I'm currently working with the expression vector pET32b+ (Novagen). I have been trying the insert a DNA sequence into the vector in sillco, without success. After trying once and again, I downloaded the vector manual and saw that ver a, b and c differ in the MCS, as shown below.
http://www.helmholtz...T-32a-c_map.pdf
As shown in the manual, ver b has one less letter in the MCS. This in turn is changing the ORF entirely and is causing my insert not to have the His-Tag at the end.
I'm using RE Nco I and Sal I.
Do I take the reading frame as lined up against a ?? or do I take the Nco I site as indicated in the manual (CCATGGC)?? The normal RE Nco I is CCATGG
Can someone help?
Hi there,
The issue is not the NcoI site as it is exactly in the same frame in the three plasmids (cc ATG g) but how SalI site is arranged towards yfg reading frame and the downstream his tag reading frame. Basically the cloning can be done in any of the three plasmid but each of them would require a specific reverse primer (defining the common reading frame for the orf and the his tag sequence). For instance with pET32b, SalI sequence is in frame with his tag downstream sequence which means the cloned orf has to be in frame with SalI site (GTC GAC). For pET32a the frame in SalI has to be xxG TCG ACx and for pET32c xGT CGA Cxx.
So basically...I have to add the correct primers to my DNA segment so it lines up with the ORF of the vector? I can choose what ever RE in the MCS??
My fragment is
GCTCAGCACTACTCTGTTGCCTGGTCTTCCTGGCTGGGGTGGCAGCCAGCCGAGATGCGAGCACCCTGTCTGACAGCAGCTGTATCCACTTGCCAACCAGCCTGCCCCACATGCTGCGGGAGCTCCGAGCTGCCTTCGGCGAGGCGAAGACTTTCTTTCAAATGAAGGACCAACTGCACAGCTTACTGTTGACCCAGTCTCTGCTGGATGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCGGAAATGATCCAGTTTTACCTGGAAGAGGTGATGCCACAGGCTGAGAACCACGGGCCTGACATCAAGGAGCACGTGAACTCACTGGGGGAGAAGCTGAAGACCCTGCGGCTGCGGCTGCGGCGCTGTCATCGCTTTCTGCCCTGCGAAAACAAGAGCAAGGCGGTGGAGAAGGTGAAGAGAGTCTTCAGTGAGCTCCAAGAGAGGG
In that segment, I want to express the gene
ATGAAGGACCAACTGCACAGCTTACTGTTGACCCAGTCTCTGCTGGATGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCGGAAATGATCCAGTTTTACCTGGAAGAGGTGATGCCACAGGCTGAGAACCACGGGCCTGACATCAAGGAGCACGTGAACTCACTGGGGGAGAAGCTGAAGACCCTGCGGCTGCGGCTGCGGCGCTGTCATCGCTTTCTGCCCTGCGAAAACAAGAGCAAGGCGGTGGAGAAGGTGAAGAGAGTCTTCAGTGAGCTCCAAGAGAGGG
I'm using the pET32b+ vector
Hi again,
The principle of MCS is that you can use any of them but if they are in an open reading frame you have to clone so that this reading frame is respected. In your case, SalI site is in frame with the downstream Histag sequence in pET32b so it's pretty easy to design the primers:
For the NcoI primer you will have to add a codon starting with G right after ATG because NcoI cutted sequence is CCATGG and if ATG is defining the frame the next codon will be GXX (which is not the case in your gene). Usually Glycine (GGX) or alanine (GCX) encoding codon is added. So for your gene I would go for NNNCCATGGXX(ATG)AAGGACCAACTG...
For the SalI primer I would go for NNNGTCGAC +sequence complementary to end of your gene (avoiding stop codon). I noticed there is no stop codon at the end of your gene and if using ATG coding frame there is an extra G (after AGG final codon) at the end of the sequence you gave so be careful (if you have cloned exactly this sequence in pET32b, this extra G is shifting the frame right at the end of the insert and therefore no Histag can be associated to your encoded protein...) .
Good luck.
There is no end codon at the end of my gene because I was expecting to insert the gene into the MCS so that it fuses with the His Tag. If you notice, the end of the His Tag has a stop codon.
If I cut from, let's say BamHI and HindII from the vector, and I shift the frame (cut out on A from the HindIII site)
And I insert the entire insert (with the gene in the middle). I get the exact result I'm looking for, with the His Tags at the end:
MLRELRAAFGEAKTFFQMKDQLHSLLLTQSLLDDFKGYLGCQALSEMIQFYLEEVMPQAENHGPDIKEHVNSLGEKLKTLRLRLRRCHRFLPCENKSKAVEKVKRVFSELQERELAAALEHHHHHH
(ATGAAGGACCAACTGCACAGCTTACTGTTGACCCAGTCTCTGCTGGATGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCGGAAATGATCCAGTTTTACCTGGAAGAGGTGATGCCACAGGCTGAGAACCACGGGCCTGACATCAAGGAGCACGTGAACTCACTGGGGGAGAAGCTGAAGACCCTGCGGCTGCGGCTGCGGCGCTGTCATCGCTTTCTGCCCTGCGAAAACAAGAGCAAGGCGGTGGAGAAGGTGAAGAGAGTCTTCAGTGAGCTCCAAGAGAGGGAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCAC).
The only thing that is mis-aligning my protein is that extra A in the pET32b+ vector. The frameshift for the version b, shifts the ORF.
I added the GCX sequence to my primer as indicated...however, I still don't get the protein I mentioned above.
I've been at this for a while and at this point, I'm pretty frustrated.
So what is your cloning strategy at the end? You first said about NcoI/SalI and now you are into BamHI/HindIII... Anyway, I don't understand what you are doing with the HindIII site (cutting off one A... Why? How to get sure to get only one base off? ). As I already told you the DNA sequence you intend to clone contains an extra G at its 3' end (if translating the whole sequence into protein respecting the ATG frame will give a final Arginine which is encoded by AGG codon which means the third G of the GGG triplet stands for nothing). So if using HindIII site which is in frame with 6His sequence in pET32b, the sequence at the cloning junction should be CCAAGAGAGGAAGCTTGCGGCCGC... So incuding HindIII cutting site in frame with the gene sequence in your reverse primer should be fine.
Mr. Dominique...you are absolutely right. When I remove the G on the 3', the gene is in frame. I appreciate your patience and your help.
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