DESIGNING A SYNTHETIC ORGANISM

Asfa A S (HT080934L)

Vasanth Natarajan (HT081073M)

Department of Chemical & Biomolecular EngineeringNational University of Singapore

SYNTHETIC BIOLOGY

Origin of Life

Designer Cells

Minimal Genome

Recreate Life

Vision of “SYNTHETIC BIOLOGY “

Synthetic Biology

Synthetic biology is an ambitious and relatively new field of biology that hopes to recreate life. The first and foremost challenge in creating 'life in lab' lies in identifying the minimum essential components that can take on the essential properties of a living organism

What Defines LIFE

LIVING CELL

Autonomous Replication

Darwinian Evolution

Continued growth and division dependent on input of small molecules and energy

Genetic and phenotypic variation for survival and reproduction

Bottom Up Approach Top Down Approach

Design a protocell

Semi-synthetic

Current Strategies

Synthesizing cell from scratch

Strip down the genes of an existing cell to bare minimum enough to sustain life

Synthesizing Life – Bottom Up Approach

Assembly of single lipid molecules/micelles

Gradual growth

Environmental factors to control division

RNA - store information

RNA – RNA polymerase – replicate its own sequence

2 RNA molecules – simplest cell

Szostak et al. , Nature 2001

Minimal Genome Concept

Aims to strip down a present day bacterium to its minimum essential components pertaining to replication, transcription and translation machinery.

Understand the basic components of the cell that makes it living.

Provides a template genome that can be used to recreate life

A less complex cell that can be reliably modeled and engineered to meet our requirements.

Essential Genes – A Comparative Study

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Studies on Minimal Genome

Ess

enti

al G

enes

Craig Venter, 2005

Ehrlich SD,2003

Gil,2004

Koonin,1996

Mycoplasma genitalium – 482 protein coding genes – smallest genome

Craig Venter,2005 – 382 protein coding genes + 5 paralogous families – Transposon mutagenesis

Ehrilch SD, 2003 – 271 essential genes in Bacillus subtilis – Gene knock out by non replicating plasmid

Gil, 2004 – 206 essential genes – Comparison of Endosymbioints - Predicted

Koonin, 1996 – 256 essential genes – Comparison of M.genitalium and H.influenzae - Predicted

Functional Groups

Main Roles Craig Venter, 2005

Ehrlich SD, 2003

Koonin, 1996

Gil, 2004

Intermediary metabolism 4 2 0 2 Transport and binding proteins

35 35 7 32

Protein fate 24 16 3 10 Transcription 12 7 1 3 Cell envelope 35 35 23 35 Hypothetical proteins 47 47 43 45 Unknown function 37 36 20 29 Nucleosides and nucleotides 16 8 1 8 Energy metabolism 29 21 1 10 Protein synthesis 95 11 7 10 cell/organism defense 1 1 1 1 Synthesis of cofactors and carriers

7 2 1 3

Cellular processes 6 5 0 2 Fats and phospholipid metabolism

6 3 2 5

Regulatory function 2 2 1 2 DNA metabolism 25 12 3 11

Comparison of Functional Groups

Essential Genes – A Conclusive List

Different studies come up with a different number of essential genes.

Computation - Underestimates minimal genes - accounts only those genes that have been conserved in evolution.

Transposon mutagenesis - Over estimates the genes – Classifies genes that slow down growth as essential and essential genes that tolerate mutation as non essential.

Antisense RNA - limited success rates

Most mutants produced are single mutants – synthetic lethality may not be accounted

Construction of a single cell with systematic combination of all the mutations in a single

strain is beyond the scope of present day technology.

Designing a Synthetic Organism

Determine the minimal genes

Synthesize and assemble the

genome

Genome Transplantation

SYNTHETIC ORGANISM

Transposon Mutagenesis

Gene knockout using non replicating plasmid insertions

Antisense RNA

Computationally Predicted

Into a suitable propagating cell that can take up the genome

STRATEGY

Engineer the genome/add new

functions

Success so far…

Infectious Virus Completely Synthesized – World’s First Artificial Organism - 2002

3026 bp 2682 bp1895 bp

cDNA - T7 RNA polymerase promoter constructed from 3 overlapping DNA fragments.

Each fragment - overlapping 400-600 bp.

Each segment – 69 nt of + and – sequences

cDNA transcribed – Infectious RNA

Infection demonstrated in mice.

SYNTHETIC RNA => TRANSLATED => REPLICATED =>ENCAPSIDATED INTO NEW COAT PROTEINS

Cello et al. Science, 2002

In the Future…

Mycoplasma laboratorium

Synthetic Genome

Only essential 382 genes

Complete synthesis, cloning and sequential assembly

Immortal synthetic organism

Military Purpose – Pentagon

Self killing switch

Synthetic Algae

Biofuel

Synthetic Genomics

Proposed Applications

Biofuel – A dream in the making

Goals

Seeks alternatives to fossil fuels Sustainability Cost reduction

Challenges

Microorganisms can be designed to make useful materials from renewable materials (Sustainability)  - to seek alternatives to fossil fuels.

In this case, designing a set of chemical pathways which allows conversion of natural or waste materials for the production of Biofuels .

Biofuel – A dream in the making (contd.)

ZM - Z.mobilisSC - S.cerevisiaeEC - E.coli

adh,pdc,pfl

Genes that are important for ethanol production

How to design a synthetic organism by adding new functions to the existing genome ?

Biofuel – A Strategy for Designing synthetic organism

Identified Essential gene list

Add new genes to the

existing prototype and

assemble genome

Screen for viability of

cell , maximum replication and higher

ethanol production

Adh,pdc,pfl

Synthetic organism

which produces

ethanol with minimal genes

Genome Transplanta

tion

success

NO YESWhat next ?

Add few more imp

genes

Biomedical Applications

Devices- For example, for tissue regeneration or tissue repair complex molecular devices can be developed.

- Another example could be development of macromolecular assemblies to sense the damage in blood vessels and repair them.

Novel Drug Release Technology

Smart Drugs -----> Synthetic molecular ensemble

Encapsulates drug in an inactive form.

Sensing disease indicators

The programmed module will make a decision

Activates the drug . (Active only in cells affected by disease)

Inactive form

Inactive form

Active form

Programmed ModuleDisease

Indicators

Therapeutics

Genetic code expansion

Environmental Applications

Bioremediation:

Treatment of environmental contaminants via biological systems.

Rational modification of bacteria and other microorganisms to eliminate toxic waste from soil.

For certain chemicals for which clean up is difficult, novel organisms with specific wiring can be used.

Biosensing : Detect biotoxins Helps in detecting toxin levels in environment

The Hindering Factor

How to overcome ? Bio engineered systems

remains noisy

Not easier to predict accurately how a new system will behave

Engineered organisms capable of self replication and evolution

Expensive , Unreliable and adhoc biological systems

Obstacles

FORSEEN RISKS

Some of the risks are indefinable at present – we cannot anticipate certain risks at this early stage

Accidental release of harmful organism - Extinction of existing species - Endemic - Damaging/Disrupting the habitat ( Upset natural balance)

Purposeful Design and release of harmful organism – Bioterrorism

Bio-hacker culture

Control Measures

To educate and train a responsible generation of bioengineers and scientists

Working with approved research facilitiesControls and regulations can be imposed on part

suppliers (eg . screening of oligonucleotides )Strict laws and policies to be imposed. Incorporating novel genetic codes for high risk

organisms to avoid tampering.

Conclusion

Synthetic Biology – Greatest existing challenge

Synthetic and semi-synthetic approaches.

Discerning the minimal genome enhances better understanding of cells

Engineered organism can be used for various applications in fields of biomedicine and environment

Potential risks and hazards not clear

Key References

Cello J et al. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template. Science(2002), 297

Smith et al. Complete Chemical Synthesis, Assembly and Cloning of a Mycoplasma genitalium Genome. Science(2008), 319

Koonin et al. A Minimal Gene Set for Cellular Life Derived by Comparison of Complete Bacterial Genomes. PNAS(1996),93

Venter C.J et al. Essential Genes of a Minimal Bacterium. PNAS(2006),103

Szostak et al. Synthesizing Life. Nature(2001),409

Ehrlich SD et al. Essential Bacillus subtilis genes.PNAS(2003),100

Gil et al. Determination of the Core of a Minimal Bacterial Gene Set. Microbiology and Molecular Biology Reviews(2004),68

Thank You!