“Engineering modular and orthogonal genetic logic gates for

robust digital-like synthetic biology”

Baojun Wang, Richard I Kitney, Nicolas Joly & Martin BuckJamal Elkhader & Queenie Chan

22 February 2012

Background

AND gate:

Background

NAND gate:

Background

Terms:o Digital system: on/offo 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. ClassificationII. Design AND gateIII.Test ModularityIV. repeat II and III for NAND gateV. Compatibility

Classification: Forward Engineering

• 3 different promoters: Plac, PBAD, Plux

• 3 test conditions:1. promoters' responses, varying chassis (E. coli

MC4100, E. coli MC1061)

IPTG --> lacArabinose --> BADAHL --> lux

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

Classification: Forward Engineering

• 3 different promoters: Plac, PBAD, Plux

• 3 test conditions:1. promoters' responses, varying chassis (E. coli

MC4100, E. coli MC1061)2. promoters + RBSs responses with varying RBS's

IPTG --> lacArabinose --> BADAHL --> lux

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

Classification: Forward Engineering• 3 different promoters: Plac, PBAD, Plux

• 3 test conditions:1. promoters' responses, varying chassis (E. coli

MC4100, E. coli MC1061)2. promoters + RBSs responses with varying RBS's3. Promoters + RBS's responses with varying

temperatures

IPTG --> lacArabinose --> BADAHL --> lux

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

Circuit Design: AND Gates

Gate 1

Gate 2

Testing for Modularity

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

Gate 230ºC

37ºC

NAND gates: Design & Modularity Test

Gate 3

Gate 4

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

Gate 3 Gate 4Predicted

Results

NAND gate FACS data

Both inputs added -->

off

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

• Compatibility of AND gate with chassis

• Top: Gate 1 (Plac + PBAD)

• Bottom: Gate 2 (Plux + PBAD)

Compatibility ✗✗ ✗ ✗

✗

✗

Classification Design AND gate

Test Modularity NAND gate Compatibilit

y

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

SignificanceModular:

• 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 Plac

PBAD

Plux