“Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology” Baojun Wang, Richard I Kitney, Nicolas Joly & Martin.

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Presentation transcript:

“Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology” Baojun Wang, Richard I Kitney, Nicolas Joly & Martin Buck Jamal Elkhader & Queenie Chan 22 February 2012

Background AND gate:

Background NAND gate:

Background Terms: o Digital system: on/off o Forward engineering: classifying/evaluating components before choosing the parts to build the system o Modular: can change inputs and outputs and logic gate will still work o Orthogonal: no cross-talk

Project Overview Purpose: to engineer a digital-like system using modular and orthogonal parts via forward engineering I.Classification II.Design AND gate III.Test Modularity IV.repeat II and III for NAND gate V.Compatibility

Classification: Forward Engineering 3 different promoters: P lac, P BAD, P lux 3 test conditions: 1.promoters' responses, varying chassis (E. coli MC4100, E. coli MC1061) IPTG --> lac Arabinose --> BAD AHL --> lux Classification Design AND gate Test Modularity NAND gateCompatibility

Classification: Forward Engineering 3 different promoters: P lac, P BAD, P lux 3 test conditions: 1.promoters' responses, varying chassis (E. coli MC4100, E. coli MC1061) 2.promoters + RBSs responses with varying RBS's IPTG --> lac Arabinose --> BAD AHL --> lux Classification Design AND gate Test Modularity NAND gateCompatibility

Classification: Forward Engineering 3 different promoters: P lac, P BAD, P lux 3 test conditions: 1.promoters' responses, varying chassis (E. coli MC4100, E. coli MC1061) 2.promoters + RBSs responses with varying RBS's 3.Promoters + RBS's responses with varying temperatures IPTG --> lac Arabinose --> BAD AHL --> lux Classification Design AND gate Test Modularity NAND gateCompatibility

Circuit Design: AND Gates Gate 1 Gate 2 Testing for Modularity Classification Design AND gate Test Modularity NAND gateCompatibility

Gate 2 30ºC 37ºC

NAND gates: Design & Modularity Test Gate 3 Gate 4 Classification Design AND gate Test Modularity NAND gateCompatibility

Gate 3Gate 4 Predicted Results

NAND gate FACS data Both inputs added --> off Classification Design AND gate Test Modularity NAND gateCompatibility

Compatibility of AND gate with chassis Top: Gate 1 (P lac + P BAD ) Bottom: Gate 2 (P lux + P BAD ) Compatibility ✗ ✗ ✗ ✗ ✗ ✗ Classification Design AND gate Test Modularity NAND gateCompatibility

Conclusions AND and NAND gates are modular Sigmoidal response property allows system to be robust (minimal background noise) Forward engineering and engineering-by- parts works! Crosstalk can be avoided by picking promoters that are not native to chassis

Significance Modular: Can use other inputs and outputs and AND / NAND gate will still work Parts-based engineering approach Control: New genetic logic devices can be engineered predictably to generate desired behaviours in response to specific extra- or intra- cellular signalling inputs Improves sensing specificity and accuracy of biological control

Supplementary Slides P lac P BAD P lux