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My Kitchen Table PCR Sophomore Year of High School

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

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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

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

PCR Primer

Page 3: 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

Primer-Defined Changes to the PCR Copies

∆⁻ +

Page 4: 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

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
Page 6: 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

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

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

Plasmid Uptake by Calcium-Assisted Heat Shock

Asif et al, Front Microbiol 2017

Page 9: 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

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

DNA-Programming w/ Plasmids

Page 11: 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
Page 12: 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

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

Page 13: 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

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
Page 15: 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

Machines in the Microbial Factory

Escherichia coli

Image Credit: David Goodsell

Chromosome

Inner Membrane

Outer Membrane

Cell Wall

Flagellum

Page 16: 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

Drug Selection of Transformed E coli Cells

Page 17: 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

Drug Selection of Transformed E coli Cells

Penicillium mold

Inner Membrane

Cell Wall

Page 18: 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

Drug Selection of Transformed E coli Cells

Beta-lactamase ( Bla/ampr )

Penicillium mold

Inner Membrane

Cell Wall

Page 19: 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

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

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

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

Beta-lactamase ( Bla/ampr )

Beta-galactosidase ( lacZ )

DNA-Programming w/ Plasmids

Page 23: 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

Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)

Hierarchy of Protein Structure

Page 24: 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

Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)

Hierarchy of Protein Structure

Page 25: 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

Cyclic-AMP Receptor Protein (CRP) Homodimer (CRP-CRP)

Hierarchy of Protein Structure

Page 26: 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

Modularity in Motifs and Domains

Page 27: 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

Solving for Structure: Cryo Electron Microscopy

2.2-Å

1.6-Å 20-Å

Page 28: 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

Solving for Structure: X-Ray Crystallography

2.2-Å

1.6-Å 20-Å

Page 29: 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

Color-Based Screens with Beta-galactosidase (lacZ)

Page 30: 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

Genetically-Encoded Green Fluorescence

Aequorea victoria

Image Credit: Roger Tsien Nobel Lecture

Page 31: 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

wild-typeS65T

Chromophore Engineering

Image Credit: Roger Tsien Nobel Lecture

Page 32: 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

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

Page 33: 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

Protein Pigments

Acropora millepora

Page 34: 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

Protein Pigment Pellet Palette

Page 35: 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

Chromophore

Creating the Chromophore Colorwheel

ConservedSimilarDivergent

Page 36: 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

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)

Page 37: 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

Copying and Changing DNA with Polymerase Chain Reaction

Page 38: 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

Point Mutagenesis with KLD (Kinase, Ligase,DpnI) Mix

∆

⁻

+

Page 39: 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

Gibson Assembly Mechanism

Page 40: 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

Gibson Assembly for Mutagenesis and Drug Selection Swap

PvuII

PvuII

Page 41: 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

(Bonus) Primer Fitness Prediction

Shift -6ntsShift -12ntsShift -18ntsShift -24nts

Page 42: 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

Alternative Antibiotic Selection

protein

Page 43: 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

Plasmid Uptake by Calcium-Assisted Heat Shock

Asif et al, Front Microbiol 2017


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