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

Aug 19, 2022 181 likes •657 views

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

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COLUMBIA COOPER iGEM 2011

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Environmentally friendly manufacture

  • f quantum dots in E.coli

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COLUMBIA-COOPER iGEM 2011

COLUMBIA COOPER iGEM 2011

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OUR RESEARCH FACILITES THE COOPER UNION

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OUR RESEARCH FACILITES GENSPACE

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WHAT ARE QUANTUM DOTS?

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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

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PROJECT GOALS

MAIN DIAGRAM

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Engineer E.coli to nucleate quantum dots from heavy metal salts dot

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MAIN DIAGRAM

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Use antibiotic selection to tune system for specific colors

PROJECT GOALS

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CHEMICALLY SYNTHESIZED QUANTUM DOTS Served two purposes

  • Characterization of Blue Light Promoter
  • Control vs. biologically produced Quantum Dots

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CHEMICALLY SYNTHESIZED QUANTUM DOTS

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CHEMICALLY SYNTHESIZED QUANTUM DOTS

  • Cadmium
  • Selenium
  • tri-n octylphosphine
  • trioctylphoshine oxide
  • 1-octadecene
  • Reaction temperature: 225°C.

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CHEMICALLY SYNTHESIZED QUANTUM DOTS

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STANDARDS FOR BIODOTS

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CHARACTERIZATION VIA FLUORESCENCE SPECTROSCOPY

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Low Energy Production Possibility for Self Assembly Greater Biocompatibility WHY BIOLOGICALLY MADE QDs ARE EVEN COOLER?

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BIOLOGICAL MECHANISM

Metallophilic peptides stabilize heavy metal wurtz- ite crystals

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BIOLOGICAL MECHANISM

Peptides Coat the Crystal

  • Stabilize the structure
  • Control dot size
  • Coat with biocompatible organic

material

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OUR EXPERIMENT OUTLINE

Express and characterize QD nucleating peptides Use blue QDs to activate blue light sensing promoter Use blue light sensing promoter to transcribe antibiotic resistance

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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 Qi, George Georgiou, Brent Iverson, and Angela M. Belcher

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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.

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METHODS - OLIGO ANNEALING A7 - N-SLTPLTTSHLRS-C Z8 - N-VISNHAESSRRL-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C

  • Tiny sequences of 70 bp or less. We

used oligo annealing with sticky ends for ligation into biobrick vector pSB1C3 A7 - N-SLTPLTTSHLRS-C Z8 - N-VISNHAESSRRL-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C

  • Tiny sequences of 70 bp or less. We

used oligo annealing with sticky ends for ligation into biobrick vector pSB1C3 A7 - N-SLTPLTTSHLRS-C Z8 - N-VISNHAESSRRL-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C

  • Tiny sequences of 70 bp or less. We

used oligo annealing with sticky ends for ligation into biobrick vector pSB1C3 A7 - N-SLTPLTTSHLRS-C Z8 - N-VISNHAESSRRL-C J140 - N-TGCAACAACCCGATGCACCAGAACTGC-C

  • Tiny sequences of 70 bp or less. We

used oligo annealing with sticky ends for ligation into biobrick vector pSB1C3

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METHODS - OLIGO ANNEALING

  • Used “Gene Synthesis

Optimization Program”,

  • riginally developed by

the 2006 iGEM team from Davidson College

  • Ordered 4 overlapping

primers which were then annealed

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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.

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METHODS - LIGATION AND EXPRESSION

CDS7 pET-28

METHODS - LIGATION AND EXPRESSION

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AND ZINC SULFIDE

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INCUBATION WITH CADMIUM CHLORIDE AND SODIUM SULFIDE

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Use antibiotic selection to tune system for specific colors

PART 2: THE BLUE LIGHT SENSOR

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NEGATIVE FEEDBACK INHIBITION YcgF/YcgE System

  • YcgF is sensitive to blue light
  • Release of YcgF/YcgE dimer allows

for transcription

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DIMERIZATION CAUSES RELEASE OF INHIBITOR

In the presence of blue light, chloramphenicol resistance is transcribed when YcgF/YcgE dimerizes and releases.

DIMERIZATION CAUSES RELEASE OF INHIBITOR

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...WHICH ACTIVATES ANTIBIOTIC RESISTANCE

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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

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DESIGN OF BBa_K643001 Used psB1A3 instead of the psB1C3 backbone

  • Prevents redundant Chloramphenicol

resistance, which would have made the part impossible to characterize

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BLUE LIGHT BOX

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DESIGN OF BLUE LIGHT BOX

  • Dimensions 8X6x5 inches
  • Light tight
  • Interior lined with aluminum

foil

  • 72 blue LEDs (emitting at 472

nm)

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RESULTS - OD 600nm CONFIRMATION

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HUMAN PRACTICES: MAKER FAIRE

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Editor’s Choice

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WHY QDs ARE GREAT FOR IGEM

Can be easily combined with ligands to make composites. High color specificity within a wide

  • range. Good for multiple site tagging.

QDs don’t photobleach like fluorescent proteins.

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APPLICATIONS - Adding to the SynBio toolbox

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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)

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WE ARE GRATEFUL FOR YOUR SUPPORT!

Stanley Lapidus and Allan Kuchinsky

Our Moms and Dads

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