I want to clone gene fragment into the vector pT1NX in order to intracellularly express it. According to the instructions provided by BCCM/genecorner plasmid collection, for intracellular expression the fragment can be cloned into the unique BglII site. However, in this way of cloning (using one restriction enzyme) there is the risk of the fragment being cloned in a wrong direction (in about 50% of clones). Can anyone experienced with this vector tell me if I can clone my gene fragment (with a functional Ribosome Binding Site or RBS) into the vector using two restriction enzymes BglII and SpeI (which removes usp45 signal sequence and SpaX fragments from the vector) instead of cloning it into the unique BglII site in order to get a intracellular expression and circumvent the risk of cloning in the wrong direction?
Enclosed I have attached the vector map
I have no experience with this vector, but I would use a method that does not rely on digestion/ligation such as Gibson assembly.
Yes, you can clone between the BglII and SpeI site if you are only wanting cytoplasmic expression assuming your fragment has the RBS site on it, which you say it does.
So many thanks for your answer dear Dr. Michael J. Benedik.
Yes, I also think that we can do that by cloning between BglII and SpeI and using these two restriction enzymes seems more logical for intracellular expression but I wonder why the instructions provided by BCCM have not recommended it and instead have stated cloning with one restriction enzyme i.e. BglII. What could the possible reason be?
Thus far I know pT1NX include Nae site complementary to GCCGGC sequence which should be introduced in your forward primer of your insert. By the way you should introduce a TAA stop codon, immediately followed by a restriction site ( BglII is recommended here) this will make a directional cloning finally.Probably the reason that company recommended a certain restriction site is considering the Nae site in the backbone.
Thanks a lot dear Mayam Seifi Kalhor for your interesting comment,
Yes, as you mentioned the vector contains GCCGGC sequence which is a target site for NaeI, but we do not aim to perform a PCR reaction for cloning our gene into pT1NX. Also we are not going to insert it between BglII and NaeI sites. On the other hand, I do not think amplifying a gene fragment already inserted into pT1NX using PCR with a primer containing NaeI restriction site might interfere with its cloning. We just want to clone a synthetic gene sequence including BglII and SpeI restriction sites into the vector and wonder why this way of cloning (i.e. using two different restriction enzymes BglII and SpeI) has not been recommended in the instructions of cloning using this vector.
Dear Mohsen, because this expression vector normally is used by gateway cloning approach by which you should first introduce your insert in an intry clone containing GCCGGC . This is the manual for this cloning defined by the company. However using different methods ( that you are also using) are optional and I dont think it makes any trouble for you.
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