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Minimal Genome: Synthetic Biology

This document discusses efforts to synthesize minimal bacterial genomes. Researchers first identified the minimal number of genes needed for a free-living organism by studying Mycoplasma genitalium, which has the smallest known genome. They found around 100 non-essential genes in M. genitalium through transposon mutagenesis. Researchers then aimed to synthesize a minimal bacterial genome containing only the essential genes, estimated to be around 256 genes. In 2010, scientists assembled the first self-replicating synthetic bacterial genome, a modified M. genitalium genome, using a multi-stage assembly process. While synthetic genomes offer promise for useful applications, they also raise risks regarding synthetic pathogens and genetic transfer that warrant consideration.

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66 views25 pages

Minimal Genome: Synthetic Biology

This document discusses efforts to synthesize minimal bacterial genomes. Researchers first identified the minimal number of genes needed for a free-living organism by studying Mycoplasma genitalium, which has the smallest known genome. They found around 100 non-essential genes in M. genitalium through transposon mutagenesis. Researchers then aimed to synthesize a minimal bacterial genome containing only the essential genes, estimated to be around 256 genes. In 2010, scientists assembled the first self-replicating synthetic bacterial genome, a modified M. genitalium genome, using a multi-stage assembly process. While synthetic genomes offer promise for useful applications, they also raise risks regarding synthetic pathogens and genetic transfer that warrant consideration.

Uploaded by

sandraudg1993
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Minimal genome

Synthetic biology

Daniel G. Gibson, et al. Science 319, 1215 (2008)

Daniel G. Gibson, et al., Science 329, 52 (2010)

synthetic genomes to use as


biofactories

1. Know the minimal genome


2. Synthesize the minimal genome

2002- Synthesis of full length genome, infectious polio virus


(Cello et al., Science. 9;297:1016-8)
2003- Synthesis genome of X174 bacteriophage synthesis
5386-base
(Smith et al., PNAS 100: 1544015445)

Minimal genome?
The minimal genome approach seeks to estimate the
smallest number of genetic elements sufficient to build a
modern-type free-living cellular organism. (Mushegian)
Essential set of survival genes

Knowledge of existing genomes


Define shorter list of key players
Protein sequence similarities

Homology
is the basic concept of any evolutionary
analysis

Glass et al 2006 PNAS 103 (2): 425430

Why Mycoplasma?

Wall-less
Obligate parasites

Smallest known genome of any free living


organism capable of growing in axenic culture
Evolved by massive genome reduction
Lack genomic redundancy

species name

number of genes

size (Mbp)

Pelagibacter ubique

~150

0.13

Candidatus Hodgkinia cicadicola Dsem

169

0.14

Candidatus Carsonella ruddii PV

182

0.16

Candidatus Sulcia muelleri GWSS

227

0.25

Candidatus Sulcia muelleri SMDSEM

242

0.28

Buchnera aphidicola str. Cinara cedri

357

0.4261

Mycoplasma genitalium G37

475

0.58

Candidatus Phytoplasma mali

479

0.6

Buchnera aphidicola str. Baizongia


pistaciae

504

0.6224

Nanoarchaeum equitans Kin4-M

540

0.49

Non-essential genes
Transposon mutagenesis to systematically disrupt genes
2,462 transposon insertion sites

transposon mutagenesis

Non-essential genes
Transposon mutagenesis to systematically disrupt genes
2,462 transposon insertion sites

mutants that survived after 4 weeks

Detected about 100 non-essential genes

Statistical saturation mutagenesis

100 nondispensable M. genitalium genes

What genes are nonessential?


48% of genes found were hypothetical proteins or encoded proteins
of unknown function
None of the genes encoding the key enzymes of DNA replication
were disrupted

Some of those that were identified


DNA metabolism, transporters, recombination, DNA repair

may be essential for long term survival


Found more genetic redundancy than previously thought

100 genes are not essential, but


In combination?
Need to be able to efficiently assemble reduced genomes with
combinations of these genes missing

100 nonessential genes


382 protein coding genes
3 phosphate transporter genes
43 RNAcoding genes

MINIMAL GENOME

Theorically 256 genes

complete system of translation


DNA replication machinery,
Rudimentary system of recombination and repair
A simple transcription apparatus with four RNA polymerase subunits
a single factor and three transcription factors
large set ofchaperone-like proteins
Energy producing metabolism
NO amino acid biosynthesis
Limited repertoire of metabolite transport systems

Low percentage of essential genes

100 genomes shows that only 63 genes are ubiquitous:


Non included genes that slow down growth
Alternative pathways in different specie
Failure of laboratory conditions

Zang and Wang, 2010. Protein Cell 1: 427434

Smith et al., PNAS 100: 1544015445

One lethal error per 500bp

M. genitalium JCVI-1.0
582,970base pair Mycoplasma Genitalium genome:
Contains functional copies of all wild type genes except MG408
MG408 disrupted by antibiotic marker to block pathogenicity and
allow selection
Watermarks in intergenetic regions (~1000 pb)
458 protein code genes
43 rRNA, tRNA, strcutral RNA genes
Watermarks

Five Stage Assembly

25 cassette 5 to 7 kilobases (kb)


Partion the genome in 101 pieces
(Overlaps 50-80 nucelotides)

Assembly of cassettes by in vitro recombination

Assembly of cassettes by in vitro recombination

Yeast TAR Vector

Great promise . . .

Renewable fuel sources


Pharmaceuticals
Chemical detoxification
Environmental control
Beneficial microbes

. . . or great risk?

Synthetic pathogens
Genetic transfer (similar to GMO arguments)
Economic risk
Patent/ownership

http://www.youtube.com/watch?v=iQ1VNEgcWE8

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