Join GFP to my insert without mutagenesis - (Mar/06/2006 )
Hi everyone!
I have GFP within a plasmid (pTarget) and another inserted sequence within another palsmids. (pTarget).
I wonder how I can tag GFP to the carboxil terminus of my insert. If I use Restriction Digestion procedure, my insert has a stop codon so the GFP sequence will not be expressed. Is there a easy way of achieving this without using mutagenesis?
Mutagenesis kits are really expensive. Would it be easier for me to get one of these plasmids which insert GFP into Carboxil terminus following a quite simple protocol?
I'm looking forward to hearing new ideas and suggestions from this forum of experts.
Thank you.
I don't really know how you did with restriction digest and where your stop codon is.
Lets imagine that the sequence that you want to attach GFP to, is of 2kb for example. GFP sequence thats within the plasmid is of 2kb if I remember correctly. so you can try fusion pCR and then restriction digest. In the sense, design two primers for your insert forward and reverse primer. The end of the sequence where you want to attach GFP to, that will be reverse primer, will be designed in such a way that 18nt bases will act as primer for your sequence and you will attach part of GFP seuquence to this primer, which will actually be forward sequence of GFP primer.
So reverse primer of your insert+GFP forward primer will whole make your reverse primer for insert.
Insert restriction sites at forward primer of your insert and reverse primer of your GFP sequence.
You will amplify GFP as well as insert seperately.
Once both are amplified, check quickly on gel using an aliquot, if you haven't amplified anything else.
Once you see one band, mix portions of both the amplified products and run them in PCR machine with only polymerase for 5 cycles then add forward primer of your construct and reverse primer of GFP sequence and amplify whole stretch to obtain a fusion product.
Once you have this fusion product, cut the plasmid and insert this amplified product there.
Now you still have to watch where that stop codon sits and if its really required or you can PCR mutagenise using glycine sequence or something like that.
Let me know if I am clear enough and this is what you were looking for
I am sure that approach will work and I will keep it in mind. However, I'm going to explain my idea and let me know what you think:
As my GFP is in a plasmid it has incorporated various restriction sites I can use.
Then my plan is to try to find one of these restriction sites at the end of my sequence, before the stop codon. Must cut my sequence only one site to be successful. If I don't find any then, design a primer with an unique restriction site incorporated as a tag. This primer will cleavage my sequence just before the stop codon and subsequently I will get my sequence with a new restriction site and without any stop codon. Then I can join my sequence to the GFP using restriction enzyme diggestion.
I would apprecite your comments and also by the rest of experts.
Thanks
As my GFP is in a plasmid it has incorporated various restriction sites I can use.
Then my plan is to try to find one of these restriction sites at the end of my sequence, before the stop codon. Must cut my sequence only one site to be successful. If I don't find any then, design a primer with an unique restriction site incorporated as a tag. This primer will cleavage my sequence just before the stop codon and subsequently I will get my sequence with a new restriction site and without any stop codon. Then I can join my sequence to the GFP using restriction enzyme diggestion.
I would apprecite your comments and also by the rest of experts.
Thanks
Hi gsamsa,
It's pretty unlikely you will find an appropriate RE site in your sequence, and there's no need for you to incorporate one just to remove your stop codon - you can do it much more easily by PCR (exactly as described as JiangM). No need for restriction digests, and avoids beard-pulling over ligation problems etc... Just design the downstream fusion-PCR primer for your insert to target the 18bp immediately upstream of the stop codon (don't include it though) then include a sequence 'tag' from the start of the GFP sequence on the 5' end of this primer. The stop codon will be eliminated before you 'fuse' the 2 PCR products.
BTW, I wouldn't use Taq polymerase for this, as the error rate of standard Taq (~1 wrong base per 1000kb extended) may leave you with unexpected mutations in your final fusion gene... Use a proofreading polymerase (i.e. Vent from NEB, or similar) or - even better - use Klenow or T4 polymerase to make the initial dsDNA fusion product (from the 2 mixed/annealed PCR products) then amplify this with a high-fidelity polymerase before you clone it.
hope that helps - post back if it's not clear.
Del.
Clear as crystal. Del and Jing have convinced me completely. Just a little last question, the restricition sites added to the primers. Do I really need them? My plan is inserted the fusion construct into pTarget which is TA cloning vector. I think there are some high-fidelity polymerases with the ability to add A to the end. In that case, can I use that high-fidelity polymerase to get the fusion product?
I'm extremely grateful to both.
Thanks a lot.
gsamsa, the apprentice
Once you have this fusion product, cut the plasmid and insert this amplified product there.
Clear as crystal. Del and Jing have convinced me completely. Just a little last question, the restricition sites added to the primers. Do I really need them? My plan is inserted the fusion construct into pTarget which is TA cloning vector. I think there are some high-fidelity polymerases with the ability to add A to the end. In that case, can I use that high-fidelity polymerase to get the fusion product?
I'm extremely grateful to both.
Thanks a lot.
gsamsa, the apprentice
Hi gsamsa,
No, you don't really need the restriction sites in the final 'outer' primers, unless directional cloning of your fusion gene is important. Due to the problems of cutting at RE sites located close to the ends of PCR products, many people would clone the PCR product anyway, before attempting to cut it at the introduced sites (see posts elsewhere in this forum). I think the high-fidelity polymerases which give 3'-A overhangs are actually mixtures of proofreading polymerases (presumably responsible for most of the polymerisation) and some normal Taq, so the products have a mixture of blunt and 3'-A ends. Should be much better than common-or-garden Taq alone, but maybe not quite as good as a proofreading enzyme ... ?
I would be inclined to use Vent or Deep-Vent polymerase then blunt-end clone the products. If you're on a budget, use an SmaI-cut / dephos. pUC vector and ligate overnight at 4oC in a rapid ligation buffer (see http://130.15.90.245/rapid_ligation_buffer.htm for recipes / links) to improve your chances. If you can buy/beg/borrow an aliquot of Invitrogen Zero Blunt TOPO vector, then it's even easier - 5min benchtop ligation. Now that I think about it, you can also directionally clone your product in this vector just by adding a 5'-CACC sequence to one of the primers; no need to restrict at all. In our hands these TOPO vectors work like magic, so highly recommended...
good luck!
Del.
Hi,
I am glad I've found this post because I have the same problem.
I want to clone my insert (1,7 Kb) into GFP-N1 vector (so GFP would be in C-terminal position).
In the ATG extreme of my insert there is a suitable RE to clone into GFP, but I've failed twice in creating a reverse primer containing a restricition site that eliminates stop codon.
I designed these reverse primers as follows: about 15-18 bp of my insert, but eliminating stop codon and creating the restriction site os my interest at the same time, and about 10 bp more which were not complementary with the region after stop codon of my insert . I analyze primers to asure this final tag of the primer could not form dimers or hairpins. This final tag of the primer had not the same sequence as GFP (except for the restriction site) because my priority was designing a primer wich wasn't complementary at all with the sequence of my insert after stop codon.
PCR worked relatively well.
I've tried two different primers with two different RE and the problem seems to be that there is no full diggestion of the PCR product althought the restriction site is created (I 've sequenced the PCR product) and the enzymes, in theory, only need 1 bp extra to the recognition site! Ligation with GFP plasmid never worked.
I've never heard this system of mixing two different PCR products...Any advice more on designing this reverse primer and do the PCR?
I've not fully understood how to amplify GFP, and which primers to use... 
hi EG,
I found the paper where fusion pcr is described pretty well http://www.biotwiki.org/twiki/pub/Main/MyP...lerCarr2003.pdf but I don't really have a picture of it in my mind yet. Another good hint seems to be the same method described in another way in this post here http://www.protocol-online.org/forums/inde...9&hl=fusion+pcr . I'll find it out - somehow - perhaps discussing it with my collegues. For me its always good to have it drawn down and I'll get it faster 
In my specific case I want to fuse 3 PCR products and clone these afterwards in a prepared plasmid. Otherwise I could try to do a ligation with 3 inserts and one vector part but I want to use this method as an alternative way.
hope I could help you somehow......
Hi
I would like to thank for advices given in this topic. Namely I have initially tried to fuse my protein of interest to gfp and insert into plasmid by triple ligation procedure. None of approaches was successful. I have find out that my two inserts form homodimers and large concatemers which than do not ligate with plasmid. To solve this i applied fusion PCR with some annealing temp. optimization and joined two inserts. Final product was than digested and successfully ligated into plasmid.
I recommend this technique for such experiments, will use it in future as first choice.
Very appreciated!