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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
April 15, 2023 3
April 15, 2023 4
Concept
Marginal Distinction
April 15, 2023 5
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
April 15, 2023 6
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.
April 15, 2023 7
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?”
April 15, 2023 8
IF YES?
THEN:
“WHAT WOULD IT TAKE TO CREATE A SYNTHETIC GENOME?”
April 15, 2023 9
April 15, 2023 10
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!
April 15, 2023 11
Figure 2: ɸx-174 plaques on E. coli culture lawn by chemically synthesized
genome
WHOLE GENOME TRANSPLANT APPORACH
April 15, 2023 12
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
April 15, 2023 13
Fig. 3: False color scanning electron micrograph of M. genitalium cells
April 15, 2023 14
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
April 15, 2023 15
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)!”
April 15, 2023 16
April 15, 2023 17
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
April 15, 2023 18
Fig. 6: PFGE images of intact circular genomic DNA (in well) and nicked genome
at 1.25 Mbp position
April 15, 2023 19
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
April 15, 2023 20
Recipient Cell Surface Markers Changed
Figure 9: Snapshot ; Genome transplantation(Science, 3-August-2007, Vol. 317)
April 15, 2023 21
Genome Transplant : Transforming Species
Towards Chemical Synthesis of “Minimal Artificial Genome”
April 15, 2023 22
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
April 15, 2023 23
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
April 15, 2023 24
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)
April 15, 2023 25
Fig. 10: One step isothermal recombination for DNA fragment ligationAssembles long fragments (>100kb) with overlaps
April 15, 2023 26
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
April 15, 2023 27
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.
April 15, 2023 28
Scanning Electron Microscopy Proteome Analysis
Morphological and Proteomic Comparison
Fig. 15: DNA fragmentation and RFLP analysis of synthetic and wild type genome
April 15, 2023 29
DNA Polymorphism Analysis
April 15, 2023 30
Fig. 16: Snapshot: Cell with total synthetic genome (Science, 2-July-2010, Vol. 329)
Total Synthetic Genome Towards Minimal Cells: Roadmap Established
April 15, 2023 31
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
April 15, 2023 33
Fig. 17: Plos One adaptation logo on 2014 issue of Synthetic Biology collection depicting bacterial lawn as editable circuitry
Pharmaceuticals and MedicineSemi Synthetic Artemisinin
April 15, 2023 34
– 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)
April 15, 2023 35
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.
April 15, 2023 36
Fig. 19: Artemisia annua L. grown in plant tissue culture facility for artemisinin isolation.
April 15, 2023 37
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
April 15, 2023 38
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
April 15, 2023 39
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)
April 15, 2023 40
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.
April 15, 2023 41
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).
April 15, 2023 42
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
April 15, 2023 43
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)
April 15, 2023 44
Fig. 23: Genetic arrangement in mevalonate operon of E. coli for FPP production
Synchronous co-expression of mevalonate operon and amorphadiene synthase genes
April 15, 2023 45
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!
April 15, 2023 47
Fig. 26: Summary pathway scheme optimized for Artemisinic acid production to produce Artemisinin
VaccinesType B Meningococcal Vaccine
(Bexsero)
April 15, 2023 48
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)
April 15, 2023 49
Fig. 27: False color SEM of Neisseria meningitidis
Neisseria meningitidisGlobal prevalence
April 15, 2023 50
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
April 15, 2023 52
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
April 15, 2023 53
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
April 15, 2023 54
Fig. 29: Schematic Representation of OMV conjugate presented in dossier to FDA by innovator
April 15, 2023 55
OMV Conjugate Model
Post Marketing Status
• First Report submitted to EMA in 2014• Current status:
UNDER ADDITIONAL MONITORING (FDA, EMA)
April 15, 2023 56
Disease MechanismsAgammaglobinaemia
April 15, 2023 57
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
April 15, 2023 58
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
April 15, 2023 59
April 15, 2023 60
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
April 15, 2023 61
Fig. 33: Simplified genetic switch of translational control
Switches“Gene expression under binary modulator”
Oscillators“Reversible Gene expression, regulated by
modulator concentration”
April 15, 2023 62
Gene Switches• Gene networks under “Binary Modulation”e.g; Lambda PR switchModulator: RecA (DNA Damage)
April 15, 2023 63
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.
April 15, 2023 64
• 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.
April 15, 2023 65
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)
April 15, 2023 66
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
ReferencesHeyden E. C. Is the $1,000 genome for real? Nature (2014); 1: 14530-14535.
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).
April 15, 2023 68
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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Thank You
April 15, 2023 70
Questions?Ask at: faisal786.btc@gmail.com
April 15, 2023 71
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