[DIYbio] Re: *experienced* beginner is creating a bioluminescent plant - both synbio and old-fashioned way

Hi everyone. You may remember the old thread with a similar name, *some* time ago :D

https://groups.google.com/forum/?fromgroups=#!topic/diybio/MhOcaOT09lA

 

 

There's news.

 

As you surely have heard, Genome Compiler is launching a kickstarter- crowdfunding campaign (very soon), to raise money and have DNA synthesized that will be put into plant (synthetic constructs for both nucleus / chloroplast) and makes it glow in the dark. (Everyone can place designs and the best ones will be elected by a comitee and synthesized. See http://www.glowingplant.com/ for additional info). Gonna post the link to kickstarter as soon as it is online. You gotta look at their video - it's awesome!

 

We at the Ars Electronica Biolab will be under the lucky ones who will get the synDNA then. Also BioCurious and other interested labs get the DNA.

 

 

 

Additionally, we got the idea to use the meantime to do an old-fashioned, albeit  long-winded, approach: Do a PCR of a chloroplast promoter, of RBS + Lux operon, of Kanamycin resistance, of a terminator and clone it into pGreenII. I can/will also post the sequences, however if one wants to make an *awesome* glowing  plant, getting the DNA from Genome Compiler would be more advisable...(The primers alone we'll order cost more than 50 Euros, while one gets glowing plant seeds as a gift for donating 40$ to the kickstarter campaign.)

We would put that insert into pGreenII, pGreenII into Agrobacterium (containing also pSoup), and place them on a plant leave piece -> regenerate a plantlet out of it on selective medium.

According to this http://www.ncbi.nlm.nih.gov/pmc/articles/PMC554353/  paper, Chloroplast transformation by Agrobacterium works. However, there is not a single other paper having done that a second time.

Now we are curious to see, if that is really true.

 

 

 

 

Why would we do an additional hard-work, old fashioned restriction-ligation approach while also getting the synthetic DNA, which - by the way - will also glow much stronger, due to better promoters, optimal 5' and 3' UTR, due to genes from other species enhancing light output e.g. FMNH availability, antenna proteins enhancing light output dramatically by shifting the light into less-energetic wavelengths (less-energetic wavelength -> more photons, better visible), .... ?

 

I guess the simple answer is curiosity. And is there a better waste of time than ligate together DNA pieces for a glowing plant (also when just weakly glowing)? :D See if we can do the restriction-ligation of four PCR products, select for transformants the "annoying" way by X-Gal. Then see if the agrobacterium-plastid transformation paper is true (But why should De Block et al. lie to us? :D ). And if that works,  whether the transplastomic plants will glow strong enough to be seen with naked eye in the darkness after all...

(Here was also mentioned Dr. Krichevski once, who inserted a lux-operon into chloroplasts, which was/is visible to the naked eye, but only after 5 minutes of eye-adaption)

 

But, If the synthetic DNA arrives before we got the old-fashioned approach ready, it will be paused, and perhaps/probably even cancelled.

You see, the synthetic DNA is even more exciting, because it is designed to be worlds brighter, especially because you don't have limits when designing, don't need to design extra primers for each gene, get a template, …  Last but not least: no error-prone restriction work

 

So, in best case, we'll have two plants, one labeled "traditional genetic engineering" , weakly blue glowing and one "synthetic DNA" – which will hopefully be very bright yellow or green or red, well be seen without eye-adaption. Hopefully bright enough to read a book when laying under it - but I'm positive ;)

 

 

Maybe (probably :D ) there will be some detail-related questions… ;) Like how to get the chloroplast DNA from a leave, does it need clean-up before PCR, etc. :)

We thought of just mechanically damaging a leave piece and then 90°C for 20 minutes... 

Best,

Andreas