Contents • Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Team Information Jasmine Dr Leonard Panthaki Pattenden (The Mad Candice Raeburn Candice Raeburn Michelle Chayeb Scientist) (The Small One) Flora Yuen Breegini Danielle Culas Kamato (The Shopaholic) Muriel Aldunate Sebastian Ramov (The Bully) Lyna Thach
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Where we come from? www.cityofmelbourne.com.au
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
The Issue Too much electricity use is a global concern, causes fossil fuel to be burnt
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
The Solution Our Fossil Electricity Green House Machine Fuels Gas Emission Biological Lighting System Global Warming
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Applications - Manufacturing Christmas House Hold Commercial Street Tree Lights lighting Lighting Lighting
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Results • Making a part • Problems and limitations • Next year • Acknowledgements
Components of Our Machine • Fluorescent Proteins • Cell Free Chassis • E. Coli Cell Free System • Wheat Germ Cell Free System
• Fluorescent Proteins • Cell Free Chassis • E. Coli Cell Free System • Wheat Germ Cell Free System
Fluorescent Proteins GFP YFP Cherry BBa_E0030 BBa_1712028 VIC_Green BFP Blueberry BBa_E0040
Structure Of Fluorescing Protein β -strands α helix
How Do They work?
Ser65 Tyr66
• Fluorescent Proteins • Cell Free Chassis • E.Coli Cell Free System • Wheat Germ Cell Free System
CELL FREE SYSTEMS •What Is a Cell Free System? n o i t a c i n o S Centrifuged supernatant Cleared Lysate
• How Does A Cell Free System Work? • reactions that occur in a cell take place in a cell free system with out the complex interactions found in the whole cell http://genome.imim.es/
• Types of Cell Free Systems www.hondaireland.ie Endogenous mRNA= Manufacturing, >protein synthesis sustainability •Wheat Germ; safer alternative - non toxic
• Why A Cell Free System? From: Collections of Henry Ford P189.16783 Potential ordered and sequential control
•control the machine •components and toxins •Non propagating and non replicating •E.coli ideal for coupled reactions, simple •ethicals less complication at control of initiation means better synthesis • not optimised - scale limited • Synthetic biology; cloning is defined and standardised • Can optimise the chassis and move to scale
Repeated 3 times Centrifuged 2000rpm Resuspended Sonicated Resuspended pallets S30 A pallets S30 B buffer buffer S30 Buffer: Potassium Acetate, Magnesium Centrifuged Sulphate, MQ water pH of 7.8 20000 x g S30 A contained 2mercaptoethanol Supernatant was E.coli Extract
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
How does the Machine Work?
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Where to next? • Switching on and off • Resonance Energy transfer • Fluorescence Resonance Energy Transfer (FRET) • Bioluminescent Resonance Energy Transfer (BRET) • Scale up • Dialysis • Continuous Exchange Cell Free (CEFC) • Continuous Flow Cell Free (CFCF)
Resonance Energy Transfer •Distant dependant energy transfer • FRET- external illumination •BRET- luciferase (oxidative enzyme) Energy Transfer with the emission of a photon
Dialysis
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Making the Part • iGEM fluorescence protein Biobricks in many colours but no blue • Different strategy of cloning: Quikchange Mutagenesis: point Mutations
Quikchange Mutagenesis Mutant Strand Synthesis: Mutant Strand Synthesis: Plasmid DNA is •Denature DNA template methylated •Anneal mutagenic primers •Extend primers Seg Cyc Temp time 1 1 95 1 min 95 50 sec 60 50 sec 2 18 68 1 min 3 1 68 7 mins Dpn I Digestion of Template Dpn I Digestion of Template •Digest parental methylated DNA Transformation: Transformation: •Transform mutated plasmid
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Results • Expressing BFP, GFP,YFP and cherry • Quick Change Failed • No visible fluorescence in machine • Fluorescence still visible after 8 weeks
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next Year • Acknowledgements
Problems!!
• Experimental Problems • Leaving PCR reaction on the sink over the weekend • Mix up in what reagents were added to the reactions • Pipetting • clumsiness
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
Next year… • First year in iGEM • Learnt how things are run and how things should be done • That means we will be back next year with our aims set at gold
• Team information • Who are we? • Where are we from? • The Idea • The issue • The solution • Application in society • The machine • Components of the machine • How the machine works • Further ideas? • Making a part • Results • Problems and limitations • Next year • Acknowledgements
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