my kitchen table pcr sophomore year of high...
TRANSCRIPT
![Page 1: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/1.jpg)
My Kitchen Table PCR Sophomore Year of High School
![Page 2: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/2.jpg)
PCR Primer
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Primer-Defined Changes to the PCR Copies
∆⁻ +
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Copying DNA with Polymerase Chain Reaction
![Page 5: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/5.jpg)
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Chromophore
Creating the Chromophore Colorwheel
ConservedSimilarDivergent
Acropora millepora
![Page 7: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/7.jpg)
Gibson Assembly for Mutagenesis and Drug Selection Swap
PvuII
PvuII
![Page 8: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/8.jpg)
Plasmid Uptake by Calcium-Assisted Heat Shock
Asif et al, Front Microbiol 2017
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A Primer for This Week
![Page 10: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/10.jpg)
DNA-Programming w/ Plasmids
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NextGenSynth Assignment:DNA-Programmed Coral Color Changes
Acropora millepora
Think like a molecule. -George Church
Acropora millepora ChromoProtein
Liljeruhm et al, J Biol Eng. 2018
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NextGenSynth Assignment:DNA-Programmed Coral Color Changes
Acropora millepora ChromoProtein
Think like a molecule. -George Church
![Page 14: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/14.jpg)
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Machines in the Microbial Factory
Escherichia coli
Image Credit: David Goodsell
Chromosome
Inner Membrane
Outer Membrane
Cell Wall
Flagellum
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Drug Selection of Transformed E coli Cells
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Drug Selection of Transformed E coli Cells
Penicillium mold
Inner Membrane
Cell Wall
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Drug Selection of Transformed E coli Cells
Beta-lactamase ( Bla/ampr )
Penicillium mold
Inner Membrane
Cell Wall
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Beta-lactamase ( Bla/ampr )
Beta-galactosidase ( lacZ )
DNA-Programming w/ Plasmids
![Page 20: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/20.jpg)
Beta-lactamase ( Bla/ampr )
Beta-galactosidase ( lacZ )
DNA-Programming w/ Plasmids
![Page 21: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/21.jpg)
Beta-lactamase ( Bla/ampr )
Beta-galactosidase ( lacZ )
DNA-Programming w/ Plasmids
![Page 22: My Kitchen Table PCR Sophomore Year of High Schoolfab.cba.mit.edu/classes/S66.19/S66.19/slides/HTGAA...1.6-Å 20-Å Solving for Structure: X-Ray Crystallography 2.2-Å 1.6-Å 20-Å](https://reader034.vdocument.in/reader034/viewer/2022042313/5edc97c8ad6a402d666752c1/html5/thumbnails/22.jpg)
Beta-lactamase ( Bla/ampr )
Beta-galactosidase ( lacZ )
DNA-Programming w/ Plasmids
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Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)
Hierarchy of Protein Structure
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Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)
Hierarchy of Protein Structure
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Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)
Hierarchy of Protein Structure
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Modularity in Motifs and Domains
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Solving for Structure: Cryo Electron Microscopy
2.2-Å
1.6-Å 20-Å
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Solving for Structure: X-Ray Crystallography
2.2-Å
1.6-Å 20-Å
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Color-Based Screens with Beta-galactosidase (lacZ)
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Genetically-Encoded Green Fluorescence
Aequorea victoria
Image Credit: Roger Tsien Nobel Lecture
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wild-typeS65T
Chromophore Engineering
Image Credit: Roger Tsien Nobel Lecture
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Fluorescent Spectrum & StructuresDiscosoma Arabidopsis thaliana
Image Credit: Roger Tsien Nobel Lecture
Dou et al, Nature 2018
Image Credit: Roger Tsien Nobel Lecture
Chapman et al, PNAS 2008
110 amino acids + 1 nucleotide
84 nucleotides
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Protein Pigments
Acropora millepora
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Protein Pigment Pellet Palette
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Chromophore
Creating the Chromophore Colorwheel
ConservedSimilarDivergent
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Part Map and Sequence for Assignment
J23106: Promoter (Start DNA -> RNA)RBS: Ribosome Binding Site (RNA -> Protein)amilCP: Chromoprotein Coding Sequence (CDS)Terminators: Transcription Stop (Stop DNA -> RNA)
VF2: Fwd Primer Site (PCR)VR: Rev Primer Site (PCR)
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Copying and Changing DNA with Polymerase Chain Reaction
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Point Mutagenesis with KLD (Kinase, Ligase,DpnI) Mix
∆
⁻
+
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Gibson Assembly Mechanism
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Gibson Assembly for Mutagenesis and Drug Selection Swap
PvuII
PvuII
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(Bonus) Primer Fitness Prediction
Shift -6ntsShift -12ntsShift -18ntsShift -24nts
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Alternative Antibiotic Selection
protein
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Plasmid Uptake by Calcium-Assisted Heat Shock
Asif et al, Front Microbiol 2017