In this vector our gene will be under the control of lac promoter, so will it be active in plant system? Anybody have experience working with this vector?
No, the lacZ promoter drives the expression of the lacZ alpha fragment in bacteria, so you can evaluate cloning of fragments into the multiple cloning site using X-gal. Your gene of interest, along with the corresponding promoter (you could use CaMV35S for example) should be cloned into the MCS. pCAMBIA1302 is a reporter vector, allowing the expression of GFP controlled by the 35S promoter, but you could also clone another gene (and promoter) into the multiple cloning site.
Thanks Gustavo. It means i need to clone desirable promoter also along with my gene of interest. I had an impression that this vector is useful for high level gene expression in plants.
I'm not completely sure, but I think CAMBIA vectors don't have a version with the MCS downstream of 35S promoter for over-expression of a gene of interest. You may find many options of over-expression vectors based on the Gateway technology here: http://gateway.psb.ugent.be/
Gustavo i have gone through the website of gateway vectors, can you pls tell me how exactly we can use these vectors (any experience working with the same).
Panchsheela, the system is based on recombination reactions, so you need to include the matching sites in your insert. It is usually done by designing primers for PCR amplification of your sequence of interest, which also include the recombination sites, so the PCR product can be used directly in the reaction. In my experience, the system works quite well, and there is no problem trying to find compatible restriction sites between the vector and your insert. Here you can see a better explanation of the system: http://www.plantphysiol.org/content/145/4/1144.full
Of course you will need a kit for the reaction, which you can get from Invitrogen. Here you can check the manual for the sequences you need to include when designing the primers: http://tools.invitrogen.com/content/sfs/manuals/gatewayman.pdf
Hope it helps.
I'm aware this is an old thread but I just found it and maybe I can share my experience.
I'm using pCAMBIA1302 for the expression of an E. coli gene in microalgae and found that this vector work well for this purpose, the only problem is the CaMV35S promoter expression is not equal in all the species, (in my case it works great in Scendesmus but doesn't work so well in Chlorella).
The Lacz is located in the MCS but its not in frame with any of the 2 promoters it has, so you have to add a complete operon in the MCS or clone directly downstream of any of the two CaMV35S promoters it has, with the consequent consequences that the only way to do it is by either substitution cloning (it looses the GFP or the Hygromicin resistance gene) or by one fragment end ligation with SpeI or NcoI if you wanna keep the GFP (in this case you get a fused protein).
I found out that the only way to clone in this vector directly is by using NcoI and BstEII (you either fused your gene with the GFP if you digest with only one of this enzimes or excise the GFP completely if you double digest).
Hope it helps
Alejandro Gomez Mejia, do you have any info on transforming Scendesmus and Chlorella?
Just saw this thread and that peaked my interest!
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