Synthetic Biology and iGEM Cambridge iGEM2007 team 27 July 2007

Synthetic Biology '...a new and rapidly emerging discipline that aims at the (re-)design and construction of (new) biological systems.' Synthetic Biology 3.0 Simple receiver biobrick AgrC AgrA Membrane-bound histidine kinase AIP sensor Response regulator Constitutive promoter P2 (activated by AgrA-P)‏ PoPS out

'Combines science and engineering in order to design and build novel biological functions and systems.' Wikipedia + Synthetic Biology

AN ENGINEERING PERSPECTIVE Engineers view biology as a technology Heavy emphasis on developing foundational technologies International Genetically Engineered Machine competition (iGEM) ‏ Registry of Standard Biological Parts Synthetic Biology

iGEM "Can simple biological systems be built from standard, interchangeable parts and operated in living cells? Or is biology just too complicated to be engineered in this way?" Randy Rettberg, Director of the iGEM competition 'Dunno. Let's try to build some devices...'

iGEM – BioBricks Library of standardized parts (called BioBrick standard biological parts) ‏ Use them Design your own Contribute to the registry B0034 RBS

iGEM – The Registry

iGEM BROADER GOALS Enable systematic engineering of biology Promote the open and transparent development of tools for engineering biology Help construct a society that can productively apply biological technology

Synthetic Biology ENGINEERED BIOLOGICAL SYSTEMS Maintain and enhance human health and our environment Fabricate materials and structures Produce energy Provide food Process information

POTENTIAL APPLICATIONS – Health Synthetic Biology Sepsis - An overwhelming systemic immune response to toxin-producing bacteria in the bloodstream Designed feedback loop Inhibits signaling cascade at MyD88 (a 'weak spot') ‏ 26 new BioBricks Type of artificial immunotolerance

Synthetic Biology POTENTIAL APPLICATIONS – Materials & Information Processing

Synthetic Biology POTENTIAL APPLICATIONS – Energy Carbon sequestration Bacteria-produced ethanol Bacteria hydrogen production

Our Team A mix of biologists, engineers and physicist. Shared enthusiasm of synthetic biology Front row, from left: Narin Hengrung, Yi Han, David Wyatt Second row, from left: Zhizhen Zhao (Jane), Stefan Milde, Dmitry Malyshev, Xinxuan Soh (Sheila) Third row, from left: Yi Jin Liew, John Crowe, Lovelace Soirez, Stephanie May, Yue Miao Back row, from left: James Brown (PhD student mentor), Jim Haseloff (faculty), Gos Micklem (faculty)

Our Ideas Gram Positive bacteria, B. subtilis Signalling—two- component peptide signalling system Bacterial Amplifier PoPS out PoPS in Amplifier

B. subtilis General background Gram-positive soil bacterium Genome ~4100 genes Class I contaminant Often used as a model Gram-Positive organism Codon usage differs from E. coli

Why use B. subtilis? Model Gram-positive organism, not yet used in iGEM Better at secreting substances than Gram-negative bacteria Easy to transform Adds new biobricks and new capabilities to Registry

Our aims Culturing B. subtilis Transformation methods Promoters and shuttle vectors Assembling a system

The agr system of S. aureus Oligopeptide-based quorum sensing agrD agrB agrC agrA P2 promoter AgrB membrane AIP AgrC AgrA P

Peptide signalling: importance “Bacterial Maze” Another independent signalling system for the Registry  and a paradigm for more? Aid to research into S. aureus countermeasures

Peptide signalling: roadmap Transfer agr system into B. subtilis, then into E. coli AIP sender device AIP receiver device Permeability device Senders and receivers in B. subtilis Senders and receivers in E. coli Natural agr operon Mutant fepA gene B. subtilis biobrick chassis

Biological Amplifier To build a standard amplifier which can be used in any synthetic (biobricked) system It should take a standard PoPS input and give a standard PoPS output

Biological Amplifier Amplifier construct PoPS in PoPS out RBS activator Promoter (induced by activator)‏

Biological Amplifier – Further uses Repressor Signal divider

Biological Amplifier – Further uses Logic gates – XOR PoPS 3 PoPS 1 PoPS 2 Activator 1 Activator 2 Activator 3 Promoter 1 Promoter 2 Promoter 3

Biological Amplifier – Further uses Logic gates – AND PoPS 1 PoPS 2 Activator 1 Activator 2 Activator 3 Promoter 1 Promoter 2 Promoter 3 PoPS 3

Biological Amplifier Overall this system could be widely used within synthetic biology given its flexibility and the range of possible basic constructs