[subroutine]

Adding a poly(A) tail when engineering rna plasmids is so common that it's part of the standard feature library of most plasmid editors.

Here I have a screenshot of the ApE editor displaying one plasmid I made for a neurobio experiment involving the overexpression of two chimeric proteins (actin and profilin, respectively linked to green and red fluorophores; note the editor has autotagged the polyA tail feature):

https://ibb.co/XSCSKC9

[userbinator]

Looks almost like a hex editor.

[samus]

It kind of is. We are not quite advanced enough to have the equivalent of true programming languages for genetics.

[digitaltrees]

But we will. GitHub for genetics would be awesome

[sleepydog]

It reminds me of Wireshark

[koeng]

ApE is one of those tools that is so old-school but just works. I used to hate using it but it's really grown on me - Snapgene is just too darn expensive and benchling isn't very power user friendly

[x-shadowban]

Can you add any number of A's, or just 1, or shorten it by 1, and it still is functional?

[subroutine]

The number of adenine repeats that confer functional properties is quite variable but it definitely needs to be more than "just 1". I've seen anywhere from 25-250 used in designed plasmids. The exact number people use in their engineered sequence is based on a number of factors, not all of them scientific in nature (e.g. companies charge per basepair synthesize a bespoke polypeptide; e.g. you copied the sequence from a previous clone into ApE and that sequence used 30 repeats and worked fine).

[kevviiinn]

Iirc there's a protein that adds 12-15 slowly then another protein comes in and adds another 200+ when it detects the start of the tail. Not sure about the details about how or why it stops tho. At least when considering mRNA getting prepped for nuclear export

[subroutine]

You are probably right, I'm not an expert on this process. But there's likely a difference between the innate eukaryotic cell polyadenylation process vs. how coronavirus accomplishes polyadenylation because coronavirus rna never enters the nucleus.

[kolinko]

In this case it probably works most reliably with around this number of As, and less/more would decrease reliability, butnitmwould still be functional.

Most of genetics is like that.

[kevviiinn]

With more As you risk running into the upper limit on sequence length for the virus shell and with less you run the risk of quicker degradation and not enough expression

[subroutine]

The first point being only a concern if you are a virus. If instead you are engineering say, an rna vaccine, you could package your transcript into lipofectamine instead of a coronavirus envelope ;) but point taken, there's always an upper limit.

[kevviiinn]

Also a concern when engineering viruses for research or other treatments such as the AAVs

[subroutine]

True. I should have said "if you are using a virus"