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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).

Hyaluronic Acid and Curdlan Synthesis
Hyaluronic Acid (HA) is an industrially important biopolymer, whose production has been shown to increase in situations where glycolysis is inhibited inside the cell. Inhibition of glycolysis redirects the cellular resources towards HA synthesis, but in turn decreases cell viability. Under conditions of glyolysis inhibition, our construct will help provide the energy required to maintain cell viability as close to normal as possible. This emerges as a novel method to optimize carbon source flux within the cell, by directing as much of it as possible towards the exopolysaccharide synthesis of HA.
Exopolysaccharide biosynthesis is a process in which the ATP demand is a significant proportion of total cellular ATP demand. One such exopolysaccharide of high industrial relevance is Curdlan or beta-1,3-glucan. Therefore our construct, which enhances the ATP production in the cell, is expected to supplement the ATP requirement during the synthesis of Curdlan and increase its rate of production for commercial purposes.

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.