3 ms·

Their claims around Type IIS assembly are also suspect. eg in Golden Gate assembly, you choose Type IIS that reach over and cut, so the restriction site is abse

Their claims around Type IIS assembly are also suspect. eg in Golden Gate assembly, you choose Type IIS that reach over and cut, so the restriction site is absent from the final assembled product. "Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible" https://en.wikipedia.org/wiki/Golden_Gate_Cloning https://en.wikipedia.org/wiki/Golden_Gate_Cloning ---- The choice of focusing on a particular RE pair also smells of p-hacking. Their claim that BsaI/BsmBI makes for easy mixing/matching genomes doesn't make sense in this day and age, when you can use other techniques to make hybrids more effectively (eg, you are not restricted to the natural location of those restriction enzyme sites)

throwawaymaths 4y ago
The argument is about the negative space of the RE. Regardless of what the article says, To do golden gate/Gibson/etc. best practice is to cut the template first (with RE) then assemble against the open ends, so to do this you must ablate the existing re sites. The alternative is to linearize by round the horn pcr. At 33kb, it's not impossible but why bother with the pain when it's much easier to snip.
tripletao 4y ago
Their description of the assembly strategy as "Golden Gate" seems like incorrect terminology. The WIV has at least published papers using BsaI and BsmBI, though. https://twitter.com/jbkinney/status/1583267221047869441 https://twitter.com/jbkinney/status/1583267221047869441 https://twitter.com/jbkinney/status/1583248052969562112 https://twitter.com/jbkinney/status/1583248052969562112 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006698 https://journals.plos.org/plospathogens/article?id=10.1371/j... EDIT: Kinney also asserts here that they left the sites in a final assembled genome (i.e., the genome of a replication-competent virus). I'm still trying to figure out if that's true, though.
emsign 4y ago
Just because there are more modern techniques doesn't mean there haven't been older ones used, or techniques used incorrectly.

throwawaymaths 4y ago

The argument is about the negative space of the RE. Regardless of what the article says, To do golden gate/Gibson/etc. best practice is to cut the template first (with RE) then assemble against the open ends, so to do this you must ablate the existing re sites. The alternative is to linearize by round the horn pcr. At 33kb, it's not impossible but why bother with the pain when it's much easier to snip.

tripletao 4y ago

Their description of the assembly strategy as "Golden Gate" seems like incorrect terminology. The WIV has at least published papers using BsaI and BsmBI, though. https://twitter.com/jbkinney/status/1583267221047869441 https://twitter.com/jbkinney/status/1583267221047869441 https://twitter.com/jbkinney/status/1583248052969562112 https://twitter.com/jbkinney/status/1583248052969562112 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006698 https://journals.plos.org/plospathogens/article?id=10.1371/j... EDIT: Kinney also asserts here that they left the sites in a final assembled genome (i.e., the genome of a replication-competent virus). I'm still trying to figure out if that's true, though.

emsign 4y ago

Just because there are more modern techniques doesn't mean there haven't been older ones used, or techniques used incorrectly.