BMEN Journal Club
Francis Crick (1916-2004)
• Nobel Prize, Medicine, 1962
–
–
–
–
Physicist turned Biologist
DNA struct co-discoverer
Proposed “Central dogma”
Worked at Cavendish, Cambridge
• Quotes
– “If you want to understand function, study structure.”
– “There is no form of prose more difficult to
understand and more tedious to read than the
average scientific paper.”
– “A busy life is a wasted life.” -- My favorite! ☺
BMEN Journal Club | Jan 25th 2011 | Nitin Singh
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Welcome!
• Name
– How about “Crick Club”?
• Aim
– Discussing Science for fun.
• Format
– Presentation: High quality, recent, slightly generic,
rigorous, interactive.
– Gossip: Any recently read paper.
• Benefits
– Knowledge, critical thinking, presentation skills.
– Free snacks – thanks to Prof. Sagar & Laranda. ☺
BMEN Journal Club | Jan 25th 2011 | Nitin Singh
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Distributed Biological Computation
Crick Club Meeting
Nitin Singh, Bioengineering
Outline
• Introduction (3 mins, 3 slides)
– Logic Gates, Transcription regulation, Recombinant
DNA
• Current Work (25 mins, 10 slides)
– State-of-Art, Cellular logic gates, What is new, XOR
gates
– Modeling section
• Conclusion (2 mins, 1 slide)
• Discussion (10 mins)
– Questions, Observations, Future work
Crick Club | Jan 25th 2011 | Nitin Singh
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Logic Gates
• Fundamental constructs in computation
• Based on binary Boolean logic
• Example: NOR gate
A
B
Q
0
0
1
0
1
0
1
0
0
1
1
0
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Activator
Promoter
Gene
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Activator
Ligand
Activator
Promoter
RNA Poly
Gene
Ligand, Transcription factor and RNA polymerase.
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Activator
Ligand + Activator
Promoter
RNA Poly
Gene
Ligand binds to activator.
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Activator
Hi RNAp!
RNA Poly
Ligand + Activator
Promoter
Gene
Ligand + Activator assembly binds to Promoter region, and attracts RNAp.
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Activator
RNA Poly
Ligand + Activator
Promoter
Gene
RNA Poly is recruited. Transcription is now on!
Crick Club | Jan 25th 2011 | Nitin Singh
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Transcription Regulation
• Repressor
RNA Poly
Stay away!
Ligand + Repressor
Promoter
Gene
RNA Poly is recruited. Transcription is now on!
Crick Club | Jan 25th 2011 | Nitin Singh
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Recombinant DNA
• Desired Genes and Promoters can be inserted
to host organism using a carrier called
plasmid.
• Though these genes are not part of host DNA,
they are transcribed and translated normally.
• Fluorescence proteins are used to measure
gene expression level.
Crick Club | Jan 25th 2011 | Nitin Singh
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Current Work: Description
State-of-Art
• Cellular logic gate implementation is standard
practice now. This 2002 Science paper4
appears to be seminal.
• Advance logic artifacts such as switches, pulse
generators, oscillators exist.5
• Examples: Next Slide
Crick Club | Jan 25th 2011 | Nitin Singh
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Cellular Logic Gates
a) NOR gate and truth table b) NOR gate layout on DNA
c) Input, OR and NOR gate implementation.2
Crick Club | Jan 25th 2011 | Nitin Singh
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What is new
• Existed
– Logic gate constructs within
single cells1
– Cell communication mechanisms
such as quorum sensing6
• Novel2, 3
– Multi-cellular design using
extracellular communication
molecules as ‘wires’!
• Impact
– Overcomes interference
between single cell circuits
– Modular design
a) Single Cell constructs b) Multi-cellular
computation1
Crick Club | Jan 25th 2011 | Nitin Singh
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XOR Gate with ‘wires’
a, Four colonies—each composed of a strain containing a single gate—are arranged such that the computation progresses from left to right, with the result of each layer communicated by
means of quorum signals. The inputs (Ara and aTc) are added uniformly to the plate. b, Spatial arrangement of the colonies. c, Each colony responds appropriately to the combinations of
input signals. Fluorescence values and their error bars are calculated as mean ± s.d. from three experiments. d, Cytometry data for the XOR gate (cell 4). Ref[1]
Crick Club | Jan 25th 2011 | Nitin Singh
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More ‘wired’ Gates
Crick Club | Jan 25th 2011 | Nitin Singh
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Current Work: Modeling
Modeling
• Single promoter
– Ligand binds Transcription factor (TxF)
– Ligand-TxF Complex (L-TxF) binds DNA
– Model Response
• Tandem promoter
– Modeling Tandem Promoters
– Tandem Promoter Interference
Crick Club | Jan 25th 2011 | Nitin Singh
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Ligand binds TxF
C
0
= C + C
C = C
F
L
0
K
n
d
n
+ Ln
C0 : Total TxF concentration
CF : Free TxF conc.
C : Ligand-TxF complex conc.
L : Ligand conc.
K : Dissociation const.
n : Cooperativity const.
Standard Hill Function based model
Crick Club | Jan 25th 2011 | Nitin Singh
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L-TxF complex binds to DNA
PLas
K1 + K 2 C
=
1 + K1 + K 2 C
PLas : Probability of transcription
Crick Club | Jan 25th 2011 | Nitin Singh
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Model Response
Crick Club | Jan 25th 2011 | Nitin Singh
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Modeling Tandem Promoters
PD
PU
GFP Gene
dX
= a U .bU . PU + a D .b D . PD − γ . X
dt
X : GFP conc.
a : Max. transcription rate
b : GFP production rate from mRNA transcript
Gamma: GFP degeneration rate
At Steady state
'
U
'
D
X = α .PU + α .PD
Crick Club | Jan 25th 2011 | Nitin Singh
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Tandem promoter Interference
Crick Club | Jan 25th 2011 | Nitin Singh
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Conclusion
• Multi-cellular logic gates with molecular
‘wires’ present modular design much needed
for designing complex cellular circuits.
• Looks promising!
Crick Club | Jan 25th 2011 | Nitin Singh
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What I did not cover ☺
• Comparison of Liquid and Plate based gate
results which covers:
– Modeling diffusion-reaction
– Effect of distance b/w colonies
• Experimental details
– Plasmids, strains, methods etc.
Crick Club | Jan 25th 2011 | Nitin Singh
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References
1.
2.
3.
4.
5.
6.
Li B., You L., Synthetic biology: Division of logic labour, Nature, 2011.
Tamsir, A., Tabor, J. J. & Voigt, C. A., Robust multicellular computing using
genetically encoded NOR gates and chemical ‘wires’, Nature, 2011.
Regot, S. et al., Distributed biological computation with multicellular
engineered networks, Nature, 2011.
Guet, C. C., Elowitz, M. B., Hsing, W. & Leibler, S. Combinatorial synthesis
of genetic networks, Science, 2002.
Purnick, P. E. M. & Weiss, R., The second wave of synthetic biology: from
modules to systems, Nature Rev. Mol. Cell Biol., 2009.
Brenner, K., Karig, D. K., Weiss, R. & Arnold, F. H. Engineered bidirectional
communication mediates a consensus in a microbial biofilm
consortium. Proc. Natl Acad. Sci. USA, 2007.
Crick Club | Jan 25th 2011 | Nitin Singh
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Thank you!
Discussion
• Molecular computing
– Why,
– Will it compete with Si-computers?
Crick Club | Jan 25th 2011 | Nitin Singh
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