Looking for explination (layman's terms)

[T.J.]

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

[CodonAUG]

Promoters = Short stretch of DNA that cell machinery(proteins) recognizes and attach to start the transcription (DNA to RNA) of the gene.  Genes often have promoters that are unique to them or shared with a couple other genes.  

It is sort of like an on/off switch.  If the switch is gone, the gene won't work.  If the switch is there but off, it won't work.  If the switch is there and on, the gene works.

Organisms change the combinations of genes that are turned on and off in a cell depending on its type (ex: heart vs. liver, stem vs. root).  By using the chloroplast promoter to control the Lux genes, only cells that have the chloroplast gene activated will glow.

If you are not familiar with the techniques, I would recommend buying a molecular biology lab book on Amazon (used should be around 20).  It'll walk you through all of the molecular biology techniques you will need -- from cloning to PCR.

[Mega [Andreas Stuermer]]

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.

[Mega [Andreas Stuermer]]

http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-61779-558-9_35

Sometimes in a paper, the authors email is given. You could then email them and kindly ask for a plasmid. They will ask you for your university affiliation. Also make sure that you offer them a fee for handling and shipment and for their efforts.

On the other hand, if you can do PCR, you could also just amplify a chloroplast DNA fragment (see in the literature, there was one that naturally contains a MunI restriction site).


Btw, my hybrid promoter will also express in E.Coli, so at least you get glowing bugs :D But could have done that with pVIB too, just cheaper :D