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Team:IIT Madras/Project/Device
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The energy advantage provided to cells due to GPR expression increases the growth rate of PR expressing cells. This difference in growth rate has been hypothesized to be used for screening transformed cells. We propose a plasmid vector for screening, where the antibiotic resistance gene is replaced by Proteorhodopsin generator. The following is the modified protocol for screening: <ul> | The energy advantage provided to cells due to GPR expression increases the growth rate of PR expressing cells. This difference in growth rate has been hypothesized to be used for screening transformed cells. We propose a plasmid vector for screening, where the antibiotic resistance gene is replaced by Proteorhodopsin generator. The following is the modified protocol for screening: <ul> | ||
<li>Clone the part of interest into the <b>pREXa</b> cloning vector’s MCS.</li> | <li>Clone the part of interest into the <b>pREXa</b> cloning vector’s MCS.</li> | ||
| - | <img src="https://static.igem.org/mediawiki/2011/b/bf/Image1sunscreen.png"/> | + | <img src="https://static.igem.org/mediawiki/2011/b/bf/Image1sunscreen.png" style="float:left;"/> |
<li>Transform bacterial cells (using standard protocol).</li> | <li>Transform bacterial cells (using standard protocol).</li> | ||
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<li>Plate cells on Minimal media agar containing Retinal (10uM).</li> | <li>Plate cells on Minimal media agar containing Retinal (10uM).</li> | ||
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<li>Place the plate under green light (525 nm) and incubate at <b>37 C</b>.</li> | <li>Place the plate under green light (525 nm) and incubate at <b>37 C</b>.</li> | ||
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<li>Pick transformed colonies after 12 hours of incubation.</ul></li> | <li>Pick transformed colonies after 12 hours of incubation.</ul></li> | ||
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Revision as of 01:32, 6 October 2011
Device
We have designed a “Carbon Stress Buster” device that rescues E.coli cells from substrate depletion conditions, and/or situations where the glycolytic pathway (the metabolic pathway that converts glucose to usable energy) is inhibited. Our device has the following subparts:
- Green-light absorbing Proteorhodopsin (GPR) generator.
- Carbon stress induced promoter upstream of the PR generator.
- A system for providing the Chromophore “Retinal” for light absorption activity of GPR.
The device is induced by carbon stress in terms of decrease in substrate concentration in the media, and GPR is generated. Proteorhodopsin with retinal is expressed on the bacterial membrane. When green light of wavelength 525 nm is shone on the bacterial culture, Proteorhodopsin’s proton pumping activity is initiated, and the PMF (Proton Motive Force) increases. Through ATP synthase which is constitutively expressed on the bacterial membrane, the PMF drives ATP synthesis and rescues the cells in substrate stress conditions.
We propose using this device for a variety of applications including enhancing recombinant protein yield for low substrate conditions (Project Artemis) and for light-based screening for positive clones (Project Sunscreen).
Project Artemis
Artemis is the Greek Goddess of Light, and is known as the “Protector of the Vulnerable”. Our team has designed an Expression vector based on the Carbon Stress Buster device, for improving yield of Recombinant protein in substrate limiting conditions.
Project Sunscreen
The energy advantage provided to cells due to GPR expression increases the growth rate of PR expressing cells. This difference in growth rate has been hypothesized to be used for screening transformed cells. We propose a plasmid vector for screening, where the antibiotic resistance gene is replaced by Proteorhodopsin generator. The following is the modified protocol for screening:
- Clone the part of interest into the pREXa cloning vector’s MCS.
- Transform bacterial cells (using standard protocol).
- Plate cells on Minimal media agar containing Retinal (10uM).
- Place the plate under green light (525 nm) and incubate at 37 C.
- Pick transformed colonies after 12 hours of incubation.
