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Here's a thread telling the story of our new technique for genetic recording of lineage history during long term evolution experiments. The work was co-led with
@alex_nguyen_ba and@ivana_cvijovic in@MichaelMDesai's lab. Read the paper here:http://nature.com/articles/s41586-019-1749-3 …Show this threadThanks. Twitter will use this to make your timeline better. UndoUndo -
José Ignacio Rojas Echenique Retweeted
My lab, in collaboration with
@betulland at U Arizona, has a new preprint. The work was led by my fantastic postdoc@s_venkataram. https://www.biorxiv.org/content/10.1101/850644v1 … He describes the main results in this thread:https://twitter.com/s_venkataram/status/1198702231878619136 …Show this threadThanks. Twitter will use this to make your timeline better. UndoUndo -
José Ignacio Rojas Echenique Retweeted
This week on the Nature cover: Waves of mutation. Barcode system images evolutionary dynamics of laboratory yeast. Browse the issue here: https://go.nature.com/336w2ip pic.twitter.com/pe16RxYmiG
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José Ignacio Rojas Echenique Retweeted
If you haven't yet read
@alex_nguyen_ba 's paper, https://www.nature.com/articles/s41586-019-1749-3#Sec5 … perhaps you'd like to read the blog post I wrote on it? http://www.phagefood.com/an-in-depth-look-at-yeast-evolution-suggests-theoretical-models-need-updating/ …Thanks. Twitter will use this to make your timeline better. UndoUndo -
José Ignacio Rojas Echenique Retweeted
A renewable barcoding system reveals the evolutionary dynamics of laboratory budding yeast, showing that fitness changes over time in a travelling wave of adaptation that can fluctuate owing to leapfrogging events, according to a Nature paper. https://go.nature.com/32NssJG pic.twitter.com/KH1aRQ68yS
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José Ignacio Rojas Echenique Retweeted
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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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.
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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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@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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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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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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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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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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@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.Show this threadThanks. Twitter will use this to make your timeline better. UndoUndo -
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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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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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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Our technique is an attempt to recombine two existing technologies, high resolution DNA barcoding (see http://nature.com/articles/nature14279 …), and gene stacking (e.g. http://nature.com/articles/nbt.2825 …).
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José Ignacio Rojas Echenique Retweeted
Notice here that Alex uses the two L spelling of travel[l]ing while Ivana opted for the single L. Is it a deep cultural difference between wet lab / dry lab? Will
@jireva side with Ivana and the traditional American spelling or Alex and the British journal's favourite spelling?https://twitter.com/alex_nguyen_ba/status/1194683107871313920 …Thanks. Twitter will use this to make your timeline better. UndoUndo -
José Ignacio Rojas Echenique Retweeted
What do you expect evolution to look like in a microbial population? Check out our new study offering a view of that over 1000 generations in yeast in the lab. co-led by
@alex_nguyen_ba,@jireva, in@MichaelMDesai 's labhttps://www.nature.com/articles/s41586-019-1749-3 …Show this threadThanks. Twitter will use this to make your timeline better. UndoUndo
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