How to design a Plasmid? Any DIY tutorials out there?

[Jarrad]

I was wondering if there were any tutorials for diybio on designing your own plasmid's ? (preferably with a tool like benchling, ape, etc)

I'm new to Synbio but would love to see a tutorial series sort of like an arduino/electronics tutorials. That shows you how to make simple circuits and builds upon the knowledge.

From what I've been reading it certainly seems like this field has matured enough to be able to pull this off?

I'd also be happy to commission this kind of tutorials if that helps spark any interest!

[Nathan McCorkle]

Do you mean how to use a plasmid editor? Or conceptually, how adjacent
sections need to align and where they should be placed for optimal
function?

To begin, you could simply use a pen and piece of paper to develop the
block diagram of what elements you want the plasmid to posses, then
you import those sequences into a plasmid editor or custom software
(i.e. python using biopython). I've done things with plasmid elements
like search all the sequences for restriction sites, then remove all
the common restriction sites from a list of available restriction
enzymes (like a product page from a supplier), then with the remaining
restriction enzymes, sorted them by price. Now you have a price sorted
list of restriction sites that won't interfere with any of the DNA
you're working with. If you had some 'favorite' restriction enzyme,
then you'd need to mutate the DNA so the restriction pattern was
disrupted, but that's not just plasmid design, that's plsamid/DNA
mutation.

P.S. I made this intro presentation for electronics/arduino a few months ago:
https://docs.google.com/presentation/d/1O3-jwrJn4zr42MAQcltEDqjT0Hx80D7JD2zUPDE4o3A/edit?usp=sharing

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-Nathan

[Mega [Andreas Stuermer]]

I don't think so because mostly you just have your insert cloned into a standard plasmid.

Depending which lab you're in they use pUC19 or the retro-version pBR322. Some hipsters may even use IP-free vectors. Btw, patents on pBR322 should have expired too?

All have in common colE1/pUC origin and ampicillin resistance...

[Cathal Garvey]

Hi Jarrad,
I've designed at least one plasmid entirely from scratch, and others
partially or by copy/paste.

It depends what you mean by "design", because in practise plasmids are
quite modular. You can copy/paste directly from wild plasmids the
replication proteins and target sites, and copy/paste a selection system
of your choosing, and copy/paste a cloning site or desired gene, and
that'll probably work OK.

Going deeper than that is where things get a little hairy, and less
predictable. There's no software that I know of that'll tailor plasmids
at a level below copy/paste automatically, largely because every
replicon, even closely related ones, are quite different. At that level,
you just have to manually dig into the literature that's available on
the replication system you want, and closely related ones, and using
knowledge and faith you can manually tailor it and click "order".

In my case, the plasmid I ordered worked partially but did not appear
segregationally stable. However, I don't think this was due to my
modifications to the replicon, but rather due to leaky transcription of
the selection system, which was a protein that acts like a development
trigger in the target cell; even tiny amounts could have triggered a
fatal developmental bias towards poorly-replicating cells.

In other words, my own escapade in intricate plasmid design was a
partial success but ultimately didn't work, though I think that was
because of naive payload design rather than unsuccessful plasmid design.
I got greedy and ordered the novel plasmid *with* the novel payload,
which is in hindsight a bit stupid.
Still planning to revisit that project someday and revive it with a
tighter gene regulatory system and a more conventional "backup"
selection system to assist in testing..

I've been meaning to write a blogpost describing the experience of
tinkering at that level for about, oh, three years now. :) Someday I
will, but right now I'm too busy with a very similar project I'm hoping
to share with ye all soon, which will benefit from the above experience
(I'm more conservative and careful these days and I'm sick of
overspeculation..).