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Building biological systems I: Genome synthesis and genome editing

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Industrial Biotechnology

Universidade de Manchester

4.7 (284 classificações) | 12K alunos inscritos

Fossil fuels have been the primary energy source for society since the Industrial Revolution. They provide the raw material for the manufacture of many everyday products that we take for granted, including pharmaceuticals, food and drink, materials, plastics and personal care. As the 21st century progresses we need solutions for the manufacture of chemicals that are smarter, more predictable and more sustainable. Industrial biotechnology is changing how we manufacture chemicals and materials, as well as providing us with a source of renewable energy. It is at the core of sustainable manufacturing processes and an attractive alternative to traditional manufacturing technologies to commercially advance and transform priority industrial sectors yielding more and more viable solutions for our environment in the form of new chemicals, new materials and bioenergy. This course will cover the key enabling technologies that underpin biotechnology research including enzyme discovery and engineering, systems and synthetic biology and biochemical and process engineering. Much of this material will be delivered through lectures to ensure that you have a solid foundation in these key areas. We will also consider the wider issues involved in sustainable manufacturing including responsible research innovation and bioethics. In the second part of the course we will look at how these technologies translate into real world applications which benefit society and impact our everyday lives. This will include input from our industry stakeholders and collaborators working in the pharmaceutical, chemicals and biofuels industries. By the end of this course you will be able to: 1. Understand enzymatic function and catalysis. 2. Explain the technologies and methodologies underpinning systems and synthetic biology. 3. Explain the diversity of synthetic biology application and discuss the different ethical and regulatory/governance challenges involved in this research. 4. Understand the principles and role of bioprocessing and biochemical engineering in industrial biotechnology. 5. Have an informed discussion of the key enabling technologies underpinning research in industrial biotechnology 6. Give examples of industrial biotechnology products and processes and their application in healthcare, agriculture, fine chemicals, energy and the environment.

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Avaliações

4.7 (284 classificações)

5 stars
199 ratings
4 stars
71 ratings
3 stars
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2 stars
1 ratings

XL

Jun 30, 2019

Synthetic biology, industrial fermentation and green chemistry are integrated into the course and it is well organized and is a nice interdisciplinary course.\n\nThanks.

JN

May 21, 2018

The lectures were divided into modules that were appropriate in length/tie. Speakers were very easy to comprehend and the outline of the topics were well presented.

Na lição

Methods in Systems and Synthetic Biology

Recent advances in our ability to read and write genome sequences on a large scale have led to an ambitious vision for a new generation of biotechnology, often referred to as Synthetic Biology. Synthetic Biology aims at turning biology into an engineering discipline, in which organism engineers use computational tools to design biological systems with novel valuable functionalities, which are then built using advanced high-throughput genetic engineering, and tested by rapid screening technologies that collect diagnostic molecular profiles to drive improved designs in an iterative design-build-test cycle. This module will introduce the engineering concepts that inform Synthetic Biology and the cutting-edge technologies that underlie our dramatically increasing ability to construct living systems with custom-made functionalities. All stages of the design-build-test cycle for novel biosystems will be discussed, with a special focus on their integration in a unified bioengineering platform. Examples will focus on the application of Synthetic Biology as an enabling technology for the bioindustry, especially for the improved microbial production of high-value chemicals and drugs. A section on responsible research and innovation will explore the transformative potential of this innovative technology within a broader socio-economic context, creating awareness of the ethical and political implications of research in this field.

Introduction to Synthetic Biology: Visions for Biotechnology 2.06:27

Designing biological systems: Computational modelling and systems biology9:10

Building biological systems I: Genome synthesis and genome editing9:25

Controlling and engineering pathways in Synthetic Biology9:00

Testing Biological Systems Biological debugging using metabolomics8:12

Deploying biological systems: Perspectives on Responsible Research & Innovation and bioethics I5:24

Conclusions and Outlook Systems and Synthetic Biology6:40

Ministrado por

Prof. Nicholas Turner
Deputy Director
Prof. Nigel Scrutton
Director

Transcrição

My name's Adrian Jervis.

I'm a senior experimental officer here at the Synthetic Biology Research Center in

the Manchester Institute of Biotechnology, University of Manchester.

My key responsibilities are chassis and pathway engineering for

the production of microbial strains to produce speciality and fine chemicals.

Two of the key technologies, genome synthesis and

genome editing, which allows us to produce efficient strains for chemical production.

This is what we're going to talk about today.

Genome synthesis and genome editing are core technologies that sit in the build

stage in design build test cycle for chassis engineering.

But crucially fed by information from the genome scale modeling in the design stage.

Since the advent of DNA sequencing in the 1970s,

we've been deciphering the genetic code.

Initially, this was sequencing of single genes, but as the technology, capacity,

and cost of sequencing has developed over the last 30 years, we can now sequence

a whole human genome in just a few days and at a cost of just $1000.

Now industrially relevant microorganisms have a typical genome size of less than

half of 1% of a human genome, and

over 65,000 microbial genome sequences are now available.

This ability to read DNA has provided with us with

books telling us how an entire organism functions.

Although we have learned a vast number of words in these books, we are not yet

able to really understand the story:

how the highly complex gene networks produce a viable organism.

Newer technology, DNA synthesis, has now given us the ability to write DNA.

DNA synthesis technology has also undergone rapid development and

reduction in cost.

And now the first fully synthesized genomes have been completed.

Simple viruses and bacteria, and is close to the completion of the first complete

eukaryotic genome, that of the baker's yeast, the common microbial work horse.

Like DNA sequencing, the new technological targets to

be able to synthesize a whole genome for just $1,000.

And it's projected that this will be possible for bacteria by 2025, and for

yeast by 2035.

This ability to read and

write entire genomes allows researchers to begin to understand the blueprint of life,

the underlying principle of "what I cannot create I do not understand".

The understanding of the basic requirements for a genome,

a minimal set of genes, will allow us to design purpose built cell factories.

These chassis can be streamlined for the production of a wide range of chemical

compounds to replace the petrochemical industry, drug discovery,

development of bioremediation strategies, and many more applications.

There are two main approaches to producing designer genomes.

The first is to employ DNA synthesis in a bottom-up approach to build the genome

from scratch.

This is achieved by synthesizing small modules of the desired genome and then

combining these in an iterative process until a full genome is constructed.

The second approach is to use genome editing and the top down approach.

This starts with a naturally occurring genome of a useful species,

such as a bacteria or yeast, and

editing that genome so as to delete any unrequired genes and pathways.

These unessential genes may include those that have evolved to increase survival in

specific environmental conditions they will not experience in the laboratory.

The biggest landmark in the creation of a synthetic genome was achieved in 2010,

when the full synthesis and activation of a bacterial genome gave birth to the first

designed self-replicating organism, known as Synthia.

To do this, the team of Craig Venter at the J Craig Venter institute in

the United States chose a template bacterial species

with one of the simplest genomes described, Mycoplasma mycoides.

Synthesis began by chemically synthesizing short

oligonucleotides of around 100 basis.

These were annealed together to produce over 1,000 cassettes of DNA of

approximately 1 kilobase in size.

And then simultaneously introduced 10 of these cassettes into yeast, and

exploited a natural process called homologous recombination.

To combine the fragments into ten kilobase assemblies.

This was followed by two more rounds of assembly with increasing size until

the full one megabase chromosome was constructed.

This chromosome is then isolated and

transferred into an existing recipient cell.

The synthetic genome essentially had the same architecture and sequence as

the wild type strain other than 14 non-essential genes which were omitted.

And the inclusion of several watermark sequences to allow them to

distinguish the artificial genome from the natural genome.

They also included a selection marker that would turn any cells blue

that were replicating using the new chromosome.

Phenotypic analysis of the new strain showed that, other than the blue color,

it was indistinguishable in appearance and growth to the original wild-type strain,

and Synthia still remains the only synthetic bacteria constructed to date,

and will serve as a template for further chassis development.

For many years, researchers have been able to edit genomes.

However, until 2009, most of the technologies employed

were typically only well-developed for a limited number of species.

They're often also laborious and have limitations, such as the use of

antibiotic resistance markers, the introduction of unwanted scar sequences.

Or the ability to only make a single change in any one genome.

Here we can see the typical process for gene knockouts, knock ins, and in bacteria,

although similar strategies exist for many cell types.

To create knockouts, a target gene, which is shown in blue, is typically interrupted

with an antibiotic resistance gene, which is set in red.

To knock in genes, shown in green, they're also included in the resistance cassette,

and are likewise carried on to the genome.

The antibiotic resistant cassette can then be subsequently removed, but

it will leave a scar sequence.

This kind of editing has been used for many years to great success.

But new powerful genome editing technologies have now been

developed that allow complete user-defined alterations,

even allowing the change of a single letter of the genome.

These new techniques including the CRISPR-Cas R system employ nucleases,

which can be thought of as DNA scissors.

Cas9 is a nuclease, which binds guide RNA.

This guide RNA carries a short recognition sequence that perfectly matches the target

on the genome, and directs the Cas9 nuclease to bind and cut at this site.

With a cut genome, cells cannot replicate and will die.

However, cells have a repair system, so they can rejoin the cut ends of the DNA,

which will result in the addition to the substitution of a single base cut site,

thus modifying the sequence, and any survivors will also include an edit.

Donor DNA is also provided, which matches the two cut ends.

It can also be incorporated during the repair process, or

by cutting at two sites.

The DNA in between can be deleted.

It's even possible that multiple modifications can be made simultaneously,

and the CRISPR-cas R system. The CRISRP-Cas9 system has revolutionized the speed and

accuracy by which the genome editing can be implemented.

Particularly in organisms that have been traditionally difficult to manipulate.

As well as the successful booting up of Synthia, a number of other key studies

have been carried out in the field of designer genomes Viruses

typically have the smallest genomes of just a few kilobases and so

unsurprisingly the first synthetic genomes produced were viral.

The Poliovirus in 2002 and the bacteriophage in 2003.

In 2006 an E.coli genome editing study was successful in the stepwise

reduction of the genome by 15 percent

without affecting the fitness of the strain in the laboratory.

This provided an indication of how much additional genetic material some organisms

need to survive outside the favorable conditions provided in the laboratory and

also identified many nonessential genes.

In 2009, an automated system for

engineering genomes developed known as MAGE,

in which a robotic platform can perform multiple rounds of multiplexed traditional

genome editing massively speeding up and targeting genome engineering.

In 2013 genome editing technology has also been used to recode the E.coli genome

resulting in a free code which can then be used to incorporate non natural amino acids

and other modifications into proteins.

Eukaryotes are a much more difficult task with larger and

much more complex chromosomes.

But the first yeast chromosome was synthesized in 2014 in

an ongoing international consortium known as SE 2.0.

This is now nearing completion of the other 15 chromosomes and

will soon produce an entirely synthetic eukaryotic genome for the first time.

It's clear that we now have the technologies for

building a synthetic genome via DNA synthesis or genome editing.

But this is still a significant challenge only achievable by teams of

skilled scientists with access to the correct technologies.

However, these technologies will continue to develop and

inevitably actually being a relatively routine process.

The real rate limiting step in designing

a full genome is our level of understanding of its complexity.

Even the small genome Mycoplasma which is 500 genes operates in complex networks and

pathways and our ability to understand this complexity is still in its infancy

Despite these hurdles, genome synthesis and editing have allowed a leap in the engineering of microbial chassis,

and in the coming years we should see an increase in the sophisticated design of genomes.

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