Laboratory Techniques �in Synthetic Biology

SynBio4ALL Beginner Course: Week 5

Instructor: Cholpisit Ice Kiattisewee

November 29^th, 2023

Recording: https://youtu.be/OBGXDvzRsgo

Overview of Today’s Lecture

1. General Workflow and Biosafety
2. DNA as a basis of Synthetic Biology
1. DNA Amplification and Modification (in vitro)
2. Assembly of DNA in a tube (in vitro)
3. Using microbe to make DNA (in vivo)
4. Designing and Analyzing DNA (in silico)
3. Solving real-world problem
• Insulin/Artemisinin biosynthesis by microbe (Week 1&3)
• NETLANTIS iGEM 2022 Overgraduate Grand Price Winner

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Biosafety Levels

• There are 4 levels of biosafety (microorganisms)
• For Synthetic Biology, we usually work with BSL-1 organisms (safest)
• E. coli or Baker’s yeast
• Sometimes we may work with BSL-2 organisms if the project is related to human health.

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See https://www.cdc.gov/orr/infographics/biosafety.htm

Biosafety Levels

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Can usually work on a bench but should autoclave all waste

Need to work on laminar flow hood

Biosafety Levels

5

Need positive pressure face coverage

Need a full-body suit and proper cleaning

Most biological information were stored in the form of DNA

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Lecture 2 by Steven : Central Dogma of Molecular Biology

Plasmid

DNA

DNA as LEGO pieces

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Lee et al. ACS Synth. Biol. 2015, 4, 9, 975–986

DNA as LEGO pieces

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From Lecture 4 by Prince : Golden-Gate Assembly

How to synthesize DNA pieces?

1. In vivo DNA Replication
2. Polymerase Chain Reaction
3. Chemical synthesis

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No modification!!!

– cheap

Illustrations from JoVE and Study.com

How to synthesize DNA pieces?

1. In vivo DNA Replication
2. Polymerase Chain Reaction
3. Chemical synthesis

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Can modify DNA

– relatively cheap

Illustration from wikimedia.org

How to synthesize DNA pieces?

1. In vivo DNA Replication
2. Polymerase Chain Reaction
3. Chemical synthesis

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Commercially available in small amount – high cost

Illustration from Oligonucleotide Synthesis|TCI AMERICA

Polymerase Chain Reaction (PCR)

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Illustration from Polymerase Chain Reaction (PCR) (genome.gov)

3 steps of PCR

Setting up the molecular experiment

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It’s like transferring clear liquid

over and over and over!!!

Polymerase Chain Reaction (PCR)

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Illustration from Addgene: What is Polymerase Chain Reaction (PCR)

Thanks! Alyssa

After PCR then what?

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PCR

Purify

DNA in a tube

Assembly

New DNA

Restriction/Ligation – Cut/Stitch DNA

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Illustration from Addgene: Plasmid Cloning by PCR

Using specific sticky ends enable directional joining

BioBrick Assembly Strategy

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Illustration from J5 Design Manual (jbei.org)

BioBrick strategy enables simple protein fusion

Overlapping Sequence Assembly

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Illustration from Gibson Assembly®|NEB

Long overlapping ends enable higher accuracy

Overlapping Sequence Assembly

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Illustration from Sharebiology

PCR-based Modifications of DNA

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Illustration from Q5 Site Directed Mutagenesis Kit - New England Biolabs

PCR-based methods enable simple modification

Plasmid Architectures

• At their most basic level, plasmids are small circular pieces of DNA that replicate independently from the host’s chromosomal DNA
• General elements of a plasmid are
• Origin of replication
• And its replication elements
• Homologous region (integrative)
• Selectable marker
• Gene of interests
• Multiple Cloning Sites (MCS)

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Plasmid

DNA

Recap from Lecture 3 and Lecture 4

Putting plasmid into bacteria is relatively simple!!!

Putting DNA into bacteria (or yeast)

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Illustrations from Plasmids 101 (addgene.org) and Ren et al. Appl. Microbiol. Biotechnol. 2019

Transformation can be achieved by heat-shock or electric-shock

Validation of new DNA: Sequencing

DNA sequencing could be performed by different technologies including

1. Sanger sequencing (cheap),
2. Next-gen sequencing (massively parallel)
3. 3^rd-gen sequencing (long read)

Illustration from ECNUAS - iGEM 2022

Each nucleotide yield different signal (color). Then, they can be aligned to that of template or expected sequence for validation

In silico analysis of DNA sequence

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Benchling®: Free for academic users

A more powerful version available for Enterprise user

SnapGene Viewer: Free

A more powerful version available as SnapGene®

Let’s do a DEMO!!!

SynBio4ALL Benchling Project

https://benchling.com/

https://www.snapgene.com/snapgene-viewer

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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Make different DNA fragments by PCR

Links to DNA sequences:

Desired sequence: ColE1-AmpR-sfGFP plasmid

1) sfGFP_part, 2) AmpR_part, 3) ColE1_part

DNA fragments could also be made by

1) restriction digest reaction or

2) ordered for chemical synthesis

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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Make different DNA fragments by PCR

Links to DNA sequences:

Desired sequence: ColE1-AmpR-sfGFP plasmid

1) sfGFP_part, 2) AmpR_part, 3) ColE1_part

DNA fragments could also be made by

1) restriction digest reaction or

2) ordered for chemical synthesis

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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Make appropriate sticky-ends for ligation

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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Make appropriate sticky-ends for ligation

Jump to SynBio4ALL Benchling Project

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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White

Green

Assembly of 3 Fragments into a plasmid – ColE1-AmpR-GFP

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White

Green

Converting GFP into RFP

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PCR

Or DNA Synthesis

Digest

Assembly

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

Converting GFP into RFP

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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 SynBio4ALL Benchling Project

Converting GFP into RFP

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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

Converting GFP into RFP

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PCR

Or DNA Synthesis

Digest

Assembly

Green

Red

Links to DNA sequences:

Starting ColE1-AmpR-sfGFP plasmid

🡪Digested ColE1-AmpR

mRFP template

🡪PCR mRFP insert

Real-world Example: Insulin

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Illustration from BioNinja|Universality

From Lecture 3 by Ryan

https://www.ncbi.nlm.nih.gov/

https://www.uniprot.org/

Real-world Example: Artemisinin

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Ro, et al. Nature, 2006

FPP

Yeast enzymes

Plant enzymes

Yeast cell

Artemisia annua

Artemisinin

From Lecture 1 by Heidi

Real-world Example: NETLANTIS

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Illustrations from UCopenhagen (igem.wiki)

Problem:

Recalcitrant plastic net

Inspiration:

Strong Natural Fiber

Design:

SynBio Engineered Biodegradable Fiber

Real-world Example: NETLANTIS

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Illustrations from UCopenhagen (igem.wiki)

Real-world Example: NETLANTIS

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Illustrations from https://2022.igem.wiki/ucopenhagen/parts

Useful Resources

• Plasmids 101 eBook from Addgene
• Addgene has a lot of education materials including YouTube tutorials
• iGEM parts registry – parts.igem.org
• All DNA parts for iGEM projects will be deposited on this website
• General plasmid toolkits articles
• Lee et. al. 2011 – BglBricks characterization of E. coli plasmids
• Silva-Rocha et. al. 2012 – SEVA: Standard European Vector Architecture developed mainly for broad-host-range Gram-negative bacteria (e.g. Pseudomonas putida)
• Radeck et. al. 2013 – The Bacillus BioBrick tool (Bacillus subtilis is a Gram-positive bacteria)

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That’s it~~~

Any questions?