Coolest open Source vector ever - anyone experienced at gibson assembly?

[Mega [Andreas Stuermer]]

Hi everyone, 

Just wondering if there is anyone with a lab (more or less) experienced with gibson assembly, who would like to participate in creating an open source vector. It is ought to be the coolest cloning vector ever. 

I got my hands on a quite ancient piece of DNA (which is nowhere near outdated/obsolete). 

Since the aec lab doesn't have competent coli in stock, nor ligase, nor gibson stuff, it would be great to outsource this "job". 

Basically it should be quite easy, replacing a piece of pUC19 with my DNA. So 2 PCRs, and an assembly reaction. If we dare, we can even insert 2 pieces (making it 3 PCRs) to make the plasmid even better. 

Just shoot me a mail if you have the free capabilities ;) 

[Michael O.]

Hi Andreas,

Maybe you should take a look at http://parts.igem.org/Plasmid_backbones - there's no need to make yet another open source vector...

best,

michael

[Andreas Stuermer]

Hi Michael, this one will be cisgenic selection to make it legal to use outside a certified lab in Europe, that's the clue ;) 

Best,

Andreas

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to a topic in the Google Groups "DIYbio" group.
To unsubscribe from this topic, visit https://groups.google.com/d/topic/diybio/IZsG2l6KhVs/unsubscribe.
To unsubscribe from this group and all its topics, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/46f50c4d-e227-415e-8f0e-523c5b0bea0c%40googlegroups.com.

For more options, visit https://groups.google.com/d/optout.

--

---------------

http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf

[Nathan McCorkle]

*sigh* if only that website didn't suck so bad... I can't find any
useful information from that link... there is no table of vectors that
I can see there, with species/ORI specifications and other features
listed.

Anyway, the last time I tried finding their vector list, I seemed to
see very limited choices, or maybe the website was just as terrible
then and I have just been missing the trove of information.

[Mega [Andreas Stuermer]]

Might I add, it's a shame that GFP is illegal in Europe, as it counts as S1 and S1 is restricted to commercial labs

[Mega [Andreas Stuermer]]

I meant certified, not commercial

[Koeng]

Thats not open. Last time I asked personally for one of their vectors they didn't respond. 

In addition, their plasmids lack complex features, common MCSs, and their standard sucks *unless* you have the parts registry which is about 3500$. The plasmids have lots of common restriction enzyme sites in the backbone (XhoI for example) which makes making compatibility layers for new parts very difficult.  The construction of said vectors to make a complex part takes a very long time, about 3 sequential cloning reactions from basic parts. While their plasmids do have some features that are useful like the terminators around their MCS, they didn't include the M13 primers, they included their own primers which companies don't have, making sequencing very large amounts of DNA more difficult. In addition, while there are negative selection methods they could use to simplify plasmid construction, they don't usually use them.

Pretty much, other than when competing iGem, their plasmids are a pain to work with. A lot of the projects there actually couldn't get prizes because their constructs didn't go in the standard plasmid so well. It's difficult to actually modify the backbone to fit your special needs unless you use gibson or the like. Plus they wouldn't accept plasmids in other formats. 

Because of these limitations, no labs I know of actually use it for normal cloning. They *all* use traditional cloning or golden gate, but mostly gibson. There's definitely need for an open source plasmid that actually fits all specifications well enough to be used.

-Koeng

[Cathal (Phone)]

Facilitating gibson with high-efficiency, pre-defined entry-points was a design goal for indieBB. As was M13 sequencing sites for potential "library" use and construct validation.

I still think there's room for a well engineered DIY-first vector, so go for it Andreas! :)

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Mega [Andreas Stuermer]]

Wow that are many features. I guess it is best to focus on the 2 features to make version 1.0. 

Else I'd have to synthesize all

[Mega [Andreas Stuermer]]

I mean, at the moment the design says no element will be synthesized, because synthesis is still too expensive

[BraveScience]

Hi,

Still in need of extra help?

In my lab we usually use fusion PCR instead of gibson assembly. That with the combined use of restriction sites allows you more "last minute" changes apparently.

If that's interesting let me know.

[Andreas Stuermer]

Hi! Thanks a lot for the offer, but it already is on its way. Though might I ask for an efficient fusion PCR protocol? ;)

--

---------------

http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf