Re: [DIYbio] Re: Translating bacterial DNA into plant DNA

Dne neděle, 30. září 2012 19:08:48 UTC+2 xmort napsal(a):

There is no selection process involved, just statistics. In these transient assays you typically use quite large amounts of agro. With such an overkill pretty much every mesophyl cell which can be transformed really is  transformed. Considerable proportion of these cells get transformed with multiple constructs, quite many with all of them. And you usually dont need the whole leaf or plant to express your genes, several hundred cells per leaf will usually be sufficient. You only need some  suitable assay to detect the positive cells, which in your case should be quite easy due to luminiscence. You might need some sort of microscope though. May be also a digital camera with proper filter set and sufficiently long exposure will do as well.   This is just an quick an dirty method to check, that you have all genes necessary for a particular function and that all of them work in concert in plant environment. Once you are sure about this, you might start putting them all in the plant at once. 

There is one more point to think about - plant cell is quite a lot more complicated than bacterial cell. It might be that some of the metabollites needed for the pathway are in different cell compartments, than you might need to try different sorting signals  on some of your  proteins. Say that gene A might need to be tested in cytoplasm or mitochondria, gene B cytoplasm, ER, golgi or mitochondria and gene C cytoplasm or chloroplast.  It can get to quite a large number of possible combinations relatively easily (16 in above mentioned example).  It is relatively easy to mix different agrobacteria and then choose the best combination as opposed to prepare 16 different megaconstructs.