Dot Dot Dot COLUMBIA COOPER Environmentally friendly manufacture - PowerPoint PPT Presentation

Dot Dot Dot COLUMBIA COOPER Environmentally friendly manufacture iGEM 2011 of quantum dots in E.coli dot columbia cooper iGEM

COLUMBIA-COOPER iGEM 2011 Dot Dot Dot COLUMBIA COOPER iGEM 2011 dot columbia cooper iGEM

OUR RESEARCH FACILITES THE COOPER UNION dot columbia cooper iGEM

OUR RESEARCH FACILITES GENSPACE dot columbia cooper iGEM

WHAT ARE QUANTUM DOTS? dot columbia cooper iGEM

WHY QUANTUM DOTS ARE COOL Fluorescent up to1000x brighter than GFP detection is unsophisticated Resistant to photo bleaching long lasting - can be shipped or used in live cells over time Narrow emission wavelength allows multiple colors - great for identification of many contaminants simultaneously Quantum Confinement applications may include qbits in quantum comupters dot columbia cooper iGEM

PROJECT GOALS Engineer E.coli to nucleate quantum dots 1 from heavy metal salts MAIN DIAGRAM dot columbia cooper iGEM

PROJECT GOALS Use antibiotic selection to tune 2 system for specific colors MAIN DIAGRAM dot columbia cooper iGEM

CHEMICALLY SYNTHESIZED QUANTUM DOTS Served two purposes • Characterization of Blue Light Promoter • Control vs. biologically produced Quantum Dots dot columbia cooper iGEM

CHEMICALLY SYNTHESIZED QUANTUM DOTS dot columbia cooper iGEM

CHEMICALLY SYNTHESIZED QUANTUM DOTS • Cadmium • Selenium • tri-n octylphosphine • trioctylphoshine oxide • 1-octadecene • Reaction temperature: 225°C. dot columbia cooper iGEM

CHEMICALLY SYNTHESIZED QUANTUM DOTS dot columbia cooper iGEM

STANDARDS FOR BIODOTS dot columbia cooper iGEM

CHARACTERIZATION VIA FLUORESCENCE SPECTROSCOPY dot columbia cooper iGEM

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WHY BIOLOGICALLY MADE QDs ARE EVEN COOLER? Low Energy Production 1 1 Possibility for Self Assembly 2 Greater Biocompatibility 3 dot columbia cooper iGEM

BIOLOGICAL MECHANISM 1 Metallophilic peptides stabilize heavy metal wurtz- ite crystals dot columbia cooper iGEM

BIOLOGICAL MECHANISM 2 1 Peptides Coat the Crystal - Stabilize the structure - Control dot size - Coat with biocompatible organic material dot columbia cooper iGEM

OUR EXPERIMENT OUTLINE Express and characterize QD nucleating peptides 1 1 2 Use blue QDs to activate blue light sensing promoter 3 Use blue light sensing promoter to transcribe antibiotic resistance dot columbia cooper iGEM

STRATEGY CDS-7 (Cadmium Sulfide) Biosynthesis and characterization of CdS quantum dots in genetically engineered Escherichia coli (2011) Congcong Mi, Yanyan Wang, Jingpu Zhang, Huaiqing Huang, Linru Xu, Shuo Wang, Xuexun Fang , Jin Fang, Chuanbin Mao, Shukun Xu, A7 (Zinc Sulfide) Z8 (Zinc Sulfide) J140 (Cadmium Sulfide) Viral assembly of oriented quantum dot nanowires (2003) Chuanbin Mao, Christine E. Flynn, Andrew Hayhurst, Rozamond Sweeney, Jifa dot Qi, George Georgiou, Brent Iverson, and Angela M. Belcher columbia cooper iGEM

METHODS - CDS-7 N-GDVHHHGRHGAEHADI-C • Ordered from Invitrogen • Added Nco1 site (containing start codon) and BamH1 site for ligation into pET28, flanked by biobrick ends. dot columbia cooper iGEM

METHODS - OLIGO ANNEALING A7 - N-SLTPLTTSHLRS-C A7 - N-SLTPLTTSHLRS-C A7 - N-SLTPLTTSHLRS-C A7 - N-SLTPLTTSHLRS-C Z8 - N-VISNHAESSRRL-C Z8 - N-VISNHAESSRRL-C Z8 - N-VISNHAESSRRL-C Z8 - N-VISNHAESSRRL-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C • Tiny sequences of 70 bp or less. We • Tiny sequences of 70 bp or less. We • Tiny sequences of 70 bp or less. We • Tiny sequences of 70 bp or less. We used oligo annealing with sticky ends for used oligo annealing with sticky ends for used oligo annealing with sticky ends for used oligo annealing with sticky ends for ligation into biobrick vector pSB1C3 ligation into biobrick vector pSB1C3 ligation into biobrick vector pSB1C3 ligation into biobrick vector pSB1C3 dot columbia cooper iGEM

METHODS - OLIGO ANNEALING • Used “Gene Synthesis Optimization Program”, originally developed by the 2006 iGEM team from Davidson College • Ordered 4 overlapping primers which were then annealed dot columbia cooper iGEM

METHODS - EXPRESSION CDS7 Did not appear on any gels, so we could not gel purify it. We assumed this could be size related and decided to design primers for PCR extraction instead. This was successful. dot columbia cooper iGEM

METHODS - LIGATION AND EXPRESSION METHODS - LIGATION AND EXPRESSION CDS7 pET-28 dot columbia cooper iGEM

AND ZINC SULFIDE dot columbia cooper iGEM

INCUBATION WITH CADMIUM CHLORIDE AND SODIUM SULFIDE dot columbia cooper iGEM

PART 2: THE BLUE LIGHT SENSOR Use antibiotic selection to tune 2 system for specific colors dot columbia cooper iGEM

NEGATIVE FEEDBACK INHIBITION YcgF/YcgE System • YcgF is sensitive to blue light • Release of YcgF/YcgE dimer allows for transcription dot columbia cooper iGEM

DIMERIZATION CAUSES RELEASE OF INHIBITOR DIMERIZATION CAUSES RELEASE OF INHIBITOR In the presence of blue light, chloramphenicol resistance is transcribed when YcgF/YcgE dimerizes and releases. dot columbia cooper iGEM

...WHICH ACTIVATES ANTIBIOTIC RESISTANCE dot columbia cooper iGEM

DESIGN OF BBa_K643001 Composite part • Blue Light Promoter Gene From BBa_K238013, designed by K.U. Leuven in 2009 • Chloramphenicol Resistance Gene From BBa_P1004, designed by Knight Lab at MIT in 2006 dot columbia cooper iGEM

DESIGN OF BBa_K643001 Used psB1A3 instead of the psB1C3 backbone • Prevents redundant Chloramphenicol resistance, which would have made the part impossible to characterize dot columbia cooper iGEM

BLUE LIGHT BOX dot columbia cooper iGEM

DESIGN OF BLUE LIGHT BOX • Dimensions 8X6x5 inches • Light tight • Interior lined with aluminum foil • 72 blue LEDs (emitting at 472 nm) dot columbia cooper iGEM

RESULTS - OD 600nm CONFIRMATION dot columbia cooper iGEM

HUMAN PRACTICES: MAKER FAIRE dot columbia cooper iGEM

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Editor’s Choice dot columbia cooper iGEM

WHY QDs ARE GREAT FOR IGEM Can be easily combined with ligands 1 1 1 to make composites. 2 2 High color specificity within a wide range. Good for multiple site tagging. 3 3 QDs don’t photobleach like fluorescent proteins. dot columbia cooper iGEM

APPLICATIONS - Adding to the SynBio toolbox dot columbia cooper iGEM

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NEXT STEPS • Fully characterize all potential quantum dot nucleating peptides. • Test additional light-sensitive promoters to improve color sensitivity tuning system • Test light-sensitive promoter system alongside quantum dot production • Conjugate QDs with antibodies (now producible in bacteria!) for one- stop-shop tagging • Experiment with combining quantum dot production with other biobrick systems and devices (like the biocircuit example) dot columbia cooper iGEM

WE ARE GRATEFUL FOR YOUR SUPPORT! Stanley Lapidus and Allan Kuchinsky Our Moms and Dads dot columbia cooper iGEM