My friend the genetics guy has a response;
So I work in a genetics lab and I've got a few questions/comments about this... not trying to be a downer, but here we go:
1. plants don't have plasmids, so you can't just mix E. coli plasmids with plant plasmids, because one doesn't exist
can be found in all three major kingdoms, Archea, Bacteria and Eukaryote" - Wikipedia
(There was a typo back a few posts where I mention plant plasmids, but I ment to say protoplasts...
but you are correct, a plasmid in a plant cell wouldn't be expressed,
so it wouldn't work.)
2. That't not how you engineer a plant. With that size of a piece of DNA you would probably want to build an artificial chromosome. So you're not going to cross bacteria with anything really.
The bacteria is just the vector. I don't intend to cross bacteria genes with anything...
The protocol I plan to used is from here...Plant Tissue Culture Concepts and Laboratory Exercises Second Edition
If that doesn't work. I'll use the same E Coli cultures
and remove the DNA with a plasmid kit and transfect with something like QIAconnect
But I'll try the other method first, as I have the materials already.
I know its not the most effective method, but it does work or could
There are two protocols I'm going to try. One is E Coli carrying target plasmids
mixed with African Violet protoplasts (almost said plasmids again) and restriction enzymes and heat shocked.
The other protocol is basically, take the DNA from the E Coli, add it to the African Violet protoplasts,
15 minutes in a 42 degree sonic water bath, and plate.
Then cross your fingers and hope some DNA giggled in...
But if those don't work, and they may not, I can always fall back on a transfection kit.
3. you can't just smack a promoter onto one end of the DNA segment with so many genes running off of it. You're treating it like an operon, but that's more a bacteria thing.
While this appears to be the case, I've seen research that indicates otherwise.
Maybe I am thinking of bacteria, but I could swear the paper I saw on it was Eukaryotes.
It discussed controlling expression not only at the DNA level, but at the transcription and translation phases.
This is the promoter sequence
I'm going to use.
And you're not considering that you don't know what proteins are involved in protein folding. So you could move over all the genes sucessfully and you still might not get anything. But if you try to put in everything as an operon like that you're not going to get anything probably.
Maybe, but maybe not...
4. With DNA segments that large you're probably not going to want to use PCR to amplify the DNA, except for the small fragments.
What would you suggest then? I'm using PCR to isolate and assemble the target DNA,
the E Coli will amplify the DNA I use to transfect the African Violet protoplasts.
5. Designing primers, I'm not even sure what you're talking about because the hardest thing about designing primers is for one knowing where the gene ends and begins, which the bigger problem is knowing where the promoter ends and the gene begins, and the second is is your promoter sequence so common in the chromosome that you're going to accidently amplify other DNA segments that you don't want to?
Well, we're even here, because I don't know what you're saying either.
But you forgot you also have to make sure the primers don't hair-pin or self-dimer...
I know my promoter sequence and the gene sequences.
I can't really use primer-BLAST, but I can use gels
to make sure any unwanted fragments are left behind.
Your desire seems strong enough, but I'm afraid your knowledge of genetics isn't good enough to ever really do this.
Here we disagree again. My knowledge isn't good enough to ever
really do this?
This is my first attempt. It may fail, but I will learn from it and try again.
But won't your "genetics" buddy look like a retard if I do succeed? I've only started a few months ago.
None of this is really that out of the box, especially transfecting African Violets.
All the tools and information is out there.
You're talking about doing something very complex here, and you're talking about moving over multiple genes and you don't even really know how they work or how they are produced, folded, etc, and I don't think you realize just how impossible it is going to be for you to do this.
I understand its complex, but its not impossible.
If I have to step up my project to the next level, when PCR and Gels are no longer enough, I will.
I've already begun to looking into setting up a front company to get the FDA certification
to get the equipment I might need. I can get a used DNA sequencer on eBay for 2-5 grand...