineering

Consistent building blocks

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Standardisation - biology

Standard plasmids Interchangeable parts Standard method of assembly (Biobricks website)

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Standardisation - biology

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Standardisation - biology

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Mathematical model

Allows the ability to make and test predictions about

  • Circuit behaviour (toggle switch, repressilator)
  • Stability
  • Robustness against perturbations
  • Effect of biological noise arising from low numbers of molecules

(stochastic simulations) Design Build Test Refine

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Synthetic Biology

  • Challenges of Synthetic Biology
  • Technological advances
  • Risk and Safety
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Challenges for Synthetic Biology

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Technological Advances

  • 1. Fluorescent proteins/single cell microscopy

Time lapse movies single cells

  • follow dynamics of multiple components of

a given circuit

  • correlations between outputs
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Technological Advances

  • 1. Fluorescent proteins/single cell microscopy

Time lapse movies single cells

  • follow dynamics of multiple components of

a given circuit

  • correlations between outputs
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Technological Advances

  • 2. DNA synthesis
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Technological Advances

  • 2. DNA synthesis
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Technological Advances

  • 2. DNA synthesis

http://www.invitrogen.com/site/us/en/home/Products-and- Services/Applications/Cloning/gene-synthesis/gene-strings-dna- fragments.html#7

http://www.idtdna.com/pages/products/genes

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Synthetic Biology

Risk and safety Reputable companies check requested sequences

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Risk and Safety

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Polio virus

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Technological Advances

  • 2. DNA synthesis

1970 – chemical synthesis of one gene 207bp long (took > 1 year)

(2002) 7440 bp, 2 years, ~$10/bp Next?

(2010) 1 077 947 bp

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Technological Advances

  • 2. DNA synthesis

http://syntheticyeast.org/

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Example of use of DNA synthesis

Biofuels

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Production of methyl halides (CH3I) by microorganisms from biomass

  • precursors for petroleum
  • 1. BLAST search for methyl halide

transferases (MHT), including metagenomic data (eg seawater)

  • 2. Chemical synthesis of each DNA

encoding the gene sequences. No need to culture or even identify the

  • rganism. No cloning!
  • 3. Express proteins and assay enzyme

activity

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Learning by building: natural and synthetic gene circuits

Study DNA looping energy in vivo

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Learning by building: natural and synthetic gene circuits

300bp spacing

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Monte Carlo Fitting

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Learning by building: natural and synthetic gene circuits

1.0 10.0 100.0 1000.0 10000.0 2000 4000 6000 8000 10000

j

Separation (bp)

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Learning by building: phage lambda

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RNAP

Learning by building: phage lambda

More complex system

  • 6 operators
  • 3 promoters
  • enhancer like element (UP)
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Learning by building: phage lambda

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Perturb the system – mutants

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Learning by building: phage lambda

Small improvements to binding

  • Change in gene expression levels
  • Mimic eukaryotic enhancer elements

Development of new tools for synthetic biology

  • Incorporation of distant binding sites
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Learning by building: synthetic gene circuits Bistability – mutually exclusive states

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Learning by building: synthetic gene circuits Mixed feedback loop - bistability

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Learning by building: synthetic gene circuits Mixed feedback loop - bistability

Design Build Test Refine

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Learning by building: synthetic gene circuits

Design - Components from phage

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Learning by building: synthetic gene circuits

Mixed feedback loop - bistability Stable states where production = degradation

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Learning by building: synthetic gene circuits

Design

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Learning by building: synthetic gene circuits

Hysteresis - Characteristic of bistability Design

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Learning by building: synthetic gene circuits

Design

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Learning by building: synthetic gene circuits

Build

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Learning by building: synthetic gene circuits

Test  Hysteresis x Sharp transitions

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Learning by building: synthetic gene circuits

Refine Stochastic simulations

  • ‘Noise’ in transcription
  • Flipping from one state

to the other

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Learning by building: synthetic gene circuits

Refine

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Learning by building: synthetic gene circuits

Refine

Redesign system to reduce noise:

  • plasmid copy number
  • growth rates

But intrinsic noise in biological processes unavoidable!

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Synthetic Biology and Rational Design

  • Synthetic biology – what is it?
  • Analogy with engineering
  • Learning by building: natural and synthetic gene circuits

Funding