synthetic genome

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GENOME GENETICS Seminar on 13 th Nov 2014 SYNTHETIC GENOMES Presented by: Nethravathi R GN113011

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Page 1: Synthetic Genome

GENOME GENETICS

Seminar on

13th Nov 2014

SYNTHETIC GENOMES

Presented by:

Nethravathi R

GN113011

Page 2: Synthetic Genome

• The world is facing increasingly difficult challenges today.

• Population growth resulting in the growing demand for critical

resources such as energy, clean water, food and medicine are

taxing our fragile planet.

• To fulfill these needs we need to exploit technologies.

Can we use the genomic advances to offer

the world viable, sustainable alternatives?

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SYNTHETIC BIOLOGYHot field in life science area that involves the study of

designing and construction of new biological

entities such as enzymes, genetic circuits, and cells or whole

biological systems, and the re-design of existing, natural

biological systems for useful purposes

SYNTHETIC GENOMICS

Study of Invitro chemical synthesis of genetic material i.e.,

DNA in the form of oligonucleotides, genes, or genomes with

Computational techniques for its design.

SYNTHETIC GENOME

Artificially synthesised genome (invitro)

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PRECONDITIONS OF SYNTHETIC GENOMICS

Recent achievements in

• Whole-genome sequencing

• Development of methods of molecular tools

• DNA-sequencing, DNA-synthesis, and DNA-editing

technologies.

• Bioinformatics

• Deepening of knowledge on mechanisms of functioning of

living systems at a cellular level.

Page 5: Synthetic Genome

Guess who?

Page 6: Synthetic Genome

• John Craig Venter is a leading scientist in genomic research.

• With the invaluable contribution of Celera Genomics that he

funded in 1998, the Human Genome Project was completed three

years ahead of expected date.

• Later, he funded J. Craig Venter Institute (JCVI), with synthetic

genomics as one of the major focuses.

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• After 15 years of investigation and ~$40 million of investment,

JCVI walked out this significant step for synthetic biology.

• The synthetic genome started with design of DNA sequence,

which highly depends on the accurate sequence of

Mycoplasma mycoides genome.

• Venter group spent numerous efforts on comparing different

sequencing results, making necessary corrections, and setting

watermark sequences to differentiate synthetic and natural

genomes.

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• The rapid development of molecular biology, genetics and

the related biotechnology and bioengineering kept bringing

surprises to scientists worldwide...

• In 1990, Mandecki et al. assembled a plasmid of 2.1 kbp using

30 chemically synthesized oligonucleotides and the method of

serial cloning

• In 1995, Stemmer et al. synthesized a DNA fragment of 1100

bp containing the TEM-1 gene of ß-lactamase using 56

oligonucleotides and the plasmid of 2700 nucleotidesusing

136 oligonucleotides

• Including the cloning of Dolly (Wilmut et al., 1997) (followed

by cloning of a variety of animals)

In recent decades....(1)

Page 9: Synthetic Genome

In recent decades....(2)

• The completion of Human Genome Project (Cheung et al., 2001;

Lander et al., 2001; Venter et al., 2001) (followed by the reports of

complete genome from other species and by the spread of

personalized genome sequencing service)

• In 2002, Cello et al reported about the first synthesis of full-sized

cDNA of the poliovirus genome (about 7500 bp).

• The induction of pluripotent stem cells (Takahashi and

Yamanaka, 2006) (followed by the production of viable iPS mice

(Zhao et al., 2009).

• In 2007, Kodumal et al. obtained the largest synthetic DNA of

32,000 nucleotides containing a cluster of genes encoding the E.

Coli megaenzyme polyketide synthase.

Page 10: Synthetic Genome

• Recently, another breakthrough was brought by John Craig

Venter and his team, who achieved the first synthetic life—

Mycoplasma that only contains synthetic genome.

In recent decades....(3)

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Published synthetic DNA sequence

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First synthesis of full-sized cDNA of the

poliovirus genome (about 7500 bp)

1. Chemical synthesis and purification of oligonucleotides of

“positive” and “negative” polarity (in accordance with

direction from 5'- to 3'-ends and vice versa)

2. Assembly of DNA segments of 400 – 600 bp due to

overlapping complementary oligonucleotide sites follow by

subsequent ligation or use of PCA assembly of three

genome fragments of 2000 – 3000 bp from these segments

using the cloning methods and subsequent assembly of the

whole genome from these fragments.

Appearance of polymerase cycling assembly (PCA) was the next

step of improvement of gene assembly; PCA was initially used for

synthesis of HIV-2 Rev gene of 303 bp in length. The development

of methodological base resulted in overcoming of a psychological

barrier of 1 kbp.

In 2002 Cello et al.

steps:...(1)

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First synthesis of full-sized cDNA of

the poliovirus genome (about 7500 bp)

3. The synthetic cDNA Was transcribed into viral RNA by

means of phage T7 RNA polymerase.

4. In cell free extracts this RNA induced synthesis of viral

particles.

5. The resultant de novo viruses exhibited Infectivity and

biochemical and pathogenic characteristics typical for

poliovirus

In 2002 Cello et al.

steps:..(2)

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second full-sized genome of X174

phage (5386 bp) synthesized • For the synthesis of the full-sized genomic DNA, they used only

ligation and PCA.

• The whole synthesis took about 2 weeks.

1. All 259 chemically synthesized oligonucleotides (42 nucleoitide

each) purified by means of gel-electrophoresis

2. Covered sequences of both DNA strands were mixed in

equimolar quantities and ligated.

3. The resultant fragments of various length (ligation did not yield full-sized

DNA due to the presence of some quantities of defect oligonucleotides and lack of 100%

effectiveness of oligomer assembly into DNA duplexes) were added up to the

“genome-sized” by means of PCA and amplified.

Smith et al.

steps:..(1)

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second full-sized genome of X174

phage (5386 bp) synthesized

4. The linear full-sized genomic DNA was circularized by means

enzymatic ligation and the use of preexisted restriction sites for

preparation of sticky ends.

5. Transfection of cells with the circular synthetic genome resulted

in appearance of infective phages

Smith et al.

steps:..(2)

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POTENTIAL POSITIVE APPLICATIONS

Scientists foresee many including

•new pharmaceuticals

•biologically produced ("green") fuels

•the possibility of rapidly generating vaccines

against emerging microbial diseases

•biofactory

There is the potential for misuse and accidents!

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CONCLUSION

The ultimate goal of synthetic biology is to build novel

biological systems that have new functions or to engineer

existing biological systems to have better efficiency.

With the many challenges to the understanding of natural

biological systems, the rapid progress of emerging tools for

synthetic biology has begun to provide genomes for

applications in the areas of energy, health care, biochemicals,

and the environment.

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REFERENCES

• http://syntheticbiology.org/

• http://www.synberc.org/

• www.syntheticgenomics.com

• www.livescience.com/6486-live-organism-synthetic-genome-

created.html

• www.intecopen.com

• Cheng et al, Syntheticbiology. Reviews in advance

biomed.eng.2012,,14:155-178

• Radko et al, The synthesis of artificial genome as the basis of

synthetic biology , Biochemistry Masco supplement series DOI

10.1134/s1990750807040014

• Konig et al, Synthetic biology and Synthetic genomics

applications between hopes and concerns ,

Currentgenomics,2013,14,11-24

• Daniel et al, Synthesising a minimal genome , Journal Science,

vol286,issue5447,2087-2090 (10 December 1999)

• Monya Baker(2011), The next step for synthetic genome, Journal

Nature | vol 473 | 403-408

Page 19: Synthetic Genome

QUESTIONNAIRE

Page 20: Synthetic Genome