Team:IIT Madras/Dry lab/Modelling

GREEN LANTERN

One of the challenging task was to establish the lighting setup which would power the proteorhodopsin in presence of retinal to carry its H+ pumping activity in the carbon deficient condition. On rigorous searching we came across a research paper (reference and relevant extract mentioned below) which clearly defined the wavelenght and intensity to maintain the proton motive force (pmf).

Source

Light-powering Escherichia coli with proteorhodopsin Jessica M. Walter, Derek Greenfield, Carlos Bustamante, and Jan Liphardt . Contributed by Carlos Bustamante, December 13, 2006

Prior to this paper the role of light in powering cells containing proteorhodopsin and participation in ocean energy fluxes remained largely unclear. This paper makes an attempt to show that when cellular respiration is inhibited by depleting oxygen or by the respiratory poison azide, Escherichia coli cells expressing PR become light-powered. The paper clearly highlighted the following main points:-

1. The rotation rate was clearly stimulated even at the lowest light intensity studied (5 mW/cm2).
2. The rate increased rapidly with intensity up to 10 mW/cm2 (15 mM azide) or 20 mW/cm2 (60 mM azide) , where the effect saturated.
3. At 50 mW/cm2, there was no detectable benefit of increased illumination. (fig.2)The energy absorbed by PR through filter used for the experiment at intensities of 10–20 mW/cm2 is equivalent to the energy absorbed by PR from the sun at sea level, taking into Walter account the absorption cross-section of PR and the solar irradiation spectrum.
4. The maximum potential PR can generate by using the free energy from photon absorption (Vpr) is similar to the potential generated by E. Coli respiration. Thus, in E. coli grown at neutral pH in rich or minimal media, or in E. coli respiring aerobically by using endogenous energy stores, PR cannot pump protons. Only when the pmf falls below the maximum potential (Vpr) during respiratory stress does PR begin to pump, and the proton flux through PR increases as the pmf falls. PR is able to maintain E. Coli cellular pmf near this maximum potential (Vpr = -0.2V) with sufficiently bright illumination ( 60mW/cm2).

Instrumentation

Power density values for green light refer to the power density passed by a D540/25ϫ filter (Chroma, Rockingham, VT) and originating at a 175 WXenon bulb (Lambda light source; Sutter Instruments, Novato,CA).The sample chamber was periodically illuminated with bright green light (160mW/cm2) coinciding with the maximum of PR’s absorption spectrum, 525 nm. At 525 nm the sample was observed to show maximum absorption (fig.1).
Based on the above fact we decided to use green LED of dominant wavelenght of 525 nm.
The specifications of the setup are given below.

Diagrams

fig(1) & fig(2) Copyright@Light-powering Escherichia coli with proteorhodopsin Jessica M. Walter, Derek Greenfield, Carlos Bustamante, and Jan Liphardt. Contributed by Carlos Bustamante, December 13, 2006

Specifications

1. LM78M05 – 3 terminal positive voltage regulator
2. LED of wavelength -525 nm( colour- parrot green)
3. 9 V battery
4. 100 ohms/51 ohms/ 33 ohms/ 18 ohms resistor

LED:

OVLFx3C7 Series (green): OVLFG3C7(fig.3)

1. Characteristics
   
   • High brightness with well-defined spatial radiation patterns
   • UV -resistant epoxy lens
   • Round Through-Hole LED Lamp(5 mm)
2. Material - InGaN
3. Typical Intensity - 5200 mcd (millicandela)
4. Lens colour - water clear
5. Operating Voltage - (-40 to 85) degree C
6. Continous forward current - 20mA
7. Peak Wavelength - 521 nm
8. Dominant Wavelength - 525 nm
9. Spectra Half-Width - 25 nm



Circuit Diagram of the Setup




Observation




	Reading 1	Reading 2	Reading 3	Reading 4
Vss (V)	5.56	5.56	5.56	5.56
Vr(V)	0.5	1.0	0.33	0.66
V led(V)	5.06	4.56	5.23	4.9
I led(mA)	29.7	26.8	30.7	28.8




Calculation



• LED equivalent efficiency - 40 lumen/W
• Luminous Intensity - 5.2 cd = 65.52 lumens
• Power given (excluding heat) by 1 LED - 1.638 W
• Power by 140 LEDs -229.32 W
• Luminous Intensity -81.105359 mW/cm2
• Internal Resistance of LED - 3.4 V/ 20 mA = 170 ohms

Assuming average current across the LED to be 30 mA. This gives an Luminous Efficiency to be 135% (fig.4). So the luminous Intensity of the entire lighting setup = 109.4922 mW/cm2 The 140 LEDs were fixed in 3 breadboards . The luminous intensity calculated above is a cumulative effect of all The LED's. So depending upon how many breadboards we use, we can choose to establish a luminous intensity of 37 mW/cm2, 74 mW/cm2 and 109.4922 mW/cm2. The distance of the lighting source from the flask containing Proteorhodopsin and Retinal was strictly maintained at 15cm.