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University of Pune, INDIA Constructing Multi-State Computational Modules using Nucleotide & Protein Mediated Cell-Cell Signalling Institute of Bioinformatics & Biotechnology Institute of Bioinformatics and Biotechnology, University of

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Constructing Multi-State Computational Modules using Nucleotide & Protein Mediated Cell-Cell Signalling

University of Pune, INDIA

Institute of Bioinformatics & Biotechnology

Institute of Bioinformatics and Biotechnology, University of Pune, INDIA

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Road M ap

  • Team IBB
  • Conceptualization: Turing Machines

The Complete Construct i. Strategy 1 : Simplified Construct Design ii. Strategy 2 : Modularity Design

  • a. Protein based signaling

University of Pune, INDIA

  • a. Protein based signaling
  • b. Nucleic acid Signaling & Riboswitches

Results

  • BioBricks Submitted to the Registry
  • Our experiences
  • Acknowledgements
  • References

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Conceptualization:

Turing M achines

University of Pune, INDIA

Turing M achines

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Genesis: The Turing Machine

University of Pune, INDIA

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1 = 1 2 = 11 3 = 111

University of Pune, INDIA

3 = 111 4 = 1111

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Turing M achines - Unary Adder

University of Pune, INDIA

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Biological implementation

Cell Cell (Final State) Tape input

University of Pune, INDIA

Cell (Initial State) (Final State) Tape output Genetic constructs Medium components 96 well plate like apparatus Mechanical design Biological analogs

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Design considerations

  • Inputs (reading a tape)
  • should be synthesizable and

degradable, available, freely diffusible.

University of Pune, INDIA

degradable, available, freely diffusible.

  • State switching
  • Independence of states
  • Only one state is “ON” at any point in time.

First attempt towards demonstration in bacterial systems!

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THE CONSTRUCT THE CONSTRUCT

University of Pune, INDIA

THE CONSTRUCT THE CONSTRUCT

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State Symbol State Symbol Direction

A A R A 1 B 1 R B H 1 H B 1 B 1 R University of Pune, INDIA

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THE PROBLEM

  • Too LENGTHY
  • Many assembly steps
  • Dead end construct

University of Pune, INDIA

  • Dead end construct
  • THE SOLUTION- Try different approaches!!!

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The Solution!

  • Simplifying the construct itself in such a way

that the phenotypic output remains the same

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  • Breaking up the system into modules situated

into different strains that interact with each

  • ther.

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Strategy 1:

The SIM PLIFIED Construct

University of Pune, INDIA

The SIM PLIFIED Construct

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Simplify construct

State Symbol State Symbol Direction

A A R A 1 B 1 R B H 1 H B 1 B 1 R

State “A” and 0 = No response

University of Pune, INDIA

State “A” and 1 = Switch to state “B’ (AHL production) State “B” and 1 = auto-induced AHL production. State “B” and 0 = AHL production and degradation of Lactose

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Strategy 2:

M odular Design

University of Pune, INDIA

M odular Design

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M odular approach

a) Protein based signaling b) Nucleotide based signaling

University of Pune, INDIA

b) Nucleotide based signaling

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  • a. Protein Based Signaling

State A HaltState

University of Pune, INDIA

State B

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Export tags

  • The Twin Arginine Translocation pathway

SRRRFLK, SRRXFLX, TRRXFLX,

University of Pune, INDIA

TRRXFLX, SRRXXLK, SRRXXLA, TRRXXLK, TRRXXLA, SRRXXLT,

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  • Tor A and YcdB
  • N terminal attachment

University of Pune, INDIA

  • N terminal attachment
  • Fusion…

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Solving the Scar Problem…

University of Pune, INDIA

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  • ATGCAGTA-----------ACTCAATCTGC
  • ATGCAGTA-----------ACTCAATCTC’C’

University of Pune, INDIA

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SOPMA Analysis

University of Pune, INDIA

YcdB with the added base ’C’ YcdB without the added base ’C’ TorA with the added base ’C’ TorA without the added base ’C’

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Proof of concept

University of Pune, INDIA

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  • b. Nucleic Acid Based Signaling

University of Pune, INDIA

  • b. Nucleic Acid Based Signaling

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University of Pune, INDIA

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  • DNA uptake

– Competence related genes enable uptake of DNA.

  • DNA secretion

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  • DNA secretion

– Cell lysis – Naturally competent strains show the property of DNA secretion. – Genetically regulated ‘cell sacrifice’

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Strain A

Strain B

Riboswitch Lock & Key System

Hypothetical Unary Adder Circuit

State Symbol State Symbol Direction

A A R A 1 B 1 R B H 1 H B 1 B 1 R University of Pune, INDIA

Strain B Strain B pLac RBS Lock LuxI Term pLuxR Key Riboswitch RBS Term pLuxR RBS Lac O Lysis Term

Promoter Term RBS Com

A B B B

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SNOWDRIFT GAME

University of Pune, INDIA

Co-operators Defectors

Co-operators (B-C)/2, (B-C)/2, B-C,B Defectors B,B-C 0,0

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Co-operator Defector

pLacI RBS Term β - gal GFP

TorA

/Y

cdB

Two strains – Co-operator and Defector Strains

University of Pune, INDIA

Co-operator Co-operator Defector

Glucose Lactose β-galactosidase GFP producing Cooperator

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The Model

Assumptions

  • At time t=0;
  • There are 'k' co-operators and 'N-k' defectors
  • Medium contains 'L' mg/ml of lactose, glucose conc. (g) = 0
  • Culture is well mixed.
  • Extracellular Enzyme conc (Ec) = 0 units/ml

University of Pune, INDIA

  • Extracellular Enzyme conc (Ec) = 0 units/ml
  • The rate constant for the conversion of Lactose to glucose plus galactose is ‘k2’

Artificial assumptions

  • Glucose is consumed by all cells. Galactose is also consumed at the same rate

Gc mg/cell/min/ml.

  • The metabolic benefit due to glucose and Galactose is same. So effectively each

lactose molecule gives rise to 2 glucose molecules

  • There is no intracellular lactose metabolism (only extracellular).
  • There is no lag in enzyme production and secretion.
  • Rate of degradation of enzyme is zero.

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g = (k2)*(Ec)*(L) mg/ml/min .... (1) r = (R)*(g)*(Gc) .... (2) D(t) = D(t-1)+ r * D(t-1) .... (3) Defector population (t)

University of Pune, INDIA

k(t) = k(t-1) + (r-c) * k(t-1) .... (4) Co-operator population (t) L = L - L * Ec * k2 .... (5) G = G +(( 2* L* Ec* k2)-( N * Gc) .... (6)

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Results

  • 20

20 40 60 80 100 120 140 160 180 200 200 400 600 Lactose conc (L) mg/ml Glucose conc (g) mg/ml Extracellula r enzyme units (Ec) /ml

5000 10000 15000 20000 25000

Co-

  • perators k /

ml Defectors d / ml Total N /ml

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  • 20 0

200 400 600

100 200 300 400 500 600 0.2 0.4 0.6 0.8 1 1.2 100 200 300 400 500 600 C/D ratio

C/D ratio

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BioBricks Submitted to the Registry

University of Pune, INDIA

BioBricks Submitted to the Registry

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Biobricks

YcdB GFP Cprom RBS YcdB GFP YcdB Cprom RBS GFP BBa_K233309 BBa_K233312 BBa_K233306 BBa_K233310

University of Pune, INDIA

TorA GFP Cprom RBS TorA GFP TorA BBa_K233308 BBa_K233312 BBa_K233311 BBa_K233307 BBa_K233310

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Biobricks

Snowdrift 1 Snowdrift 2

University of Pune, INDIA

pLL LacO AND GATE 2 pT7 LacO AND GATE 3

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pLuxR LacO LacO Const. Prom LacO BBa_K233004

Biobricks

University of Pune, INDIA

pLuxR RBS-LuxI Const.P RBS LuxR Prom Const Prom RBS- mRFP TERM BBa_K233314 BBa_K233003 BBa_K233004 BBa_K233317 BBa_K233313 BBa_K233316

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Biobricks

Turing machine- Unary adder

University of Pune, INDIA

Turing machine- Unary adder AHL+Lactose Mediated Cell Lysis Turing machine cassette 2

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What we gained from iGEM …

  • Looking at bacteria from a synthetic biology

point of view.

University of Pune, INDIA

  • Awareness in our Institute.
  • Experience

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Summing up…

  • We worked on making multi-

state, signalling ,modules.

  • 26 new parts added to the registry

University of Pune, INDIA

  • A novel fusion strategy, characterisation

underway…

  • Modeling the snowdrift game
  • And a cool time ;-)

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Acknowledgements

  • To our Advisors:

– Professor B.A. Chopade – M r. Praveen K Sahu

  • IBB, University of Pune, INDIA

University of Pune, INDIA

  • IBB, University of Pune, INDIA
  • Department of Chemistry, University of Pune
  • ‘Sakal’, ‘Times of India’
  • Friends, Colleagues and Family back home
  • Meagan, Vinoo, Randy and the entire iGEM family!

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We thank our sponsors:

University of Pune, INDIA

University of Pune, INDIA

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Team IBB_Pune

University of Pune, INDIA

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References:

  • Turing, Alan M. (1936), "On computable numbers, with an application to the

Entscheidungsproblem," Proc. London Math. Soc., Ser. 2--42, 230--265.

  • Turing, Alan M. (1950), "Computing Machinery and Intelligence," Mind 59 (n.s.

236) 433--460, also The World of Mathematics 4, Simon and Schuster, 1954.

  • Spatial structure often inhibits the evolution of cooperation in the snowdrift

game,C Hauert, M Doebeli - Nature, 2004.

University of Pune, INDIA

game,C Hauert, M Doebeli - Nature, 2004.

  • David Dubnau. DNA UPTAKE IN BACTERIA ,Annual Review of Microbiology, Vol.

53: 217-244 (Volume publication date October 1999)

  • Steinmoen, H., Knutsen, E. & Havarstein, L. S. Induction of natural competence in

Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc. Natl Acad.Sci. USA 99, 7681–7686 (2002).

  • Miller, Melissa B. & Bassler Bonnie L. Quorum Sensing in Bacteria,

Annual Review of Microbiology, 2001. 55:165–99

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