Hello, I am planning to insert GOI between CaMV 35 promoter and mGFP in pCambia1302 to produce a fusion protein and see subcellular localization of the protein.
But the vector says it's membrane bound GFP so I wonder if it would affect my protein's localization with in the cell.
Hi Hyunbae
Any tag protein can affect the localization of a protein, given the size of your protein of interest and its biochemical features. If, the vector clearly states that the mGFP is going to localize to membranes then so will the protein you will tag with this mGFP.
Also, the pCambia 1302 vector does not have a MCS site for gene insertion between the CaMV 35s promoter and mGFP5. This plasmid system is a reporter system and not a tagging system, just so you know. Of course if you have the enzymes NcoI and BglIII, you can try cloning in this site. be aware of frame sifts though.
I tried reading about mGFP5 and it does not say that it is targeted to membranes though. Its only said that its a modified GFP enabling expression in plant cells. Nonetheless, avoid any tag that has a pre-disposition to localize at a specific part in a cell to study sub-cellular location of another protein of interest.
Hope this helps
Aparajita
Aparajita Lahree Thank you for your answer! I think it's not membrane bound. Like you said, m means modified. I was confused!
I think NcoI site contains start codon of mGFP5.
We don't have BgIIII and SpeI so I was thinking to cut the whole GFP with NcoI and BstIII. Then I separately produce GOI fragment and GFP fragment and ligate them by overlap extension PCR or T4 ligation.
Maybe I need to look for another vector but it really is the only choice I can use for now :0...
Hi Hyunbae Lee,
If you are going to place your GOI before the mGFP, the transit peptide from your GOI will direct the GFP to wherever it goes inside the cell.
In order to make the cloning easier, I would suggest you to use NcoI site to clone your GOI by using a method called ELIC that allows you to insert your fragment in sense cutting with only one enzyme. (Find attached the paper) In our lab we use DH5a strain and we also realized that homologous sequences around 15bp to the vector works perfectly.
Best
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