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Fluorescent labeling of recombinant proteins in living cells with FlAsH - Dimensions
Published Date: 9th October Page Count: It is based on extensive patent-pending behavioral research at Harvard University and is used by a growing number of faculty and students at different universities. To get started, register as an instructor to set up your course and adopt this or another title, try out a live demo , or contact us for more information about adopting Perusall in your course.
Skip to content. Perusall turns often-skipped solitary reading assignments into engaging collective activities students don't want to miss.
Students collectively annotate each reading — asking questions, responding to each other's questions, or sharing other perspectives or knowledge. Randomness from the probability that various molecules will meet, collide and react inside a cell comes into play much more forcefully. One must be the antithesis of the other. On paper, Briat, Gupta, and Khammash came up with a new design. In this negative feedback loop, an activator molecule would stimulate production of a desired protein.
If something perturbed the system, any error in protein levels would be corrected through a corresponding change in the rate of anti-activator production. Best of all, because the activator and anti-activator molecules would seek out and neutralize each other, this loop would work even in a noisy cell. Gupta proved mathematically that this design would result in a stable integrator for noisy cellular systems.
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But it was still completely theoretical. The trio had designed it without knowing what an antithetical pair of activator and anti-activator molecules would look like — or even if such pairs existed. And their unfamiliarity with the biology became an issue when a reviewer of their theory requested an example.
Khammash emailed a friend, the biologist Adam Arkin at the University of California, Berkeley, asking for help. Arkin quickly suggested the sigma and anti-sigma protein factors that are ubiquitous in bacteria.
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Arkin had previously used them to build a synthetic switch in cells. Nor were sigma and anti-sigma factors the only options: There were also sense and anti-sense RNAs, and various toxin molecules and their antitoxins. The theory was published in January to much excitement. Two months earlier, Khammash had asked Aoki and Lillacci to drop the design they had been working on for three years and to try out the antithetical controller instead. The duo agreed to give the design a shot, using the same pair of sigma and anti-sigma factors Arkin had suggested. But they did, and with E.
In the fall of , while his colleagues continued struggling in the lab, Gupta attended a conference in Ohio. There, he met other researchers also trying to build integrators in cells using the theory of the antithetical controller.
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All of them were struggling. Gupta thought there might be another design that would be easier to execute, making life easier for the experimentalists. Gupta found that the mathematical constraints for robust perfect adaptation were so huge, they restricted the circuit designs that would be stable in a noisy setting. All of them required an antithetical pair of controller molecules.
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Khammash and Gupta were elated at the mathematical proof that their approach, while arduous, was not just sound but inescapable. And for Aoki and Lillacci, who were already seeing signs that their cells could adapt to disturbances, the news kept them going. Finally, Aoki and Lillacci engineered a set of E.
More dramatically, in another set of cells, when they lowered the temperature of incubation from 37 degrees Celsius to 30, the cells maintained their growth rate. Olsman hopes this example will help bring a more rational, math-based approach into synthetic biology, as in engineering.
Related Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology
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