Synthetic biology is the designing of new biological systems or the modification of the existing ones that do not occur naturally. Synthetic or artificial cells organisms with minimal genomes have uses in molecular medicine, vaccines, environmental chemistry and bio-sensors. Creation of synthetic cells involve in-vitro synthesis of unitary DNA fragments of one-kilo base pairs (1kb). These unitary fragments are ligated to make ten kilo base pair (10kb) fragments, followed by tethering 10 fragments to form one hundred kilo base pair (100kb) fragments. Each step involves transformation and sequencing procedures in E. coli host cells. Ultimately, eleven of these hundred kilo base pair fragments are joined to create a “Synthetic Genome” which is maintained in yeast cells, as maximum limit of DNA transplant acceptance of E. coli is 100kb. By this approach, synthetic chromosomes can be maintained, manipulated and transplanted to an acceptor organism to create a synthetic cell. Applications of the technology include semi-synthetic approach of Artemisinic acid, which can be used to chemically synthesize anti-malarial drug Atremisinin and its therapeutically important derivatives. Second application of synthetic biology is production of meningitis vaccine against poorly immunogenic Neisseria meningitidis serogroup-B, by preparing synthetic vesicles. Third application includes disease mechanism identification of a rare-primary immunodeficiency disease “Agamaglobinemia” using reconstruction of mutant B-cell receptor components in synthetic membranes to validate a point mutation. Fourth application include environmental fixation of carbon di-oxide to produce methane by using minimal genome containing synthetic cells of Metahnococcous sp. Fifth application is production of novel biosensors which can be toggled ON and OFF using “Visible Light” as modulator. These “Gene switches” are also able to operate in mammalian cells. With potential applications and wide research domains, synthetic biology is also under ethical and religious criticism. Future of this new dimension of biological science requires scrutiny from regulatory authorities, and monetary input from funding agencies.

1. January 22, 2016 1

2. SYNTHETIC BIOLOGY
CONCEPTS AND APPLICATIONS
M. Faisal Shahid, and Humera Faraz (Ph.D.)
PCMD, ICCBS, University of Karachi
January 22, 2016 2

3. Content
• Introduction and Background
• Concept
oDevelopment Strategy for Synthetic Chromosome
oTransplantation of Synthetic Chromosome
• Applications
oCurrent Approaches and Concepts
oFuture Extensions and Dimensions
oLimitations
oConclusion
January 22, 2016 3

4. January 22, 2016 4
Concept

5. Marginal Distinction
January 22, 2016 5
Synthetic Biology
Aims to design/fabricate
biological systems
“That do not already/normally
present in nature”
Emphasis
Artificial biological systems design
using novel/perfected
experimental techniques.
Vision
De-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
Emphasis
Alteration of existing biological
systems
Using alternative approaches
Vision
Limited alteration of biological
systems, transferred in known
cellular systems.

6. BACKGROUND
(DNA Sequencing : Digitalizing Life)
• The First Complete…
– Genome sequence: X- 174 (1977)ɸ
– 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
January 22, 2016 6
Fig 1: DNA sequence reading
format by Sangar sequencing
(Still regarded Gold Standard)

7. 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.
January 22, 2016 7

8. 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?”
January 22, 2016 8

9. IF YES?
THEN:
“WHAT WOULD IT TAKE TO CREATE A SYNTHETIC
GENOME?”
January 22, 2016 9

10. January 22, 2016 10
APPROACH FOR A SYNTHETIC CELL

11. Pilot Experiment
• Total Chemical synthesis of x-174 genomeɸ
•Transplant in E. coli
Result:
• Viral proteins synthesized, cells lysed and
plaques appeared.
Proof of Concept:
BOOTABLE synthetic software in
NATURAL LIVE HARDWARE!
January 22, 2016 11
Figure 2: x-174 plaques onɸ E. coli
culture lawn by chemically synthesized
genome

12. WHOLE GENOME TRANSPLANT APPORACH
January 22, 2016 12

13. Pro-Candidate
Mycoplasma genitalium
• Smallest genome for self replicating life

Etiologic agent: Pelvic Inflammatory
Diseases in humans
• Advantages:
Transplantation is empirical
Genome Size: 0.5 mbp
482 Protein Coding Genes
43 RNA coding genes
• Disadvantages:
Long duplication time (16 hours)
Incompatible with selection marker
January 22, 2016 13
Fig. 3: False color scanning electron micrograph of
M. genitalium cells

14. January 22, 2016 14
Donor Candidate
Mycoplasma 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

15. Acceptor Candidate
Mycoplasma 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
January 22, 2016 15
Fig. 5: False color scanning electron micrograph of
M. capricolum cells

16. Methodology
Knock 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)!”
January 22, 2016 16

17. January 22, 2016 17
Problem:
Isolation of Intact Genome

18. 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
January 22, 2016 18
Fig. 6: PFGE images of intact circular
genomic DNA (in well) and nicked genome
at 1.25 Mbp position

19. January 22, 2016 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

20. Fig. 8: Surface antibody reaction experiment: Science, 3-August-2007, Vol. 317
M. capricolum membrane antibodies don’t recognize membrane proteins after genome transplant
January 22, 2016 20
Recipient Cell Surface Markers Changed

21. Figure 9: Snapshot ; Genome transplantation
(Science, 3-August-2007, Vol. 317)
January 22, 2016 21
Genome Transplant : Transforming Species

22. Towards Chemical Synthesis of
“Minimal Artificial Genome”
January 22, 2016 22

23. 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
January 22, 2016 23
IF CELL IS

24. “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 yeast
Cells and methylate
Transplant in M. capricolum (recipient) cell with
knocked out restriction system
Sub-culture transplants and whole genome sequencing
January 22, 2016 24
Transform in yeast and
sequence.
** Yeast vector has:
A) Yeast centromere
B) Multiple Cloning Sites
C) Auto integration in genome by
Homologous recombination!
* Transform in E. coli and
sequence
Transplant Acceptance Limit of E. coli = 100kb

25. Simplified Representation for Long DNA Fragments Preparation
(Gibson Assembly)
January 22, 2016 25
Fig. 10: One step isothermal recombination for DNA fragment ligation
Assembles long fragments (>100kb) with overlaps

26. January 22, 2016 26
Fig. 11: Synthetic genome annotation map
White arrow head: 1kb fragments with 80bp overlaps
Blue: 109x 10kb fragments
Green: 11x 100kb fragments in E. coli
Red: Assembled artificial chromosome in yeast
Synthetic Genome Map

27. 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
January 22, 2016 27
Fig. 12: X-Gal reaction
Product: Galactose + 5,5'-dibromo-4,4'-dichloro-indigo

28. 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.
January 22, 2016 28
Scanning Electron Microscopy Proteome Analysis
Morphological and Proteomic Comparison

29. Fig. 15: DNA fragmentation and RFLP analysis of synthetic and wild type genome
January 22, 2016 29
DNA Polymorphism Analysis

30. January 22, 2016 30
Fig. 16: Snapshot: Cell with total synthetic genome
(Science, 2-July-2010, Vol. 329)
Total Synthetic Genome
Towards Minimal Cells: Roadmap Established

31. January 22, 2016 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

32. Content
• Introduction and Background
• Concept
oDevelopment Strategy for Synthetic Chromosome
oTransplantation of Synthetic Chromosome
• Applications
oCurrent Approaches and Concepts
oFuture Extensions and Dimensions
oLimitations
oConclusion
January 22, 2016 32

33. Applications
• Pharmaceuticals and
Medicine
– Semi-synthetic drugs
(metabolic fine-tuning!)
– Vaccines
– Disease mechanisms
• Environmental
Biochemistry:
– Carbon fixation
• Bio-sensing:
– Gene switches and oscillators
January 22, 2016 33
Fig. 17: Plos One adaptation logo on 2014 issue of Synthetic Biology
collection depicting bacterial lawn as editable circuitry

34. Pharmaceuticals and Medicine
Semi Synthetic Artemisinin
January 22, 2016 34

35. – 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)
January 22, 2016 35
Fig. 18: Malarial parasite enters body by
female Anophyles mosquito bite
Malaria

36. Antimalarials
First Generation: (1820-1970s)
Quinine (from Cinchona bark)
Isolated: 1820
Total 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.
January 22, 2016 36
Fig. 19: Artemisia annua L. grown in plant tissue culture
facility for artemisinin isolation.

37. January 22, 2016 37
Artemisinin
Downstream Limitation: Low Yield
•Isolation and purification: 0.01 – 0.5%
(J. Nat. Prod., 1984, 47 (4), pp 715–717)
16 enzyme reactions in biological pathway
Contributes 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: Cyclohexanone
Can 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 costs
Pathway optimization not engineered

38. January 22, 2016 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 conjugate
o R: Repeat region of P. falciparum CSP* protein
o T: Conserved T-cell epitopes of CSP in humans
o S: HbsAg conserved molecule
* CSP: Cryptosporozoite protein (42KDa)
Priming factor for parasite adhesion on human hepatocytes

39. January 22, 2016 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 artemisinin
production by chemical synthesis
* Nature Biotechnology. (2003); 21: 796-802.
Fig. 20: Structures of
Artemisinic Acid (Left), Artemisinin
(Artemisinin)

40. January 22, 2016 40
OPP
IPP
IDI
OPP
DMAPP
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
AAS
Acetyl-CoA
HMGS
ATP
MK
ATP
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
CTP
IspD
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.

41. January 22, 2016 41
OPP
H
H
1
2
13
4
3
5
6
7 8
910
11
12 CH2OH
H
H
CH2OH
H
H
CHO
H
H
COOH
H
H
COOH
H
H
H
O
O
H
H
O
O
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

42. • Metabolic Engineering in E. coli.
•Co-ordination of integrated gene circuits “In-trans”
a) Engineered Mevalonate operon
o Product: Fernasyl pyrophosphate (FPP)
b) Codon optimized Amorphadiene synthase operon
o 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 Artemisinin
Martin, V. J. J., et al. Nature Biotechnology 21 (7), (2003).
January 22, 2016 42

43. Semi-synthesic Scheme
Intra-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. coli
Substrate: 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
January 22, 2016 43

44. 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)
January 22, 2016 44
Fig. 23: Genetic arrangement in mevalonate operon of E. coli for FPP production

45. Synchronous co-expression of mevalonate operon and
amorphadiene synthase genes
January 22, 2016 45
Fig. 24:Synchronous co-expression of mevalonate pathway and ADS gene to
transform “Acetyl-CoA” to “Amorphadiene”

46. 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
46January 22, 2016 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

47. 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
Result
Identical 1
H and 13
C NMR spectra of semi-synthetic and natural
artemisinic acid!
January 22, 2016 47
Fig. 26: Summary pathway scheme optimized for Artemisinic acid production to produce Artemisinin

48. Vaccines
Type B Meningococcal Vaccine
(Bexsero)
January 22, 2016 48

49. 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)
January 22, 2016 49
Fig. 27: False color SEM of
Neisseria meningitidis

50. Neisseria meningitidis
Global prevalence
January 22, 2016 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

51. 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 glycoproteins
January 22, 2016 51

52. Solution
• Cocktail vaccine of KEY IMMUNOGENIC
FACTORS.
• Total synthetic origin vesicles
• Stabilizer: Detergent
• Buffering agent: Histidine
• Adjuvant: Al(OH)3
January 22, 2016 52

53. 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
January 22, 2016 53

54. Formulation
Active 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
January 22, 2016 54

55. Fig. 29: Schematic Representation of OMV conjugate presented in dossier to FDA by innovator
January 22, 2016 55
OMV Conjugate Model

56. Post Marketing Status
• First Report submitted to EMA in 2014
• Current status:
UNDER ADDITIONAL MONITORING (FDA, EMA)
January 22, 2016 56

57. Disease Mechanisms
Agammaglobinaemia
January 22, 2016 57
Fig. 30: False color B cell SEM
B cell receptors are unstable in AGN

58. 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
January 22, 2016 58

59. Carbon Fixation
Bio-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)
CO2
+ 8H+
+ 8e-
CH4
+ 2H2
O
January 22, 2016 59

60. January 22, 2016 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

61. Bio-sensing
Genetic switches & Gene oscillators
January 22, 2016 61
Fig. 33: Simplified genetic switch of translational control

62. Switches
“Gene expression under binary modulator”
Oscillators
“Reversible Gene expression, regulated by
modulator concentration”
January 22, 2016 62

63. Gene Switches
• Gene networks under “Binary Modulation”
e.g; Lambda PR switch
Modulator: RecA (DNA Damage)
January 22, 2016 63
Fig. 34: Lambda lysogeny to lytic cycle switch:
Phage ruptures E. coli cell on DNA damage detection

64. Establishment of Novel Light Inducible Gene Switch
Molecular BioSystems (2014); 10: 1679-1688.
January 22, 2016 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 switch
i.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.

65. 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.
January 22, 2016 65

66. 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)
January 22, 2016 66

67. 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.
January 22, 2016 67

68. References
Heyden 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.
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70. Thank You
January 22, 2016 70

71. Questions?
Ask at: faisal786.btc@gmail.com
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