Synthetic Biology and Rational Design Keith Shearwin University of Adelaide

Transcription

1 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) 2

3 (1) Understand natural circuits 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. 3

4 (2) Build useful stuff rational design Examples of applications of synthetic biology 4

5 Malaria ~ one million deaths annually, mostly children Artemisinin: Anti-malarial compound found in plant 5

6 Produce artemisinin in microorganisms 6

7 Produce artemisinin in microorganisms 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. 7

8 Early Sugar cane litre fermentors ~ tens of tonnes Sell at cost price Profit from other pathway intermediates - Farnesene (long chain hydrocarbon) 8

9 Building new circuits analogy with engineering 9

10 Synthetic Biology: analogy with engineering - modularity - standardisation - mathematical description 10

11 Modularity 11

12 Standardisation - engineering Consistent building blocks 12

13 Standardisation - biology Standard plasmids Interchangeable parts Standard method of assembly (Biobricks website) 13

14 Standardisation - biology 14

15 Standardisation - biology 15

16 Mathematical model Design Build Refine Test 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) 16

17 Synthetic Biology Challenges of Synthetic Biology Technological advances Risk and Safety 17

18 Challenges for Synthetic Biology 18

19 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 19

20 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 20

21 Technological Advances 2. DNA synthesis 21

22 Technological Advances 2. DNA synthesis 22

23 Technological Advances 2. DNA synthesis Services/Applications/Cloning/gene-synthesis/gene-strings-dnafragments.html#7 23

24 Synthetic Biology Risk and safety Reputable companies check requested sequences 24

25 Risk and Safety 25

26 Polio virus 26

27 Technological Advances 2. DNA synthesis 1970 chemical synthesis of one gene 207bp long (took > 1 year) (2002) 7440 bp, 2 years, ~$10/bp (2010) bp Next? 27

28 Technological Advances 2. DNA synthesis

29 Example of use of DNA synthesis Biofuels 29

30 Production of methyl halides (CH 3 I) 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 organism. No cloning! 3. Express proteins and assay enzyme activity 30

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32 Learning by building: natural and synthetic gene circuits Study DNA looping energy in vivo

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40 Learning by building: natural and synthetic gene circuits 300bp spacing

41 Monte Carlo Fitting

42 Learning by building: natural and synthetic gene circuits j Separation (bp)

43 Learning by building: phage lambda 43

44 Learning by building: phage lambda More complex system - 6 operators - 3 promoters - enhancer like element (UP) RNAP 44

45 Learning by building: phage lambda 45

46 Perturb the system mutants 46

47 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 47

48 Learning by building: synthetic gene circuits Bistability mutually exclusive states 48

49 Learning by building: synthetic gene circuits Mixed feedback loop - bistability 49

50 Learning by building: synthetic gene circuits Mixed feedback loop - bistability Design Build Refine Test 50

51 Learning by building: synthetic gene circuits Design - Components from phage 51

52 Learning by building: synthetic gene circuits Mixed feedback loop - bistability Stable states where production = degradation 52

53 Learning by building: synthetic gene circuits Design 53

54 Learning by building: synthetic gene circuits Design Hysteresis - Characteristic of bistability 54

55 Learning by building: synthetic gene circuits Design 55

56 Learning by building: synthetic gene circuits Build 56

57 Learning by building: synthetic gene circuits Test Hysteresis x Sharp transitions 57

58 Learning by building: synthetic gene circuits Refine Stochastic simulations - Noise in transcription - Flipping from one state to the other 58

59 Learning by building: synthetic gene circuits Refine 59

60 Learning by building: synthetic gene circuits Refine Redesign system to reduce noise: - plasmid copy number - growth rates But intrinsic noise in biological processes unavoidable! 60

61 Synthetic Biology and Rational Design Synthetic biology what is it? Analogy with engineering Learning by building: natural and synthetic gene circuits Funding 61