ChloriColight 2 This Year, We Photo-tunable ON/OFF system 3 - - PowerPoint PPT Presentation

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ChloriColight 2 This Year, We Photo-tunable ON/OFF system 3 - - PowerPoint PPT Presentation

Sep 25, 2023 374 likes •802 views

The Chinese University of Hong Kong ChloriColight 2 This Year, We Photo-tunable ON/OFF system 3 Halorhodopsin (HR) Light-driven Chloride pump Halobacterium Facilitate chloride import

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ChloriColight

The Chinese University of Hong Kong

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2

ON/OFF Photo-tunable system

This Year, We…

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Halorhodopsin (HR)

 Light-driven Chloride pump  Halobacterium  Facilitate chloride import

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(http://opm.phar.umich.edu/protein.php?pdbid=1e12)

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HR working principle

Halorhodopsin closed Halorhodopsin opened Chloride pumped in

Light OFF! Light ON!

Extracellular Intracellular

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Closed Open

E.coli

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Chlor i co light

  • E. li

Chloride

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Our Biobricks

 Constructed a Halorhodospin (HR) basic unit biobrick (BBa_K559000)  Constructed a intermediate with constitutive promoter (BBa_K559001)  Constructed a functional Halorhodopsin system (BBa_K559010)

Biobricks construction Characterization Summary 6

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We have successfully constructed the biobrick

1000bp 500bp 100bp lane 1 2 3 4 5 6 7

Biobricks construction Characterization Summary

800bp

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Optimal [IPTG] to induce HR expression is 0.1 mM

Optimal concentration of IPTG to trigger max. HR expression is 0.1 mM

Biobricks construction Characterization Summary 8

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Effect of different [NaCl] on bacterial growth

n=3, error bar: SEM

The upper limit of extracellular NaCl concentration that does not affect bacterial growth is 0.4 M

No effect

Biobricks construction Characterization Summary

Inhibit

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Effect of HR expression on bacterial growth

n=3 error bar : SEM

Expression of halorhodopsin does not affect bacterial growth

Transformation: biobrick is transformed IPTG: 0.1 mM of IPTG was used NaCl: Culture medium contain 0.4 mM of NaCl Biobricks construction Characterization Summary 10

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Essential factors needed for chloride absorption

*p<0.01

n=3, error: SEM

Both light and extrinsic addition of NaCl are indispensible for chloride absorption

IPTG: 0.1 mM of IPTG was used NaCl: Culture medium contain 0.4 mM of NaCl Biobricks construction Characterization Summary 11

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Environment: 0.1 mM IPTG 0.4 M NaCl Exposure time: ??? Light power: ??? Wavelength: ???

Biobricks construction Characterization Summary 12

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Optimal wavelength for chloride absorption

Optimal wavelength for chloride absorption is 530 nm

Same total monochromatic light power Biobricks construction Characterization Summary *P<0.05

n=3, error bar: SEM

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Optimal LASER power for chloride absorption is 25%

Optimal Monochromatic light power to induce chloride absorption

Biobricks construction Characterization Summary

n=3, error bar: SEM

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Bacteria are killed once exposure with over 25 % light power

Effect of monochromatic light on bacterial growth

Biobricks construction Characterization Summary

*P<0.001

n=3, error bar: SEM

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Optimal illumination time for chloride absorption is 210 s

Optimal light exposure time to induce chloride absorption

Biobricks construction Characterization Summary

n=3, error bar: SEM

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Environment: 0.1 mM IPTG 0.4 M NaCl Exposure time: 210 s Laser Light power: 25%

Summary

Wavelength: 530 nm Maximum chloride uptake

Biobricks construction Characterization Summary 17

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Control Gene Expression

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Using Light?

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Photo-Gene Expression Tuner

 Tune downstream gene expression

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Light Intensity Intracellular Chloride Concentration Gene Expression

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Intracellular Chloride-Sensing Cassette

 Pgad Chloride-Sensing Cassette (Jan Willem Sanders et al.,

1998)

Constitutive promoter Encodes gadR activator

gadR Cl-

Target Gene

gadR gadR Cl- Cl- Cl- Gene Expression

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Chloride-Sensing Cassette Regulates GFP Gene Expression

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EGFP expression is boosted up by chloride ion

(M) light

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Absorb/Release Cl-

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Light Source Intracellular Cl- sensor Intracellular [Cl-] Change Regulate Gene Expression Previous Projects

ChloriColight

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Features

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Tunable Amplifier Quantitative Control Universal Plug-in Tool

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Future Application: Converting light to electricity ?

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Our Biobrick can generate electricity

Bio Brick

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Mixing entropy

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How mixing-entropy works?

In fresh water (low [ions])

  • Discharging occurs
  • Electrodes release ions
  • Potential difference ↓

Sodium ion Chloride ion

Switch to sea water (high [ions])

  • Charging
  • Ions move into electrodes
  • Potential difference ↑

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Fabrication of entropy-mixing electrodes

Special property

  • Absorb and release ions

X-ray diffraction matched the publication

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Our entropy-mixing electrodes functioned properly

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Electricity generation cycle

Switch to 25 ml 0.4 M NaCl solution Switch to 25 fresh water Switch to 25 ml 0.4 M NaCl solution Switch to 25 ml fresh water

Voltage change Detect Current Detect Current Voltage change

Our electrodes generate electricity from change of mixing entropy

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Our proposed model

[Ions] reduces in soltuion

Chlorcolight function Rate of potential change ↑ Generate electricity Extracellular Cl– conc. ↓

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Experimental setup of mixing-entropy electricity generation

Experiment in progress

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Light Source Electricity Generation Bio Brick Light energy Change in entropy Electricity

Chloricolight converts light energy to electricity

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Light-driven electricity generation

Chloricolight

Light tunable gene expression system

Absorb/Release Cl-

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Light Source Intracellular Cl- sensor Intracellular [Cl-] Change Regulate Gene Expression Previous Projects

ChloriColight

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Special Thanks

  • Prof. Chan Ting Fung
  • Prof. Chan King Ming
  • Prof. Kong Siu Kai
  • Prof. Kevin Yuk-Lap Yip
  • Team Advisors from School of Life Science and Faculty of Engineering
  • The halorhodopsin gene is given as a gift from
  • P. Hohenfeld, A. A. Wegener,M. Engelhard

Authors of the paper “ Purification of histidine tagged bacteriorhodopsin,pharaonis halorhodopsin and pharaonissensory rhodopsin II functionally expressed in Escherichia coli”

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Acknowledgements

  • Mr. Chu Wing Leung
  • Prof. Douglas Yung & his group
  • Ms. Law Shuk Yee, Zoe
  • Ms. Shirley Chan
  • Honorable Dr. Ng Cheuk Yiu
  • Mr. Wang Feng
  • Dr. Mak Kin Wah
  • Ms. Kong Lai Ping
  • Mr. Emery Chan
  • Prof. Ge Wei
  • Prof. Shaw Pang Chui
  • Mr. Tang Wai Keung
  • Dr. Chan Wing Fat
  • Prof. Jimmy Yu & his group
  • HKUST iGEM team 2011 for collaborative works
  • Postgraduates at Prof. T

. F . Chan’s group, Prof. S. K. Kong's group and Prof.

  • K. M. Chan's group
  • Knowledge Transfer Unit, Chinese University of Hong Kong
  • Office of Student Affair, Chinese University of Hong Kong
  • Steering Committee for Promoting Personal Development through Social

and Civic Engagement

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Sponsors and Supporters

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Sponsors and Supporters

CUGEN Limited

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CUHK iGEM team 2011 Chloricolight

~THANKS~

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Mixing-Entropy Battery

  • Extracts energy from the concentration difference of two

solutions converting salinity potential into electricity

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Power management system

Due to the small and ripple nature of the voltage generated from the electrodes, we therefore apply introduce a power management system to stabilize more importantly makes the power generated useful.

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Appedix:

Large-scale light-mediated electricity generation