Re: [DIYbio] Re: The ODIN Needs Your Help - T4 DNA Ligase Testing - Free Ligase

http://www.sciencedirect.com/science/article/pii/S2405580815000850

Use a triton x100 and Tris-HCl buffer. It's cheap and extremely easy to make (Made some yesterday, going to make my own SLiCE friday)

Interesting is that the difference is that the cells grow 3x more concentrated in the JM109 method. May have to do with DNA repair genes getting turned on during late growth.

-Koeng

On Wednesday, October 21, 2015 at 3:52:40 PM UTC-7, Edoardo Gianni wrote:

Hi Josiah, good points. I had similar worries after the first good results.

I did DpnI treatment but no gel purification, that would be ideal especially if you have the same selection barker.

I did transform the original RFP in pSB1K3 and plated on chloramphenicol, got no colonies.

No sequence verification but we sent another part assembled this way to iGEM, which should sequence it soon. It would be interesting to see if you get some errors at the recombination site.

Efficiency is high as I am using quite a lot of DNA (100ng of vector in the ligation, 30ng used for transforming), plating all the cells from the electroporation and using highly competent electrocompetent cells. I would expect far fewer colonies in an assembly with less DNA and with chemically competent cells, I would assume a similar efficiency to a digestion/ligation - definitely not the efficiency of a Gibson superoptimised kit.

The method doesn't need electrocompetent cells, it's just that we got an electroporator donated and it works well for us. In addition we were working on an automated competent cells maker, which would simplify the process of making the cell. Regarding the primers, for a two part assembly the normal biobrick prefix and suffix are long enough to allow homologous recombination with the prefix and suffix in pSB1C3. For multiple part assembly the primers would have similar costs to a gibson assembly, although you could think of some standard ends to simplify the process. 

I didn't think about the lysis buffer, I'd like to try DIY alternatives though (i.e. soap? french press? glass beads?). On the other hand you only need to make the lysate once and it lasts for hundreds/thousands of reactions. The other part I thought would be expensive is the reaction buffer, but it comes at about 20$ for 6ml from NEB (it's the same one you are trying to test for the T4 ligase).

Btw regarding the T4 ligase the enzyme is what would worry me, more than the ATP. My understanding is it's not as stable as taq/restriction enzymes, but the london biohackers are happy to betatest if you ship overseas :)

Edo

On Wednesday, October 21, 2015 at 7:30:43 PM UTC+1, Josiah Zayner wrote:

That's an interesting idea.

A few questions:

Did you do a DpnI digest to remove the RFP pSB1K3 plasmid after the PCR or a gel purification of your PCR product?

Did you do any negative controls?

Have you sequenced or verified that it is in fact the pSB1C3 with an RFP insert?

Efficiency seems awfully high which makes me wonder if what you are seeing is just the pSB1K3 or something else.

Also, the need for electrocompetent cells, an electroporator, multiple sets of primers and a commercial lysis buffer kind of removes any sort of money savings. In fact, it makes this method much more expensive then a standard cloning reaction.

JZ

On Wed, Oct 21, 2015 at 10:36 AM, Edoardo Gianni <edo.g...@gmail.com> wrote:

Hello there,

I recently tried the SLiCE assembly at the London Biohackspace for our iGEM project and had quite good results with it. We put together some of the results on openwetware http://openwetware.org/wiki/SPLiCE and tested a version of the reaction with PEG8000, which seems to speed up the reaction (just like in the quick ligase buffer). We transformed the cells with pKD46 (which contains the lambda recombineering genes) instead of using the PPY strain and it seemed to work just fine. 

I am now trying to find the time to get a better idea of the efficiency as well as clone a biobrick version of the pKD46 as we synthesised the gam-bet-exo genes as gblocks. Feel free to add to the page if you have some comments :)

Edo

On Monday, October 19, 2015 at 3:00:01 PM UTC+1, Koeng wrote:

This reminds me of a goldengate PaperClip assembly. Very interesting however, once I get an opportunity I will read the entire paper! 

You tried the DH5a method or the SLiCE method?

-Koeng

On Monday, October 19, 2015 at 3:42:46 AM UTC-7, BraveScience wrote:

Same here. Ligation dependente cloning using biobricks sucks, given the volumes and the amount of steps you have to necessarily go through, doesn't turn out to be that efficient. 

I got interested as well in Gibson. Indeed price\quality of commercial kit vs homebrewed one is a tough one.

Anyway I stumbled across the BASIC system. Yes, you need a restriction enzyme (just one, BsaI) and work through ligation. But you can shuffle your parts around as you wish.

http://pubs.acs.org/doi/abs/10.1021/sb500356d

It's a one-pot reaction.

Fede

Il giorno sabato 17 ottobre 2015 22:14:42 UTC+2, Koeng ha scritto:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137466 

May be interesting

On Saturday, October 17, 2015 at 1:09:32 PM UTC-7, Koeng wrote:

http://www.sciencedirect.com/science/article/pii/S2405580815000850

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453199/pdf/12896_2015_Article_162.pdf

 It appears that JM109 can do SLiCE just fine. Not sure about comparisons of efficiency thou

-Koeng

On Friday, October 16, 2015 at 9:14:10 AM UTC-7, Bryan Jones wrote:

Koeng, SLiCE sounds interesting. Do you know where to get your hands on the PPY E.coli strain that they use? Is it sold anywhere (a quick google search didn't turn up anything)? I guess I could try to contact the authors or those papers. I'd like to give it a try in the academic lab I'm working in. If it works as well as they claim it might be a good alternative to both traditional restriction enzyme/ligase cloning and Gibson Assembly, especially for the DIYbiologist on a tight budget.

On Friday, October 16, 2015 at 8:27:08 AM UTC-5, Koeng wrote:

True true, EcoRI and HindIII are favorites of mine because of how much they supply. However, they are 6bp cutters... So I would actually argue that NotI and SbfI would be better for a new cloning system! I guess it depends on how much it would cost to remove the sites vs the actual enzyme cost.

I've noticed that at least at an academic lab people always use the same volume of enzyme... Like 1µl is 20 units of EcoRI but just 5 units of BtgZI... anyway it's a pretty wasteful approach, so if anyone is reading this is using restriction enzymes, go by units.

Then again, I haven't even cloned anything with classic restriction enzymes in a year or so because gibson is so good. However, gibson can cost an arm and a leg if you buy it directly from NEB (price for quality, their stuff gives me ~5-10xs better efficiency than our normal stuff). I know that SLiCE ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333860/ http://www.ncbi.nlm.nih.gov/pubmed/24395368 ) would probably be very cheap to make in a DIY lab if created in mass, which might be useful for you. I'm curious what the efficiency is, but if it's good, I'd be willing to switch my home experiments over if it meant I wouldn't have to buy mixs anymore.

-Koeng

(btw: Thanks! I knew I forgot something)

On Thursday, October 15, 2015 at 10:27:12 PM UTC-7, Josiah Zayner wrote:

Yeah it is just a resale. Most people don't have ATP I imagine so I want to make sure it works as is, ya' know?

I will look at BsaI BsmbI but they are less used enzymes so they are freaking expensive.

You can do like 10 times the cloning with NcoI/EcoRI/HindIII on price maybe even more.

Same thing with Biobricks enzymes. I am selling those for iGEM folks but they are expensive. Someone should go back and start a cloning system that uses EcoRI and HindIII, the two least expensive enzymes.

Also, Koeng, you forgot to use the coupon code so I refunded you shipping!

On Thu, Oct 15, 2015 at 10:14 PM, Koeng <koen...@gmail.com> wrote:

How about the raw ligase? I buy ATP directly from NEB to do goldengates with, and some ligase from a non-commercial source might be interesting. 

Also, how are you getting the ligase? Expression of it in house? Or resale? Either or, I'm sure it'll be a great offer, but I am curious if you had the vector in house for some in vivo ligations I'd like to test out. Anyway, useful site! It's awesome that you've gotten more enzymes up, but I think BsaI/BsmBI are also a real important couple. Once you get materials to do GoldenGate, it's a pretty wonderful system, just wish I could get around to making some basic parts for DIY vectors...

-Koeng

On Monday, October 12, 2015 at 1:37:02 PM UTC-7, Josiah Zayner wrote:

My company, The ODIN(http://www.the-odin.com) is starting to ship restriction enzymes and ligase! However, enzymes are pretty heat stable. T4 DNA ligase is another matter, it requires ATP, ATP hydrolyzes. I need a few people to test out some T4 DNA ligase and reaction buffer that has been shipped. Shipping of Taq and other Master Mixes has worked and they contain dNTPs so I assume this should work out just fine but I need testers to make sure.

If you goto the website(http://www.the-odin.com/t4-dna-ligase-5ul/) and use coupon code FREETHELIGASE I will ship you some ligase for free as long as you promise to provide feedback on how it works after shipping.

Thanks,
     Josiah Zayner
     The ODIN
     ca...@the-odin.com

--
-- 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/f4rSKxULI-g/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/557470ae-e2aa-450e-886d-d29d3377122a%40googlegroups.com.

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

--
-- 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/f4rSKxULI-g/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/d0f716b5-8276-4de4-8038-becb4b216522%40googlegroups.com.

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