Nowadays, I am studying a gene's function using RNAi and Overexpression. The plasmids used were Gateway-based (See Figures attached). The target gene was PCR-cloned with attB site containing primers. And the BP reaction was successfully finished. The pDNOR221::gene plasmid was screened out using kanamycin plate due to the Kana resistance gene in pDNOR221. The positive clone was plasmid extracted, which was then LR reacted with the destination plasmid showed in the pictures. But, I don't know which antibiotic should be used this time. The Streptomycin, chloramphenicol or Kana? And what are the functions of these genes? Thank you so much!!
According to your map, screening of bacterial cells containing your construct would be streptomycin/spectinomycin. Since Kanamycin resistance gene is within the T-DNA borders, that would be the resistance marker for transgenic plants. In fact the K in the name of the plasmid refers to the selection in plants (for this system of vectors pK is for for kanamycin, pH for hygromycin, pB for bar gene, and so on). Chloramphenicol is used for selection in the propagation of gateway vectors in the DB3.1 E. coli strain.
Thank you, Gustavo Acevedo-Hernandez. As you said, Kana was designed for resistance marker for transgenic plants, but how this gene express in plant without a promoter sequence? And if my gene replaced the ccdB fragment, Chloramphenicol resistant gene could also give this character to the host strain (like DH5alpha), right?
Kanamycin resistance gene does have a promoter, it is just not shown in the map. If you analyze the sequence (http://gateway.psb.ugent.be/file/download/363) you will find it is the NOS promoter. You could use chloramplenicol for bacterial selection if the selectable marker was not eliminated after recombination, provided that the host strain used (for example DH5-alpha) is sensitive to the antibiotic.
Thank you so much, Gustavo Acevedo-Hernandez! Yes, after the alignment analysis, the NOS promoter was observed. I now completely know this vector!
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