Re: [DIYbio] Re: Gibson vs SLiCE (single part recommendations)

I also got an MTA for the PPY strain but the shipping costs (for cold chain) was too much for me so I opted for getting a pKD46-deriven plasmid (pREDIA from Addgene). The only difference is in PPY gam and redb is integrated into the DH10B genome and reda in plasmid whereas in pKD 46 it is on the plasmid. I really would like to make a side-by-side comparison for the PPY and pKD46/DH10B in my lab.

Also there is this japanese guy Motohashi who does SLiCe from regular bacteria extracts (recA-) and has data on its efficiency with 15-19 bp overlaps between parts (https://link.springer.com/protocol/10.1007%2F978-1-4939-6472-7_23). He is using JM109 as his primary strain and uses a few more alternatives as far as i remember.

On the note, Gibson is too pricey for any lab who's on a budget, especially DIY labs and labs in developing countries. SLiCe and even CPEC has become better alternatives for synthetic biology and even simple cloning reactions - basically your only cost is bacterial reagents and primers (and PCR reagents in some cases).

On a last note, also read this article - www.ncbi.nlm.nih.gov/pubmed/26463009 - where they combine CRISPR with Lambda phage recombination system to efficiently modify e. coli genome.

Cheers!

C

On Sunday, June 18, 2017 at 6:07:48 AM UTC+3, Rob C wrote:

Not too long ago we obtained the PPY strain as a kind gift from the original makers (Zhang at Albert Einstein). 

In our hands making extracts from this strain works WAY better than traditional DH10b, DH5alpha, etc (using the CelLytic lysis B reagent...it's cheap for what you get). 

Although I don't have exact numbers to report, we have done several tests side by side with Gibson, and PPY based SLICE gives at least equal transformation efficiency. Accuracy is equally as good, and in some isolated cases it has been better. 

After several month of heavy cloning, our lab has now dropped purchase of all Gibson reagents. 

I'm convinced that if more people implement the PPY-based SLICE cloning in their lab, Gibson sales will go out of business. 

On Thursday, March 16, 2017 at 12:32:35 PM UTC-7, Rob C wrote:

Bryan, thank you very much for sharing your experiences. Sounds like you did all the right controls to get a proper assessment of the system. Logically it is a little surprising that there's no carry over of plasmid, but of course very good news and just goes to show the best way to answer a question is to just do the experiment. I tend to see too many people over thinking things to a point where they will convince themselves something is not feasible before they've even tried it, so the project stops before it even begins. :)

Cheers.

On Monday, March 13, 2017 at 11:57:49 AM UTC-7, Bryan Jones wrote:

I would imagine there is some risk of that, but I haven't seen that happen in my experiments. I carried out a negative control using just SLiCE extract with competent cells (No insert DNA), and didn't get any colonies on amp plates. Also the colonies I sequenced from my tests were all correct, no pKD46 carryover. It might be that DNAse breaks all the DNA from the SLiCE cells. Normally when you do a plasmid prep, you add EDTA to prevent this, but no EDTA was added here. I'm not positive why, but empirically, I can say that it does not seem to be an issue. 

On Tue, Mar 7, 2017 at 6:16 PM RBC <rco...@gmail.com> wrote:

Is there any worry that the amp resistant pKD46 plasmid will be extracted into the SLiCE lysate, in which case this plasmid will carry through to the final transformation? To me it would seem you can't use it to make amp resistant vectors..or maybe I'm missing something. Thanks for the clarification.

RC

On Friday, October 21, 2016 at 11:22:11 AM UTC-7, Bryan Jones wrote:

In my most recent test, without pKD46, I got 10 colonies from the SLiCE reaction, which was the same as the no SLiCE control. SLiCE with pKD46 resulted in 50-60 colonies. For comparison, NEBuilder resulted in about 100.

--
-- 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 the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/ed3ec371-5de0-49e0-a8e5-db6dbdae3fda%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.