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Agenda

Course Logistics

Read, Write, Edit Life HW Questions

DNA Construct Design/Twist

Benchling Tutorial (Interactive)

Gel Electrophoresis Lab

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Read, Write, Edit HW

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Read, Write, Edit Life HW Questions

DNA Read

What DNA would you want to sequence (e.g., read) and why? This could be DNA related to human health (e.g. genes related to disease research), environmental monitoring (e.g., sewage waste water, biodiversity analysis), and beyond (e.g. DNA data storage, biobank).

What technology or technologies would you use to perform this sequencing and why? Also answer the following questions:

Is your method first-, second- or third-generation or other? How so?
What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.
What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?
What is the output of your chosen sequencing technology?

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Read, Write, Edit Life HW Questions

DNA Write

What DNA would you want to synthesize (e.g., write) and why? These could be individual genes, clusters of genes or genetic circuits, whole genomes, and beyond. As described in class thus far, applications could range from therapeutics and drug discovery (e.g., mRNA vaccines and therapies) to novel biomaterials (e.g. structural proteins), to sensors (e.g., genetic circuits for sensing and responding to inflammation, environmental stimuli, etc.), to art (DNA origamis). If possible, include the specific genetic sequence(s) of what you would like to synthesize! You will have the opportunity to actually have Twist synthesize these DNA constructs! :)

What technology or technologies would you use to perform this DNA synthesis and why? Also answer the following questions:

What are the essential steps of your chosen sequencing methods?
What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?

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Read, Write, Edit Life HW Questions

DNA Edit

What DNA would you want to edit and why? In class, George shared a variety of ways to edit the genes and genomes of humans and other organisms. Such DNA editing technologies have profound implications for human health, development, and even human longevity and human augmentation. DNA editing is also already commonly leveraged for flora and fauna, for example in nature conservation efforts, (animal/plant restoration, de-extinction), or in agriculture (e.g. plant breeding, nitrogen fixation). What kinds of edits might you want to make to DNA (e.g., human genomes and beyond) and why?
What technology or technologies would you use to perform these DNA edits and why? Also answer the following questions:

How does your technology of choice edit DNA? What are the essential steps?
What preparation do you need to do (e.g. design steps) and what is the input (e.g. DNA template, enzymes, plasmids, primers, guides, cells) for the editing?
What are the limitations of your editing methods (if any) in terms of efficiency or precision?

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Review Papers

DNA Sequencing at 40: Past, Present, and Future (2017)

Shendure, J., Balasubramanian, S., Church, G. et al.

https://doi.org/10.1038/nature24286

DNA Synthesis Technologies to Close the Gene Writing Gap (2023)

Hoose, A., Vellacott, R., Storch, M. et al.

https://doi.org/10.1038/s41570-022-00456-9

ZFN, TALEN, and CRISPR/Cas-Based Methods for Genome Engineering (2013)

Gaj T, Gersbach CA, Barbas CF 3rd

https://pubmed.ncbi.nlm.nih.gov/23664777/

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Week 02 Lab

Gel Art

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Gel Electrophoresis

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Cut

Copy

Join

Restriction Enzymes

Polymerase Chain Reaction

(PCR)

DNA Cloning

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Restriction Enzymes

Can result in cut ends that can be blunt (blunt ends) or staggered (sticky ends)

Palindromic - enzymes can bind the DNA from either strand

https://slideplayer.com/slide/7876413/

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Restriction Enzymes

Used in many molecular biology techniques such as:

DNA Cloning (assembling recombinant DNA fragments)

Diagnostic Digests

Diagnostic Digests: restriction enzymes are used to verify that the DNA you have obtained is your desired DNA

We can predict the lengths of the resulting fragments (virtual digest) from sequence and restriction sites
We can check that the fragment lengths match our predictions

***The first recombinant insulin was made with Restriction Enzymes techniques

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https://www.aatbio.com/catalog/gel-electrophoresis

Gel Electrophoresis

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Gel Electrophoresis

DNA is negatively charged due to phosphates in its sugar-phosphate backbone

Negatively charged DNA fragments will move towards regions of positive potential in an electric field

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Gel Electrophoresis

Three factors affect the speed a DNA fragment will travel within a gel

Charge: The greater the charge of the fragment, the faster it will move in an electric field

Mass: The greater the mass of a fragment, the slower it will travel

Length: The gel has tiny pores that DNA can travel through. The smaller the DNA fragment, the faster it will move through the pore

DNA has same charge per mass for any nucleotide therefore gel electrophoresis separates DNA purely based on length

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Most biological information were stored in the form of DNA

Storage

Message

Function

Plasmid

DNA

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See guideline in

BioBootCamp session 3

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File Formats

DNA sequences are usually stored in either FASTA or GenBANK file formats

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File Formats

DNA sequences are usually stored in either FASTA or GenBANK file formats

FASTA and GenBank Files always store DNA sequences as coding sequences (5’ to 3’ convention)

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Retrieving sequences from Database e.g. NCBI, UniProt

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Retrieving sequences from Database e.g. NCBI, UniProt

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5’ to 3’ Convention

The 3’ to 5’ strand is called the template strand. RNA polymerase always binds to this strand and creates a complementary 5’ to 3’ strand of mRNA

When mRNA is translated into protein the mRNA codons are read by the ribosome in the 5’ to 3’ direction. Thus, the 5’ to 3’ strand is the coding strand: it contains the direct code for the amino acid sequence, not the complement.

https://slideplayer.com/slide/7876413/

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Import your sequence

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Select Enzymes

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Perform Digest

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DNA Substrates and Enzymes

Lambda DNA (DNA of the lambda bacteriophage)

sequence files: genbank, fasta

Restriction enzymes

EcoRI-HF
HindIII-HF
BamHI-HF
KpnI-HF
EcoRV-HF
SacI-HF
SalI-HF

Note: HF indicates “high-fidelity,” i.e. these restriction enzymes have a very low probability of cutting at off-target sites.

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Create Pictures!

The distinct bands formed from gel electrophoresis can be used to create pictures!

Latent Figure Protocol -- intro

https://vimeo.com/337656704

Paul Vanouse, https://www.paulvanouse.com/lfp.html

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Converting GFP into RFP

PCR

Or DNA Synthesis

Digest

Assembly

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

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Converting GFP into RFP

PCR

Or DNA Synthesis

Digest

Assembly

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

Jump to

Benchling Basic Project

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Converting GFP into RFP

Or DNA Synthesis

PCR

Digest

Assembly

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

Purify DNA fragment with

Agarose Gel Electrophoresis

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Converting GFP into RFP

Digest

Assembly

PCR

Or DNA Synthesis

Green

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

Red

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Recitation 02: Part 2

DNA Construct Design & Twist

Slides Content by Hark Jhajj and Patrick Han�Slight re-design: Georg Tremmel and Ice Cholpisit Kiattisewee

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Central Dogma of Molecular Biology

https://www.ck12.org/flexi/biology/central-dogma/what-are-the-steps-of-the-central-dogma/

DNA

RNA

Proteins

Transcription (DNA → RNA) occurs in the nucleus and is mediated by RNA polymerase

Translation (RNA → protein) occurs in the cytoplasm where ribosomes “read” RNA instructions to make proteins.

RNA polymerase

Ribosome

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Eukaryotic vs Prokaryotic Cells

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/translation/v/translation-mrna-to-protein

Similarities

Central Dogma holds

Differences

No nucleus

Transcription & translation happens @ same place

Multi vs. Single chromosome
Circular
Small
Faster replication

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Why do we use bacteria for DNA replication?

Advantages:

Takes less time

Cell division is faster
Each cell has single chromosome
Single circular plasmid
Each replicated cell has exact copy of DNA

Well-established protocols

to isolate plasmid

Easy method to get many copies of your desired DNA

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What are Plasmids?

https://blog.addgene.org/plasmids-101-what-is-a-plasmid

Plasmids are small circular pieces of DNA that replicate independently from the host’s chromosomal DNA.

Used to introduce foreign DNA into another cell.

Mainly found in bacteria, but also exist naturally in archaea and eukaryotes such as yeast and plants.

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How does DNA get replicated in bacteria?

Replicator

DNA sequence which allows initiation of replication within a plasmid by recruiting replication machinery proteins

Helicase

Enzymes that unwind DNA sequences to start the process of DNA replication.

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Can we replicate DNA using cell-free systems?

https://blog.addgene.org/plasmids-101-what-is-a-plasmid,

https://www.linkedin.com/pulse/steffanie-flair-3ct

Simple answer: YES, but how?

Polymerase chain reaction

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What is Translation?

mRNA → Protein

How do I know that AUG = methionine?

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/translation/a/the-genetic-code-discovery-and-properties

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What is genetic code?

3 letter code = protein sequence

Example: UCG = Serine

Why do we have multiple codons for the same protein?

Evolution…codon usage bias

(let's discuss in more detail)

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A bit more into translation…

Transfer RNA = anticodon = amino acids

Different Species = different preferences of transfer RNA

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What is codon optimization?

More stable RNA structure = increase translation efficiency

Codon Optimization = Codon usage bias

Which Codon is better for Arginine expression for E.coli?

Improve gene expression

https://www.youtube.com/watch?v=zQUJvmJ6sfU

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Workflow

Get many copies of plasmid DNA

Transform bacteria

with plasmid DNA

Design plasmid DNA with protein of interest

Twist

Cloning

mini/midi prep

Introduction of plasmid DNA to cells

Transcription & Translation happens

in cell

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New Workflow - Cell-free protein production

Produce proteins from DNA

Produce DNA cell-free

Design plasmid DNA with protein of interest

Twist

Nuclera or other platform

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New Workflow

Cell-free system to produce proteins from plasmid DNA

Get many copies of plasmid DNA

Transform bacteria

with plasmid DNA

Design plasmid DNA with protein of interest

Cell-free protein production system

Twist

Cloning

mini/midi prep

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What is molecular cloning?

Transform (Getting DNA into bacteria)

Pick colonies (To start bacterial cultures)

Inoculate the liquid cultures

(by introducing single colonies)

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-transformation-selection

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