This fusion of these technologies allows us to sequentially add random DNA barcodes into an evolving population to create a time-sorted barcode array at a specific locus in each cell. Earlier barcodes on this array identify more distant ancestors.
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In a traditional DNA barcoding applications, rare cells that acquire the ability to circumvent barcoding selection are not a big issue. In our case, these rebellious cells would be fatal, since they would have a huge advantage during the next round of barcode addition.
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The same argument applies to cells that acquire the ability to circumvent selection during gene stacking. The minor flaws of technologies in synthetic biology seem to limit our ability to create complex combinations of simpler constructs.
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@alex_nguyen_ba and I spent years tracking down rebellious cells that refused to acquire new barcodes. We analyzed how they managed to escape our selections and redesigned our system again and again to try to lead them in the right direction.1 reply 1 proslijeđeni tweet 1 korisnik označava da mu se sviđaPrikaži ovu nit -
Over the next couple of days, I'll describe some of the essential components of our technology and how they make long term lineage tracking possible, including: A novel, hyper-directional, set of Lox sites Reiterative Cre-Lox cassette exchange The splicing trap
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Our system requires that the barcodes we integrate into the genome stay there,even after hundreds of generations of evolution. Unfortunately, Cre-Lox integration is reversible. Worse yet, at equilibrium, integration substrates are favored over integrated product.pic.twitter.com/YMF4vPfrcG
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An ingenious solution to this problem takes advantage of mutant Lox inverted repeats (IRs) thought to decrease Cre binding. Since Cre binding to Lox IRs is cooperative, a site with a single mutated IR will recombine normally but a double mutant site will not.pic.twitter.com/rYtWqqrqIz
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If the product of recombination contains a doubly mutated Lox site, the reaction equilibrium shifts towards integration.pic.twitter.com/FthmNV8CDP
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@alex_nguyen_ba and I designed an in vitro assay to measure the stability of Cre-Lox integration products and tested a large number of inverted repeat mutations from the literature.pic.twitter.com/JK0RBbUJaU
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Only a single arm mutation was able to reduce the rate of excision to acceptable levels (more than 6 orders of magnitude lower than WT) while still allowing integration. It is difficult to understate importance of this result to the viability of our technique.
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The shift from a reaction with an equilibrium that favors integration to a reaction that produces effectively stable integration means the difference between a system that will work by itself and a system that can become a module in a more complex technology.
Čini se da učitavanje traje već neko vrijeme.
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