[DIYbio] Re: Curious about creating glowing plants

Hey there,

Thanks for even more informative insight. I read the paper, and I knew about this ahead of time because of my professor who told me about it. It took a few days for my post to be approved and posted on here, so unfortunately I was unable to update it.


I'm starting to think I should just wait and take a few more bio classes before I start meddling with this stuff. I understand most of what I am doing, but I dont understand HOW I do it.

The 35S promoter is here https://www.addgene.org/12588/ It was recommended to me by my biology lab professor because it will express the gene, at the expense of the plants lifespan.

That gameplan was more of me just trying to get my unorganized thoughts down on paper, I was planning on making a total step by step to the dot manual.

As for the plasmid sequencing, I was hoping I could find an already shrunken Ti Plasmid somewhere, and cut the gene into the plasmid myself.

Anyways, here are my last few questions:

How would I cut between the lux genes without restriction sites being there?

How can I stick two dna strands cut with different enzymes together?

As you can tell, I'm just fumbling around, but I have learned an amazing amount by just hypothesizing about theoretical glowing plants.




On Wednesday, April 29, 2015 at 6:38:16 AM UTC-7, Koeng wrote:

The main issue is is that the lux operon *will not* be expressed in plants under these conditions. Eukaryotes do not use polycistronic mRNA molecules (there are exceptions I know but nearly all genes). To see what would be required for proper lux operon expression, check out the paper I linked. They describe using 4 different promoter sets to express the lux proteins, which is probably the amount you would require. I'd recommend trying this with bacteria first to see how finicky biology is first; you'll be able to get a wonderful experience without putting too much out. 

Now onto some other issues. You do not have to cut out genes to PCR them, you can PCR them directly off plasmids. Next, your primers anneal to the inside of luxC and the outside of the EM7 promoter, ampilfying 241 base pairs. Also, what PCR mastermix will you be using? This matters a lot. Whether it be Q5, phusion, taq, Accura, or pfu, this is something you'd need to plan. Taq, for example, is extremely error prone and adds A to the end of amplified molecules (useful for TA cloning though). The lux cassette also would not have an EcoRI site after the entire cassette, so it would not clone properly. I don't see much info about your promoter choice either: the sequence of the plasmid isn't even defined at addgene. Does it have a polyA tail? What's its copy number in E coli? Where does the 35S promoter come from? What sequencing company will you use for your plasmid?

All-in-all, before making a game plan on how to do it, make a very detailed plan on exactly why you do each of the steps and identify what is required for expression. For example, check exactly why putting the entire lux operon from bacteria into a eukaryote doesn't work. (paper is linked in last message)

-Koeng

On Wednesday, April 29, 2015 at 5:16:08 AM UTC-7, Bijan wrote:

Thanks for the informative reply.

I've started to piece together a basic game plan, but I am actually going to work with more simple stuff first to get the hang of what I'm doing.

Here is the basic plan I made last night

Methods:

1. First, the luxABCDE gene will be isolated from the plasmid pGEN-luxCDABE via restriction enzyme PmeI + SacI, which will cut at the beginning of the luxA gene, and end after the terminator at luxE.

2. The isolated lux genes will be amplified with PCR, the required primers (CATATGAAGCTTGGTACCGGGATC)5' and (CTTTCGGGAAAGATTTCAACCTGG)3' will be obtained and added to the master solution

3. After amplification, the DNA will then be inserted into another plasmid (pBabe puro H-Ras 12V (35S)). The 35S mutation was chosen as it is a very strong promoter, and the gene is more likely to be expressed. To insert, the DNA will be cut using the restriction enzyme EcoRI which will make a cut at the end of the promoter. The lux gene will then be inserted.

4. The promoter plasmid will be transformed into e. Coli and multiplied.  It will then be extracted.

5. Agrobacterium tumefaciens will be used as the insertion vector for the lux gene into the plant. The plasmid will be integrated with the Agrobacterium at the tsite using electroporation and the bacteria will be cultured on petridishes containing Puromycin, as it is the marker for our plasmid.

6. Using the protocol outlined here: http://www.plantpath.wisc.edu /fac/afb/protocols arobitosis will be grown and infected by the agrobacterium.

https://www.addgene.org/12588/

https://www.addgene.org/44918/

Enter code here...

On Monday, April 27, 2015 at 2:52:22 PM UTC-7, Koeng wrote:

Large biosynthesis operons are usually very difficult to clone. Largely the reason is is that the fundamental methods of gene expression are different in each organism, making it pretty hard to explore their biology without high-throughput library methods to get favorable or native expression rates of each said protein. If you check out the glowing plant kickstarter, you'll notice that they haven't even completed their glowing plant. 

A few questions to consider:

How does gene expression differ between eukaryotes and prokaryotes?

What are the location differences between eukaryotes and prokaryotes and why do those matter?

What is an IRES?

What's the difference between a kozak sequence and a RBS sequence?

How do you transform plants?

Why does the lux cassette work well in E coli and not yeast?

However, after all, it is biology, so with a little more planning it technically *is* possible. Check out this paper for more details- http://femsyr.oxfordjournals.org/content/4/3/305.long . It's fairly good and goes over some issues they've had with yeast, a much easier organism to work with. 

Glowing plants have been criticized quite a bit in this community, mostly because so far (without chloroplast integration, another topic) it doesn't seem to work very well. Anyway, you could check out some of the kits from the-odin, http://www.the-odin.com/bioluminescent-e-coli-kit/ , which from my experience (with the plasmid) seem to work very well. 

Good luck!

-Koeng

On Monday, April 27, 2015 at 6:13:19 AM UTC-7, Bijan wrote:

First off, I've only taken freshman biology, I have no idea what I am doing at a professional level.

Alright, I think I have an idea for making glowing plants. I was thinking I could use this -> http://en.wikipedia.org/wiki/Agrobacterium_tumefaciens#Beneficial_uses With a 35S promoter (Which I can illegally get with some GMO food if I can find the right primers). Ill take the promotor and the Lux cassete from somewhere and insert it into the agro t-area which will inject it into the plant DNA.

What I don't know is

1.) How to get the 35S promoter primers
2.) How to stick the promotor on the luxABCD gene.
3.) How to isolate the t-area and integrate it into the phage cell.

 I just put this together myself. But it seems possible right?