Plasmid Loss Genetic Safeguards. igem Giant Jamboree 2014

Transcription

1 Plasmid Loss Genetic Safeguards igem Giant Jamboree 2014

2 1. Introduction 2. Modelling 3. Wet lab 4. Human Practices Agenda

3 First up: Introduction

4 Imagine this scenario Massive oil spill! Image credits: Wikipedia

5 What do we do about it? 1. Engineer synthetic organisms 2. Let them eat oil 3.??? 4. Profit!!!

6 But wait! Is it really safe to just have these synthetic organisms floating around? Where will they go? What will they do to the environment?

7 Biocontainment That s why we need biocontainment! Some way to clean up our synthetic organisms after they re done their job We want to avoid the rabbits in Australia situation!

8 So, how can we contain our Kill-switch A toxic gene controlled by inducible promoter Can be turned on to kill the cell bugs?

9 Actually, it s not quite so simple Sounds simple! Organisms are not simple machines They mutate! Image credits: Calgary 2012 igem team Mutate or die

10 Kill Switch: the movie Let s illustrate this with an animation Where Plasmo? He s the mutant hiding among the other cells.

11 Kill Switch: the movie We turn on the kill switch, cells start dying Plasmo has a mutation that makes him resistant to the kill switch

12 Kill Switch: the movie We turn on the kill switch, cells start dying Plasmo has a mutation that makes him resistant to the kill switch

13 Kill Switch: the movie Plasmo is the only one left! Remember: Plasmo still has the synthetic plasmid! He has escaped biocontainment

14 Kill Switch: the movie Plasmo replicates quickly, since there are no competitors Remember: Plasmo still has the synthetic plasmid! He has escaped biocontainment

15 Kill Switch: the movie Plasmo replicates quickly, since there are no competitors Remember: Plasmo still has the synthetic plasmid! He has escaped biocontainment

16 Kill Switch: the movie Now it s super easy to find Plasmo He s everywhere! Remember: Plasmo still has the synthetic plasmid! He has escaped biocontainment

17 Is there another way? Yes! Let s think about it from another angle We don t need to actually kill the synthetic organisms We really only need to get rid of the synthetic genes

18 Plasmid Loss Self-destructing plasmid Synthetic organism with synthetic genes Wild-type organism No synthetic genes

19 How is that any different? Kill switch: Imposes selective pressure against nonmutants Gives mutants selective advantage Plasmid loss Does not impose selective pressure on non-mutants Mutants have no selective advantage

20 Plasmid Loss: the movie Another animation! Where s Plasmo? He s the mutant hiding among the other cells.

21 Plasmid Loss: the movie We turn on plasmid loss, cells start reverting to wild-type Plasmo has a mutation that makes him resistant to plasmid loss as well

22 Plasmid Loss: the movie We turn on plasmid loss, cells start reverting to wild-type Plasmo has a mutation that makes him resistant to plasmid loss as well

23 Plasmid Loss: the movie All the non-mutants have reverted to wild-type Plasmo is still there, but it s still hard to find him since there are lots of wildtype around

24 Plasmid Loss: the movie Mutants are kept at a low proportion of the overall population Plasmo can t replicate quickly due to competition.

25 But wait! There s more Genetic Drift Let s say there s only enough food for half the cells to reproduce each generation

26 The die is cast! Half of the cells are randomly chosen to reproduce Plasmo was unlucky! He did not survive

27 The next generation The mutants have been lost from the population due to genetic drift Where s Plasmo? He s not there anymore!

28 The Big Picture Is the plasmid loss system foolproof? Unfortunately not. In most cases It s better than kill -switch But even in the worst case It s no worse than kill-switch Still need multiple lines of defence! (Paris Bettencourt 2012)

29 Next up: Modeling & Simulation

30 Model Buildup

31 Imagine in a synthetic biology lab

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33 Plasmid Loss... Activate!

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35 Wild Type Mutant Competing factors a, b Growth Rate: r 1, r 2 Carrying Capacity: K 1, K 2 All determined without competition

36 Wild Type Mutant a b r 1, K 1 r 2, K 2

37 Wild Type Mutant dx dt = r 1x(1 x K 1 ) dy dt = r 2y(1 y K 2 )

38 Wild Type Mutant dx dt = r 1x(1 x + ay K 1 ) dy dt = r 2y(1 y + bx K 2 )

39 Wild Type Mutant We have the model!

40 t 0 t 1 t 2 Deterministic Process Next, t 2 = 4

41 ?? t 0 t 1 t 2 Random Process(Poisson) Next, expected t 2 = 4

42 Bottleneck Effect Genetic Drift

43 Making Assumptions & Determining Parameters

44 Assumptions total initial population = carrying capcity

45 Assumptions Infinite nutrients, Infinite bottleneck effects Limited space

46 Experimentally Determined Growth Rate of wild type Carrying Capacity Parameters

47 Determined Theoretically Growth Rate of Mutant

48 Tested in a Range Competing Factors Bottleneck Effectiveness

49 Simulation

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51 The Battle Begins...

52 Initial Condition WT MUT = 9 1 Bottlenecks Mutant pop decreases each time Plasmid Loss

53 Initial Condition WT MUT = 9 1 Plasmid Loss

54 Statistics from hundreds of battles

55 Competing factors vs. Extinction time Mutant is slightly more competitive than wt, but it still goes extinct! Stable coexistence Mutant less competitive, Goes extinct quickly Mutant is much more competitive, wild-type goes extinct

56 Bottleneck Effectiveness & Initial Condition vs. Extinction time Less Severe Bottleneck No Extinction, Mutant survived More Severe Bottleneck Plasmid Loss efficiency, population ratio

57 Our system is most effective when: Less competitive mutant Effective Bottleneck High plasmid loss efficiency Conclusion

58 Next up: Wet Lab Implementation Experimental Design Problems Encountered

59 Implementation

60 Goal: inducible self-destructing plasmid Implemented with CRISPR/Cas9 Is like a Customizable Restriction Enzyme Overview Synthetic Genes

61 Building on previous work CRISPR/Cas9 part exists in the registry as pcas9 Bba_K Problem: existing part does not facilitate inducible expression Our job: Create and biobrick inducible version of CRISPR

62 Creating an inducible pcas9 Original pcas9 Our redesigned pcas9

63 Experimental Design

64 Target practice

65 CRISPR Cutting Assay Transformation 1: Set up the target White colonies -> Red colonies Transformation 2: Add CRISPR Observe results Red colonies -> White colonies Note: target plasmid and CRISPR plasmid must have different antibiotic resistances

66 Some Possible Outcomes 100% Efficiency all white 50% Efficiency half white, half red 0% Efficiency all red

67 Characterisation Anderson high/mid/low constitutive Different levels of cas9 expression pbad, plac, prha, Tet-off/Tet-on Inducible expression Leaky expression

68 Plasmid Assembly Gibson assembly promoter, cas9 + array, tracrrna, ptrc99a 4 fragment assembly

69 Problems Encountered

70 Assembly Problems Problem: All our eggs in one basket Did not get specific assembly form Gibson assembly Started biobrick assembly too late in the project cycle But we did manage to biobrick one part BBa_K tracr RNA to be used with Cas9 Image credits: Uppsala igem team via igem memes

71 Full list of parts we designed BBa_K tracr RNA to be used with Cas9 BBa_K Cas9 and crrna array BBa_K rearranged CRISPR/Cas9 system without promoter BBa_K CRISPR/Cas9 system with Anderson high-expression constitutive promoter BBa_K CRISPR/Cas9 system with Anderson medium-expression constitutive promoter BBa_K CRISPR/Cas9 system with Anderson low-expression constitutive promoter BBa_K CRISPR/Cas9 system with pbad inducible promoter BBa_K CRISPR/Cas9 system with plac inducible promoter BBa_K CRISPR/Cas9 system with prha inducible promoter BBa_K Randomized protospacer for CRISPR/Cas9 System BBa_K RFP-Targeting protospacer for CRISPR/Cas9 system BBa_K Low-copy plasmid ptrc99a (trc promoter and laci removed)

72 Our contribution to the The sequences of all the parts we designed are on the registry They are annotated Future teams can easily understand where the CRISPR components are located registry

73 Next up: Human Practices

74 A Guide on How to Start a Community Laboratory

75 Synthetic Biology Public Workshop In collaboration with DIYbio

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77 Plasonomics

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79 Attributions

80 Scientific Advisors Graphic Design Boris Steipe Naveen Venayak Anson Tse Plasonomics Kevin Lu Victor Kofia Temi Ayeni

81 Thank you for listening! Questions?