A terrible way to clone in a cut site
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Sebastian S Cocioba
Jul 16, 2015, 2:08:43 PM7/16/15
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TL;DR: Will a plasmid with two ORIs be stable enough to get colonies and miniprep?
Hi guys and gals,
Its been a while since I posted to the list. Been busy with iGem and whatnot. Anywho I've been having some trouble on one of my backburner projects that involves a modular restriction digest based cloning system for plant gene expression and transformation. At the heart of the system are four cutters: EcoRI, NcoI, SacI, and KpnI. The idea works because almost all the promoters I use do not have E or N in the sequence, most of the CDS's I use don't have N or S in the sequence, and the terminators I've isolated luckily do not have SacI or KpnI present internally.
Basically, the system is:
E-promoter-N-gene-S-terminator-K
Its not intended to be a universally modular system by any stretch of the imagination. It just happens to work for the most routinely used (and swappable) parts in my library. That being said, the issue lies with the pesky NcoI recognition sequence. I've posted a while ago asking if anyone had a plasmid with E-N-S in the MCS and many of the responses were tailored around the fact that the NcoI site has an ATG which can lead to bad things if smaller sequences are in frame. Few plasmids ever include NcoI in their MCS simple because it could express other things of its in frame. This phenomenon actually happens to be the core of my system.
If you are a follower and believer of the concept of Kozak consensus sequences, the most common RBS in plants happens to be the NcoI site itself. All I have to do is design primers that amplify out the CDS with two extra C's upstream (and ensure the first base downstream of the ATG is a G) and I can now clone in any coding sequence into my plant transformation vector with relative easy since the NcoI site is kinda rarely found inside the CDS itself. Unfortunately, the recognition sequence is mostly Gs and Cs which increase melting point significantly when designing primers and Primer 3 Plus always spits out instability flags when designing.
After combing through the hundreds of plasmids I have in my freezer, I came across one in particular that, if it works, would give me exactly the restriction site sequence I need...but there is a catch:
There is about 12,000 bases in between the three sites!
The plasmid in question is pCambia1302, my workhorse circuit for debugging transformation efficiencies and the very one I used during my preliminary DIY gene gun tests. It confers hygromycin resistance and a constitutively expressed mGFP5 protein. (www.markergene.com). It even has the NcoI site in front of the mGFP5 for the same exact reason I've stated above. The big issue is the fact that between the EcoRI and SacI site there exists the whole damn plasmid and worse yet it includes its own origin of replication!
My plan is to use my favorite amp-based shuttle vector p2KB (open biotechnology website) and clone in THE ENTIRE pCambia1302 plasmid cut with EcoRI and SacI. Then I'll swap out the giant chunks between E and N with the CaMV promoter, swap the chunk between N and S with a few genes of interest, etc until I replaced those massive pieces with a reasonable cassette for expression in plants that i can mix and match.
The question I have for all the great minds on this list is:
Will a plasmid containing two origins of replication (facing head to head but far apart) be stable enough in E. Coli that I could grow colonies and miniprep?
I've enclosed a picture of the proposed cloning simulated in SnapGene. The yellow arrows are origins of rep. The NcoI site is equally distanced in between the EcoRI and SacI sites and no, there is no way (AFAIK) to clone in the parts in chunks and still maintain the E-N-S-K sequence. Thanks!
Hi guys and gals,
Its been a while since I posted to the list. Been busy with iGem and whatnot. Anywho I've been having some trouble on one of my backburner projects that involves a modular restriction digest based cloning system for plant gene expression and transformation. At the heart of the system are four cutters: EcoRI, NcoI, SacI, and KpnI. The idea works because almost all the promoters I use do not have E or N in the sequence, most of the CDS's I use don't have N or S in the sequence, and the terminators I've isolated luckily do not have SacI or KpnI present internally.
Basically, the system is:
E-promoter-N-gene-S-terminator-K
Its not intended to be a universally modular system by any stretch of the imagination. It just happens to work for the most routinely used (and swappable) parts in my library. That being said, the issue lies with the pesky NcoI recognition sequence. I've posted a while ago asking if anyone had a plasmid with E-N-S in the MCS and many of the responses were tailored around the fact that the NcoI site has an ATG which can lead to bad things if smaller sequences are in frame. Few plasmids ever include NcoI in their MCS simple because it could express other things of its in frame. This phenomenon actually happens to be the core of my system.
If you are a follower and believer of the concept of Kozak consensus sequences, the most common RBS in plants happens to be the NcoI site itself. All I have to do is design primers that amplify out the CDS with two extra C's upstream (and ensure the first base downstream of the ATG is a G) and I can now clone in any coding sequence into my plant transformation vector with relative easy since the NcoI site is kinda rarely found inside the CDS itself. Unfortunately, the recognition sequence is mostly Gs and Cs which increase melting point significantly when designing primers and Primer 3 Plus always spits out instability flags when designing.
After combing through the hundreds of plasmids I have in my freezer, I came across one in particular that, if it works, would give me exactly the restriction site sequence I need...but there is a catch:
There is about 12,000 bases in between the three sites!
The plasmid in question is pCambia1302, my workhorse circuit for debugging transformation efficiencies and the very one I used during my preliminary DIY gene gun tests. It confers hygromycin resistance and a constitutively expressed mGFP5 protein. (www.markergene.com). It even has the NcoI site in front of the mGFP5 for the same exact reason I've stated above. The big issue is the fact that between the EcoRI and SacI site there exists the whole damn plasmid and worse yet it includes its own origin of replication!
My plan is to use my favorite amp-based shuttle vector p2KB (open biotechnology website) and clone in THE ENTIRE pCambia1302 plasmid cut with EcoRI and SacI. Then I'll swap out the giant chunks between E and N with the CaMV promoter, swap the chunk between N and S with a few genes of interest, etc until I replaced those massive pieces with a reasonable cassette for expression in plants that i can mix and match.
The question I have for all the great minds on this list is:
Will a plasmid containing two origins of replication (facing head to head but far apart) be stable enough in E. Coli that I could grow colonies and miniprep?
I've enclosed a picture of the proposed cloning simulated in SnapGene. The yellow arrows are origins of rep. The NcoI site is equally distanced in between the EcoRI and SacI sites and no, there is no way (AFAIK) to clone in the parts in chunks and still maintain the E-N-S-K sequence. Thanks!
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