[DIYbio] Re: Looking for explination (layman's terms)

Hi!

Here has been some discussion,
e.g.


https://groups.google.com/forum/?fromgroups=#!topic/diybio/MhOcaOT09lA
https://groups.google.com/forum/?fromgroups#!searchin/diybio/bioluminescent$20plant/diybio/jqyBqlbC2VM/QSpRRjbp_7YJ

The glowing plant project from Genome Compiler is going on at the moment, http://tmblr.co/ZgwW5uwmy4eF
and I'm also cutting together a lux plasmid for chloroplast expression at the same time.
At the moment I'm starting to get a new promoter synthesized (a hybrid promoting element for extended strenght out of the literature). Then ligate my Lux PCR and my kanamycin resistance PCR into it.

I can share my pGlowroplast plasmid with you when it's ready. My construct will be in pGreenII-0000, so agrobacterium shoots it into chloroplasts. People are not sure whether chloroplasts can be transfected with agrobacterium. Some say it's a hoax. I'll just try it.
->You would have to get a chloroplast integration plasmid (I didn't succed, asked a few authors of papers), then you could easily ligate my fragment (which contains all the elements to make chloroplasts glow) into it. (You're familiar with restriction digestion and ligation ?)


I can also share pVIB plasmid (google it ;) ) with you. It is from Carolina biological supply and contains the LuxR and LuxICDEABG operon. Lux quorum sensing doesn't work in chloroplast (found a German paper), so you would need to insert a promoter before Lux CDABEG. Lux I and R are regulation elements, more or less.














On Thursday, October 17, 2013 12:48:59 PM UTC+2, T.J. wrote:

In another post that I wrote, "Creating Luminous Plants", I was told of an experiment that takes a pcr machine and uses a chloroplast promoter containing P.Phosphoreum.  Here's his exact message :

"What already has been done: Make a PCR of  P.Phosphoreum (a glowing marine bacterium) which cut out the Lux Genes (they are very next to each other, so one PCR using 2 primers was enough), add a chloroplast-promoter and insert it into chloroplasts. They are able to express operons, and there we go. It was glowing (weakly, but visible) in fact.

See http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015461 "

Can I buy/make some of this stuff to try it at home?  I notice a few PCR machines on Ebay from time to time for a couple hundred.  Where can I buy these photobacterium?  Can I experiment with luciferase and other stuff as well?  What is a chloroplast-promoter and how can I insert it into the chloroplast of an organism?  I'm still interested in possibly doing this project. 


Past topic thread.
https://groups.google.com/forum/#!msg/diybio/FpIy91GmyM8/dCjqhMegpxgJ