April 15, 2023 1

SYNTHETIC BIOLOGYCONCEPTS AND APPLICATIONS

Conversion Seminar

M. Faisal ShahidPCMD, ICCBS, University of Karachi

April 15, 2023 2

Content

• Introduction and Background• Concept

oDevelopment Strategy for Synthetic ChromosomeoTransplantation of Synthetic Chromosome

• ApplicationsoCurrent Approaches and ConceptsoFuture Extensions and DimensionsoLimitationsoConclusion

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Concept

Marginal Distinction

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Synthetic Biology

 Aims to design/fabricate biological systems

 “That do not already/normally present in nature”

EmphasisArtificial biological systems design

using novel/perfected experimental techniques.

VisionDe-novo* synthesis of genome,transplanted into a artificial 

cellular systems.

* De-novo: From beginning

Molecular Biology/Genetic Engineering

“Study/Transfer of individual genes/circuits” from one cell to 

another

EmphasisAlteration of existing biological

systemsUsing alternative approaches

VisionLimited alteration of biological systems, transferred in known 

cellular systems.

BACKGROUND(DNA Sequencing : Digitalizing Life)

• The First Complete…– Genome sequence: ɸX- 174 (1977)– Bacterial genome: Haemophilus influenzae (1995)– Human genome: Haploid (2000) – Human genome: Diploid (2007)

• Advent of Shotgun Sequencing Tools– Boosted sequencing projects– Lowered cost per genome sequence

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Fig 1: DNA sequence reading format by Sangar sequencing(Still regarded Gold Standard)

SYNTHETIC BIOLOGY

DESIGN AND CONSTRUCTION OF“NEW BIOLOGICAL SYSYEMS” NOT PRESENT IN

NATURE. e.g.: Genes, enzymes, genetic circuits, genomes, and cells.

OR

DE-NOVO “RE-MODELLING” OF EXISTING BIOLOGICAL SYSTEMS

e.g.: Minimal Cells, Cells with limited proliferation rate etc.

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CONCEPT

If:Software of Life, exist as the “Genome” (DNA/RNA) in a living organism

What if:“A CHEMICALLY SYNTHESIZED SOFTWARE,

BE ABLE TO BOOT A NATURAL CELLULAR HARDWARE?”

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IF YES?

THEN:

“WHAT WOULD IT TAKE TO CREATE A SYNTHETIC GENOME?”

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APPROACH FOR A SYNTHETIC CELL

Pilot Experiment• Total Chemical synthesis of ɸx-174 genome

•Transplant in E. coli

Result:

• Viral proteins synthesized, cells lysed and plaques appeared.

Proof of Concept:

BOOTABLE synthetic software in

NATURAL LIVE HARDWARE!

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Figure 2: ɸx-174 plaques on E. coli culture lawn by chemically synthesized

genome

WHOLE GENOME TRANSPLANT APPORACH

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Pro-CandidateMycoplasma genitalium

• Smallest genome for self replicating life

Etiologic agent: Pelvic Inflammatory Diseases in humans

• Advantages:Transplantation is empiricalGenome Size: 0.5 mbp482 Protein Coding Genes43 RNA coding genes

• Disadvantages:Long duplication time (16 hours)Incompatible with selection marker

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Fig. 3: False color scanning electron micrograph of M. genitalium cells

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Donor CandidateMycoplasma mycoides

Veterinary pathogen-BSL 2(Contagious Bovine Pleuropneumonia)

Origin of replication compatible with M. capricolum cells

Duplication Time: 110 minutes Genome Size: 1.1 mbp

Fig. 4: False color scanning electron micrograph of M. mycoides cells

Acceptor CandidateMycoplasma capricolum

Veterinary pathogen-BSL 2(Contagious Caprin Pleuropneumonia)

Duplication Time: 80 minutes Genome Size: 1.01 mbp Can accommodate upto

2.2 mbp DNA transplant

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Fig. 5: False color scanning electron micrograph of M. capricolum cells

MethodologyKnock out restriction system of recipient cell (M. capricolum)

Donor genome isolation

Protease treatment

Methylation of donor genome (M. mycoides)

Transplant to treated recipient cells (M. capricolum)

 

Result:

“Transplanted Cells grew with donor genome and expressed all proteins

of the donor genome (M. mycoides)!”

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Problem:

Isolation of Intact Genome

Intact Genome Isolation Protocol(Brief)

• Resuspension of cells in agarose plugs• Cool to 4⁰C• Overnight protease treatment• Remove impurities by wash buffer• Isolate intact genome by:

Pulse Field inversion Gel Electrophoresis (PFGE)• Store in TE Buffer for transplantation at 4⁰C  

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Fig. 6: PFGE images of intact circular genomic DNA (in well) and nicked genome

at 1.25 Mbp position

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Fig. 7: Simplified transplant scheme representation(http://hyperphysics.phy-astr.gsu.edu/nave-html/faithpathh/lifelab2.html)

In-vitro methylation

(Restriction system knocked out)

Transplant Summary

Fig. 8: Surface antibody reaction experiment: Science, 3-August-2007, Vol. 317M. capricolum membrane antibodies don’t recognize membrane proteins after genome

transplant

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Recipient Cell Surface Markers Changed

Figure 9: Snapshot ; Genome transplantation(Science, 3-August-2007, Vol. 317)

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Genome Transplant : Transforming Species

Towards Chemical Synthesis of “Minimal Artificial Genome”

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Viable on mutagenesis:Gene regarded: NON-ESSENTIAL

Non-viable on mutagenesis:Gene regarded:ESSENTIAL

M. mycoides genome (1.1 mbp)WHOLE GENOME MUTAGENESIS

On each random insertion of transposon,a functional gene function disrupts.

Selection

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IF CELL IS

“Total Genome Synthesis Scheme”

Chemical synthesis of 1kb DNA fragment

Ligate 1x10 to make 10kb fragments*

Tether 10x10kb to make 100kb cassettes*

Recombine 11 x 100kb fragments to prepare 1.1 mbp SYNTHETIC CHROMOSOME

Ligate to yeast cloning vector**

Isolate and circularize synthetic chromosome from yeastCells and methylate

Transplant in M. capricolum (recipient) cell withknocked out restriction system

Sub-culture transplants and whole genome sequencing

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Transform in yeast andsequence.

** Yeast vector has:A) Yeast centromereB) Multiple Cloning SitesC) Auto integration in genome byHomologous recombination!

* Transform in E. coli and sequence

Transplant Acceptance Limit of E. coli = 100kb

Simplified Representation for Long DNA Fragments Preparation(Gibson Assembly)

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Fig. 10: One step isothermal recombination for DNA fragment ligationAssembles long fragments (>100kb) with overlaps

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Fig. 11: Synthetic genome annotation map

White arrow head: 1kb fragments with 80bp overlapsBlue: 109x 10kb fragments

Green: 11x 100kb fragments in E. coliRed: Assembled artificial chromosome in yeast

Synthetic Genome Map

Post Transplantation Result

Fig. 11: M. Capricolum cells transplanted with synthetic minimal genome of M. mycoides.Blue colony color due to utilization of X-Gal by β-galactosidase enzyme

Cells termed Mycoplasma mycoides JCVI-syn 1.0/ Mycoplasma laboratorium

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Fig. 12: X-Gal reactionProduct: Galactose + 5,5'-dibromo-4,4'-dichloro-indigo

Fig. 13: TEM of M. mycoides JCVI-syn1.0 transplanted subculture similar to Wild Type M. mycoides.

Fig. 14: 2D-Gel analysis: Identical protein patterns of transplanted cells as wild type M. mycoides.

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Scanning Electron Microscopy Proteome Analysis

Morphological and Proteomic Comparison

Fig. 15: DNA fragmentation and RFLP analysis of synthetic and wild type genome

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DNA Polymorphism Analysis

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Fig. 16: Snapshot: Cell with total synthetic genome (Science, 2-July-2010, Vol. 329)

Total Synthetic Genome Towards Minimal Cells: Roadmap Established

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Artificial / Minimal Cells

Advantages

Precisely optimized growth parameters

Better utilization of ATP

Fast duplication times

Addition of customized characteristics

Larger genetic alteration window

Disadvantages

Highly fragile to environment

Cumbersome initial design

Containment risks

Serious ethical and religious issues

Content

• Introduction and Background• Concept

oDevelopment Strategy for Synthetic ChromosomeoTransplantation of Synthetic Chromosome

• ApplicationsoCurrent Approaches and ConceptsoFuture Extensions and DimensionsoLimitationsoConclusion

April 15, 2023 32

Applications• Pharmaceuticals and 

Medicine– Semi-synthetic drugs  

(metabolic fine-tuning!)– Vaccines– Disease mechanisms 

• Environmental Biochemistry:– Carbon fixation

• Bio-sensing:– Gene switches and oscillators

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Fig. 17: Plos One adaptation logo on 2014 issue of Synthetic Biology collection depicting bacterial lawn as editable circuitry

Pharmaceuticals and MedicineSemi Synthetic Artemisinin

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– In current focus of tropical disease research – 584,000 deaths in 2013– 198 million reported cases (WHO, 2015)– Etiologic agent: Plasmodium sp.

P. falciparum P. vivax P. malariae P. ovale

• P. falciparum and P. vivax : Contribute to 95% total infections.• P. falciparum: Cause disease with highest mortality.• P. vivax: Contribute ~79% of total reported cases in Pakistan. (JPMA, 2013)

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Fig. 18: Malarial parasite enters body by female Anophyles mosquito bite

Malaria

Antimalarials

First Generation: (1820-1970s)Quinine (from Cinchona bark) Isolated: 1820Total synthesis: 1945

Synthetic Derivatives:Chloroquine: (1937)Pentaquine, Primaquine, Pyrimethamine (1940s)Limitation: Resistance development.

Second Generation: (1972- Present)(WHO Report TDRICHEMAL-SWG(4)I QHSi81.3, p. 5)

•Artemisinin, from Artemisia annua L.•Effective against MDR Plasmodium sp.

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Fig. 19: Artemisia annua L. grown in plant tissue culture facility for artemisinin isolation.

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ArtemisininDownstream Limitation: Low Yield

•Isolation and purification: 0.01 – 0.5%(J. Nat. Prod., 1984, 47 (4), pp 715–717)

16 enzyme reactions in biological pathwayContributes to 1.4% of plant dry weight(Minor constituent of plant secondary metabolites)100 gms. dry herb yields 2 mg artemisinin

•Total Chemical synthesis: <25%(USP (2014): US20140135507 A1)

10 reactions; Starting Material: CyclohexanoneCan not compete with price of natural product isolation

•Plant Tissue Culture: 0.018 ± 0.004%(Enz. & Microb. Tech. 1996; 18(7):526-530)

High tissue culture costsPathway optimization not engineered 

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Malarial Vaccine(Malaria Vaccine Initiative: http://www.malariavaccine.org , April 2015)

• Effective against P. falciparum only

• Unapproved, submitted to EMA in 2014, under EU charter 58

• Phase III trials conducted at 11 sites in Africa (n= 15,949)

• Components: RTS,S conjugateo R: Repeat region of P. falciparum CSP* proteino T: Conserved T-cell epitopes of CSP in humanso S: HbsAg conserved molecule

* CSP: Cryptosporozoite protein (42KDa)Priming factor for parasite adhesion on human hepatocytes

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Solution

• Semi-synthetic Artemisinin: Overall yield: 40%*

- Create pathway for Artemisinic Acid bio-syntheisis in E. coli (Not naturally found)

- Utilize artemisinic acid for artemisininproduction by chemical synthesis

* Nature Biotechnology. (2003); 21: 796-802.

Fig. 20: Structures of Artemisinic Acid (Left), Artemisinin

(Artemisinin)

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OPP

IPP

IDI

OPPDMAPP

PMD

CoA

O

CoA

OO

CoA

O

HOOC

OH

OH

O

HOOC

OH

OP

O

HOOC

OH

OPP

O

HOOC

OH

Acetyl CoA Acetoacetyl CoA Hydroxymethylglutaryl-CoA

AASAcetyl-CoA

HMGS

ATP

MKATP

PMK

Mevalonate Mevalonate-5-phosphate Mevalonate diphosphate

OH

O

HOOC

OH

Mevalonate

OH +

O

OP

-CO2

DXS OP

O

OH

OH

NADPH

DXR/IspC OP

OH OH

OH

CTPIspD

Pyruvate Glyceraldehyde-3-phosphate 1-Deoxylulose-5-phosphate ME-4-phosphate

OPP-cyt

OH OH

OH

4-(Cyt-5'diphosphate)-ME

OPP-cyt

OH OH

OH

ATP

IspE OPP-cyt

OH OH

OP

IspF

OH OH

OPOP

OPP

OH

4-(Cyt-5'diphosphate)-ME 2-Phospho-4-(cyt-5'-diphosphate)-ME ME-2,4-cyclodiphosphate HMB-4-diphosphate

IspG

Natural Pathway for Artemisinic Acid Biosynthesis in A. annua L.(16 Independent Enzymatic Reactions)

Fig. 21(a): Biosynthetic pathway for Atremisinin synthesis in A. annua L.

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OPP

H

H

1

2

13

4

3

5

67 8

910

11

12 CH2OH

H

H

CH2OH

H

H

CHO

H

H

COOH

H

H

COOH

H

H

H

O

O

H

H

OO

O

Farnesyl diphosphate

Amorpha-4,11-diene Artemisinic alcohol

Artemisinic aldehyde

Dihydroartemisinic alcohol

Dihydroartemisinic aldehyde

Dihydroartemisinic acidArtemisinic acid

Artemisinin

CHO

H

H

1

2

3

4

5

14

15

IPP

Natural Pathway for Artemisinic Acid Biosynthesis in A. annua L.(Continued)

Artemisinic acid pathway(16 reaction steps)

Atremisinin pathway(19 reaction steps)

Branch Points

Fig. 21(b): Biosynthetic pathway for Atremisinin synthesis in A. annua

• Metabolic Engineering in E. coli. •Co-ordination of integrated gene circuits “In-trans”

a) Engineered Mevalonate operono Product: Fernasyl pyrophosphate (FPP)

b) Codon optimized Amorphadiene synthase operono Product: Amprphadiene

c) Modified Cytochrome P450 monooxygenase from Artemisia annua L.:o Product: Artemisinic acid

Advantages:Total Reactions: 15 (11 in E. coli, 4 in-vitro)Yield (Overall: 40%, 95% purity)

Semi-Synthetic ArtemisininMartin, V. J. J., et al. Nature Biotechnology 21 (7), (2003).

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Semi-synthesic SchemeIntra-cellular Substrate Chanelling

Engineering of Mevalonate operon under Inducible Promoter

Optimization of Fernasyl Diphosphate (FPP) production by mevalonate pathway induction.

Co-expression of Mevalonate and FPP engineered plasmids Substrate: Acetyl-CoA, Product: Mevalonate + FPP

Codon Optimized Amorphadiene gene expressed in E. coliSubstrate: FPP, Product: Amprphadiene

Amorphadiene transformed by Amorphadiene Oxidase engineered in same cell to Artemisinic acid

Substrate: Amorphadiene, Product: Artemisinic acid

Culture harvest and purification of Artemisinic Acid

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Mevalonate Operon in E. coli

Top Operon (3 steps) Starter molecule: AcetylCoA

End Product: Mevalonate (Toxic at >0.4mM)

Bottom Operon (5 steps) Starter Molecule: Mevalonate

End Product: Farnesyl pyrophosphate (FPP)

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Fig. 23: Genetic arrangement in mevalonate operon of E. coli for FPP production

Synchronous co-expression of mevalonate operon and amorphadiene synthase genes

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Fig. 24:Synchronous co-expression of mevalonate pathway and ADS gene to transform “Acetyl-CoA” to “Amorphadiene”

Artemisinic Acid Synthesis from Amorphadiene

Codon Optimized and Modified Plant p450 oxidase in E. coli:

Modifications for:– Folding limitation– Post Transitional Modification (6 exons)– Membrane Specific Localization

46April 15, 2023 46

Fig. 25: Genetically engineered Intra cellular semi synthetic pathway for production of Artemisinin. Each Operon on Different Plasmid

Gene Insert Sizes:Mevalonate Operon: 16.2 kb; ADS gene: 1.79kb; Amorphadiene hydroxase-reductase: 15kb

Extraction of Artemisinic Acid from E.Coli

•Cell wash (4x) with buffer, pH 9.0 (removal of membrane bound Artemisinic Acid)•Silica Gel Column Separation•Purity Yield: 95%

Synthetic Biology Concept and Applications

47

ResultIdentical 1H and 13C NMR spectra of semi-synthetic and natural

artemisinic acid!

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Fig. 26: Summary pathway scheme optimized for Artemisinic acid production to produce Artemisinin

VaccinesType B Meningococcal Vaccine

 (Bexsero)

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Neisseria meningitidis• Gram-negative diplococci

• Meningitis in children

• Affected over 400 million children around the world from 1970-2010(WHO, 2015)

• Diagnosed “After” substantial damage to patient

• Mortality rate: 10-20%(FDA, 2015)

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Fig. 27: False color SEM of Neisseria meningitidis

Neisseria meningitidisGlobal prevalence

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Fig. 28: Global prevalence of N. meningitidis serotypes, Sero-group A, B and C prevalent in Austral-Asia, B,C and Y are prevalent in America(s) and Europe

Problem• Six sero-groups for invasive meningitis:

– A, B, C, W, X, and Y• Sero-group A: Prevalent in Asia and Africa• Vaccine available against A, C, W, and Y• Sero-group B: Prevalent in USA and EU• Sero-group B: Poorly Immunogenic in

humans Polysaccharide-antigenic structure

Resembles Human Neuronal cell surface glycoproteinsApril 15, 2023 51

Solution• Cocktail vaccine of KEY IMMUNOGENIC FACTORS.

• Total synthetic origin vesicles•  Stabilizer: Detergent•  Buffering agent: Histidine• Adjuvant: Al(OH)3

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Bexsero(Vaccine Review)

• FDA approval: 23rd January 2015• EMA Approval: 28th January 2013• Type “B” Human Meningitis: Active Immunization• Four bacterial component derived synthetic vaccine for 

children• Accepted for IM administration for children >2 months• Clinical Trials conducted in Italy (EU) and Princeton, USA 

(2004-2010) n=6427, (4843 infants, 1584 adults)• Indication: Active Immunization against Meningococcous

serogroup B

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FormulationActive Pharmaceutical Ingredient (API):

– Outer Membrane Vesicle (OMV): 25µg– 2 recombinant fusion proteins (NHBA, NadA). 50µg each

(Circumvents compliment, Involved in adhesion)

– Recombinant Niesserriea Factor H. 50µg(Prevents Antibody Production)

– PorA (Prevents opsonisation)

Excipients:– Aluminium hydroxide 1.5 mg – Sodium chloride: 3.125 mg– Sucrose : 10 mg– Histidine: 0.776 mg– Water for Injection: 0.5 ml

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Fig. 29: Schematic Representation of OMV conjugate presented in dossier to FDA by innovator

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OMV Conjugate Model

Post Marketing Status

• First Report submitted to EMA in 2014• Current status: 

UNDER ADDITIONAL MONITORING (FDA, EMA)

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Disease MechanismsAgammaglobinaemia

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Fig. 30: False color B cell SEM B cell receptors are unstable in AGN

Agammaglobinaemia

• Rare Primary Immunodeficiency

• Lack of mature B-cells

• Reconstruction of BCR gene products in Orthogonal Environment– (Evolutionary distant host cell)

• Rare Mutation Identified (Exon 3, 238C to T mutation in Igβ-gene).

• Synthetic re-construction of mutant gene products in artificial bi-layers

• Validated Target Gene mutation and Identified Disease Mechanism

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Carbon FixationBio-gas Production

Metabolism, Proliferation

Fig. 31: Wood–Ljungdahl pathway in Methanococcous sp.

a) Carbon Metabolism: CO2 molecule reduced to a methyl group bound to THMPT.

Methyl transfer to CO in the presence of CoA form acetyl-CoA synthesis (Cellular metabolsim).

b) Methanogenesis: CO2 forms Formyl Methanofuran (MFR), which form Methyltetrahydromethanoptrin MTHMPT.

Two formate dehydrogenesis fdhA, fdhB and with co-enzyme f420

reduces NAD to form Methane

Pathway Reaction (Summarized)CO

2 + 8H+ + 8e- CH

4 + 2H

2O

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Fig. 32: Automated Bio-gas production device for Methanococcous sp. (CA2724074 A1, US 20100047793 A1, 2010)

Automated Methane Production Device Using Minimal Synthetic Cells

Bio-sensingGenetic switches & Gene oscillators

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Fig. 33: Simplified genetic switch of translational control

Switches“Gene expression under binary modulator”

Oscillators“Reversible Gene expression, regulated by 

modulator concentration”

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Gene Switches• Gene networks under “Binary Modulation”e.g; Lambda PR switchModulator: RecA (DNA Damage) 

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Fig. 34: Lambda lysogeny to lytic cycle switch:Phage ruptures E. coli cell on DNA damage detection

Establishment of Novel Light Inducible Gene SwitchMolecular BioSystems (2014); 10: 1679-1688.

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• Modulator: • Red light: 660nm Switch ON• Red light: 740nm Switch OFF

• Effector: • Phytochrome B (PhyB)• PhB Interacting Factor-6 (Pif-6)• BD-repressor

• Downstream applications:• Responsive for gene expression

in MAMMALIAN ‘CHO’ CELLS.

• Empirical and precise control

• Monochromatic switchi.e. strict expression control

Fid. 35: Molecular design of the red light-responsive gene expression system.

Red light (660nm): PhyB activated to PhyB-FR. It dimerizes and binds PIF6 and BD on operator and activate genes selectively

under BD-PIF-6 operator.

Red Light (740 nm): Inactivated PhyB-Fr to PhyB (INACTIVE ). Cause dissociation from PIF6, repressing all genes under BD-

PIF-6 operator.

Limitation and Risk(s)

Phenotypic character often unpredictable.

Ever hanging risk of microbial terrorism.

• Exceptionally high startup cost.

 Tailored customization of each approach. 

 Extensive scrutiny from regulatory authorities required.

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Future Prospect(s)• Synthetic organism designer program AVAILABLE!

• Minimal yeast and eukaryotic genomes awaited.

• Genome “Defragmentation” now possible. 

• Novel enzyme synthesis now possible by rational computer mediated designs and total synthesis.

• Global synthetic biology market projected to grow by 18 bln. USD by 2018. (Current: 5.6 bln. USD).

• Strict ethical and religious opposition.

• Germ line synthetic biology banned in USA.(http://www.bbc.com/news/health-32530334)

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Conclusion

New dimension of science established

Open source technology,– Patented Applications, high value!

• PROOF OF CONCEPT available for development

• Low but rewarding success rate.

Future Transition from lab to bulk applications require Monetary and Regulatory Pivot.

April 15, 2023 67

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Class J. et. al. Essential genes of a minimal bacterium. PNAS (2005); 103(2): 425-430.

Gibson et. al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods (2009); 6: 343-345.

Gibson G. et. al. Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Science (2008); 319(2): 1215-1220.

Gibson G. et. al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science; 2010: 6(2):329, (5987):52-56.

Villalobos A. et. al. Gene Designer: A synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics (2006); 7: 285- 293.

Klayman et. al. Isolation of Artemisinin (Qinghaosu) from Artemisia annua Growing in the United States. Journal of Natural Products, 1984; 47(4):715-717

Hiruaki S. et. al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. PNAS (1992); 89: 8794-8797.

Yasanzai M. I. et. al. Prevalence of human malaria infection in Pakistani areas bordering with Iran. Prevalence of human malaria infection in Pakistani areas bordering with Iran. JPMA (2013); 63: 313-316.

Lartigue C. et. al. Genome Transplantation in Bacteria: Changing One Species to Another. Science (2008): 317; 632-638.

Ducat,D., J. C. Way,and P. A. Silver. Engineering cyanobacteria to generate high-value products. Trends in Biotechnology (2011): 29(2); 95–103.

A genome-based approach for the identification of essential bacterial genes. Nature Biotechnology (1998): 16; 851 – 856.

Essential Bacillus subtillus genes. PNAS (2003),4(100):4678-4683.

Venter J. C et. al. 2010. System and methods for anaerobic environmental microbial compartmentalized cultivation. US Patent 20100330651 A1

Alexander Krajete (2013). System and method for storing energy in the form of methane EP 2675904 A1.

Marco J. Morelli , Pieter Rein ten Wolde and Rosalind J. Allen. DNA looping provides stability and robustness to the bacteriophage λ switch. PNAS (2009); 20(106): 8101-8106.

Martain J., et. al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology. (2003); 21: 796-802.

Ferrari, S. et al. Mutations of the Igβ gene cause agammaglobulinemia in man. Journal of Experimental Medicine (2007): 204; 2047–2051.

Keasling, J. D. ACS Chemical Biology 3 (1), (2007).

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References(Continued)

Martin, V. J. J., et al. Nature Biotechnolgy 21 (7), (2003).

Konard M. et. al. A red light-controlled synthetic gene expression switch for plant systems. Molecular BioSystems (2014); 10: 1679-1688.

Resistance Development Time in Plasmodium: http://www.deduveinstitute.be/~opperd/parasites/chq_res.html

http://www.rsc.org/education/eic/issues/2006July/Artemisinin.asp

http://www.who.int/gho/epidemic_diseases/meningitis/en/

http://www.novartis.com/newsroom/media-releases/en/2013/1672036.shtml

https://www.gsk.com/en-gb/media/press-releases/2015/malaria-vaccine-candidate-has-demonstrated-efficacy-over-3-4-years-of-follow-up/

Image Reference Direct Links:

http://www.nature.com/nchembio/journal/v6/n1/images/nchembio.287-F1.jpg

http://bioquellus.studiorepublic.com/technology/microbiology/neisseria-meningitidis/

http://blogs.plos.org/everyone/2012/08/15/plos-one-launches-synthetic-biology-collection/

http://fineartamerica.com/featured/1-mycoplasma-genitalium-bacteria-sem-science-photo-library.html

http://www.seriouswonder.com/software-company-autodesk-creates-synthetic-virus/

http://es.slideshare.net/ArantxaMaiden/agalactia-contagiosa-34652773

http://www.nytimes.com/2009/07/14/business/energy-environment/14fuel.html?pagewanted=print

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