My Kitchen Table PCR Sophomore Year of High School PCR Primer - - PowerPoint PPT Presentation

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My Kitchen Table PCR Sophomore Year of High School PCR Primer - - PowerPoint PPT Presentation

Mar 31, 2023 249 likes •697 views

My Kitchen Table PCR Sophomore Year of High School PCR Primer Primer-Defined Changes to the PCR Copies + Copying DNA with Polymerase Chain Reaction Creating the Chromophore Colorwheel Chromophore ConservedSimilarDivergent Acropora

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My Kitchen Table PCR Sophomore Year of High School

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PCR Primer

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Primer-Defined Changes to the PCR Copies

∆ ⁻ +

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Copying DNA with Polymerase Chain Reaction

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Chromophore

Creating the Chromophore Colorwheel

ConservedSimilarDivergent

Acropora millepora

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Gibson Assembly for Mutagenesis and Drug Selection Swap

PvuII PvuII

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Plasmid Uptake by Calcium-Assisted Heat Shock

Asif et al, Front Microbiol 2017

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A Primer for This Week

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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
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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

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Beta-lactamase ( Bla/ampr ) Beta-galactosidase ( lacZ )

DNA-Programming w/ Plasmids

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Beta-lactamase ( Bla/ampr ) Beta-galactosidase ( lacZ )

DNA-Programming w/ Plasmids

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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-type S65T

Chromophore Engineering

Image Credit: Roger Tsien Nobel Lecture

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Fluorescent Spectrum & Structures

Discosoma 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 -6nts Shift -12nts Shift -18nts Shift -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