WELCOME ALL OUR TEAM. EMILY KWONG Biomedical Engineering DMITRY - PowerPoint PPT Presentation

WELCOME ALL

OUR TEAM. • EMILY KWONG Biomedical Engineering • DMITRY MALYSHEV Biomedical Engineering • KIRUBHAKARAN KRISHNANATHAN Control System Engineering

Content - Project idea - The Model • Defining the Model • Simulating the Model - Characterisation of the system - Implementing the Project Objective - Purpose of Project - Acknowledgement and References

Project idea Engineering E.coli strain to be responsive to multiple wavelength.

The Initial system Sensing Photoreceptor Inhibit Allow Red light - autophosphorylation autophosphorylation change state of phytochrome EnvZ-OmpR 2 component regulatory system Transcription and translation of OmpC promoter gene and LacZ reporter gene, which promotes the activity of LacZ OUTPUT LESS BLACK BLACK PRECIPITATE PRECIPITATE

Characterising the system Firstly, varying wavelengths: Dark Blue Green Red Control Control Control Control

Findings from varying the wavelength Increasing Black Precipitate • Sample in the dark produced most precipitate. Amount of Black Precipitate Decreasing • Gradual decrease of precipitate production as input wavelength increases. • All control strain has similar output. • Gene expression under red light is most inhibited. Red

Secondly, varying light intensity • The samples and controls are stored in LB Intensity Broth . (µEins/m 2 /s) • 2.5 At different intensities, measured by a light intensity probe. • Exposed in red light and incubated for 12hrs. 6 • Perform Miller Assay – to quantify the 7 activity of beta-galactocidase enzyme. • The photospectrometer used to measure the optical density of each sample. 10 • Parameters collected.

THE MODEL

The Initial system Sensing A QUICK Photoreceptor Control autophosphorylation EnvZ-OmpR 2 component regulatory system Transcription and translation of FLASH BACK OmpC promoter gene and LacZ reporter gene, which promotes the activity of LacZ OUTPUT LESS BLACK PRECIPITATE

Regulator Light EnvZ OmpR-P TRANSFORMING THE Enzymatic Black EnvZ – OmpR reaction External precipitate system (Transcription parameter (Activity of FLOW DIAGRAM TO A EnvZ and OmpR LacZ) Translation) CONTROL BLOCK GIVES Output Photoreceptor Sensor

THE 2 INDIVIDUAL COMPONENT INSIGHTS TO REGULATORY EACH BLOCKS SYSTEM EnvZ - OmpR

k 1 OmpR + EnvZ-P (EnvZ-P)OmpR k t k -1 k k k -k EnvZ + OmpR-P k 2 k p (EnvZ)OmpR-P k- 2 OmpR + EnvZ ODE’s derived; The 3 main process can be simplified as; • EnvZ + ATP  EnvZ-P +ADP - autophosphorylation • EnvZ + OmpR-P  EnvZ + OmpR + Pi 3 - phosphatase • EnvZ-P + OmpR  EnvZ + OmpR-P - phosphotransfer Assumptions behind model: • ATP concentration is constant and absorbed into the rate constant k k. • Concentration of EnvZ is divided into: [EnvZ], [EnvZ-P], [(EnvZ-P)OmpR], and [(EnvZ)OmpR-P] • Concentration of OmpR is divided into: [OmpR], [OmpR-P], [(EnvZ-P)OmpR], and [(EnvZ)OmpR-P] • Total concentration of OmpR and EnvZ is constant; therefore the cycle goes on and on. Eric Batchelor and Mark Goulian. Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system PNAS January 21, 2003 vol. 100 no. 2 691-696.

Transcription Translation DNA mRNA protein Replication Transcription state; d[mRNA]/dt = t1[OmpR-P] – d1[mRNA] TRANSCRIPTION AND t1 – transcription coefficient TRANSLATION d1 – transcription degradation / decay rate Translation state; d[LacZ]/dt = t2[mRNA] – d2[LacZ] t2 – translation coefficient d2 – translation degradation / decay rate d1 << d2, Because protein is much more stable than Lac Z

What’s the role of the photoreceptor? • It is known and verified from the wet labs that red light inhibits the process of autophosphorylation. k 1 OmpR + EnvZ-P (EnvZ-P)OmpR k t k -1 k k k -k EnvZ + OmpR-P k 2 k p (EnvZ)OmpR-P k- 2 OmpR + EnvZ

SIMULATING THE MODEL

Constant and parameters used; EnvZ = 1M, k1=0.01, EnvZP = 1M, k-1=0.01, (EnvZP)OmpR = 1M, k2=0.01, OmpRP = 1M, k-2=0.01, OmpR = 1M, kp=0.01, EnvZ(OmpRP) = 1M. kt=0.01, t1=0.1, t2=0.1, d1=0.01, d2=1. • What do we do with constants Kk and K- k? Which are varied by the intensity of red light shined on it.

We decided to vary the constants with intensity as shown below; Reason behind it: • all ODE’s tends to a steady state after some time • Without the presence of red light, the concentration of OmpRP should increase. Therefore the constant Kk which affects the rate of reaction should decrease with intensity. Whereas K-k should increase as intensity increases.

Simulation result From the graphs above we could see that at intensity 10% the amount of • From the graph above it is clear that at high intensity OmpRP LacZ activity tends to 1 more than 90%. Therefore more black precipitate concentration is less, therefore activity of LacZ should be less as is formed at 10% rather than 90%, which proves the model. well.

Summary of wet lab result + model = characterisation of Graph analysis • 3hrs- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours. the initial system • 6hrs- The system is still unstable, but relatively calmer. • 9hrs- The system is more similar to expected trend. • 12hrs- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase • The control strain has produced a similar trend to the experimental values.

3D plot with parameters intensity, time and activity of LacZ • Using system identification procedures, a prediction for a design process could be made; either intensity or time dependent.

Implementing LacZ activity Initial system the project objective

INTENTION : taking this as an example Fusing Colour of mRFP1 EGFP ECFP fluorescent protein fluorescence Excitation wavelength 584 nm 488 nm 434 nm protein to LacZ

BLUE LIGHT BLUE LIGHT 400nm. 500nm. EGFP LacZ activity 488 nm INITIAL SYSTEM FP OUTPUT 1 0 0 1 1 1

Final System The initial phytochrome Cph1 Cph8 PCB P heam PCB P pcyA Cph8 LacZ ho1 FP BBa_I150 BBa_I15010 BBa_I150 09 08 BioBrick representation

Barriers we have to overcome to achieve our main objective: 1. Choice of FP. 2. How to fuse the chosen FP to LacZ.

Purpose of the project These are the works of the authors of ‘Engineering E.coli to see light’ 1. We hope that our system can produce multi-colour images. 2. Biosensor for multiple wavelengths

REFERENCES: 1. Levskaya et al. Engineering Escherichia coli to see light Nature 24 November 2005 DOI:10.1038/nature04405 A. 2.Eric Batchelor and Mark Goulian. Robustness and the cycle of phosphorylation and dephosphorylation in a two- component regulatory system PNAS January 21, 2003 vol. 100 no. 2 691-696.

Acknowledgement Special thanks to: Prof Philip Wright, Prof Visakan Kadirkamanathan, Prof Alan Matthews, Dr Jagroop Pandhal, Dr Josselin Noirel, Tara Baldacchino and Sheffield Bioincubator.