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

re:

Sounds like it's no big deal for the lux genes, when they're all constituvely on.

It is a big deal actually. These so called "aberrant transcripts" are potent elicitors of gene silencing. So the leaky transcription could potentially lead to switching off  both genes. 

From the above suggestions I find the 2A peptide idea the most likely to work. The only drawback is that you will end up with equimolar amount of proteins from each individual transcript, which might or might not be suitable for proper function/regulation. Also the  short 2A peptide will remain attached to C-terminus of the protein , which might affect its function. 

I dont know what is your ultimate goal with the bacterial luc operon  but I would suggest fast and dirty method  which we use in our lab to test expression if mutliple genes in concert  -

1. you make several binary plasmid each expressing one of the genes. that is some cloning, however most of these can be usually done in one step directly from PCR product. 

2. transform  every binary plasmid to agro , so you will end up with six or so individual agro strains.

3. grow all agros in separate tubes

4. measure optical density , mix them all together so that you will have equal (or as desired) amount of each agro in the mix 

5. agroinfitrate the leaves of N. benthamiana, usually we start with suspension of about OD600=2

This way you can easily add / subtract genes t your  mix and to some extent to even modify the ratios of different genes in the mix by simply using more agro. 

Good luck

Tomas

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