Need some help starting a project (Agrobacteria-mediated transformation of a dicot via floral dip)

[antomicblitz]

I'm currently an undergrad studying molecular biology, and I recently joined a community lab after taking biochemistry lab at my school. In my class, we transformed a colony of E. coli using egfp and I thought it was really cool, and I've kind of been obsessed with trying it with other organisms as well, so I wanted to see if maybe I could do the same thing with a plant. After spending a few days at the library and searching through a couple of scientific journals, I found that I could do a similar procedure with agrobacteria, which can use a disarmed tumor-inducing plasmid to inject a segment of tDNA from another binary plasmid into a plant genome (I'm still not 100% sure how that works or where in the genome it gets incorporated, but I know it works at least). I would like to try and put egfp into a plant and have it expressed at detectable levels. I've had people tell me it's not too difficult to do. If any of you have done it before, or if you know what is involved, could you possibly give me some pointers on how/where I can order the appropriate plasmids, and which strain of agrobacteria I can use? I found a couple of protocols, so I have a general idea of what I'm gonna do (floral dip), but I'm still a bit iffy on how to select the right plasmids and bacteria, and where exactly on the binary plasmid I should ligate my gene of interest. Any help is much appreciated :).

[Sebastian S Cocioba]

I use agro strain GV3101 pmk90. You could probably have it shipped to you if you write to Stan Gelvin's lab over at perdue. My go to transformation plasmid is pPZP-200 series that confers kanamycin resistance as a selection marker. Floral dip sucks in my oppinion and youll be limited to arabidopsis only. Most important part of plant genetic engineering is tissue culture. You can build a laminar hood for cheap using a hepa air purifier, giant clear plastic storage bin, and some ducting. That way you could readily transform any plant (YMMV).

Just put a plant promoter like corn ubiquitin or CaMV in the mcs, them your egfp, then a terminator like the nopaline synthase one already in your plasmid downstream in the selection marker region. Then transform agro with the plasmid. Strain gv3101 is aggressive but a little hard to get rid of. It has genomic gentamycin resistance and spectinomycin in the plasmid if you are using pzp. I can send u my plasmid if you cant get it elsewhere. I transform tobacco on a daily basis and its my goto model organism. I can also send you seeds if you want to join the cult of nicotiana. Ive made some fluoroplasmids to shoot with my gene gun and results were fair. I use non optimized cometgfp and could never get my hands on egfp. Ill trade you my agro vector for yours with egfp. 😃 also feel free to ask whatever you want. I like helping plant newbies. I post all my lab stuff on fb and twitter. More than welcome to follow if you'd like. Good luck!


-Sebastian
Sent from my iPad

On Nov 27, 2014, at 6:03 AM, antomicblitz <aml...@ucsc.edu> wrote:

I'm currently an undergrad studying molecular biology, and I recently joined a community lab after taking biochemistry lab at my school. In my class, we transformed a colony of E. coli using egfp and I thought it was really cool, and I've kind of been obsessed with trying it with other organisms as well, so I wanted to see if maybe I could do the same thing with a plant. After spending a few days at the library and searching through a couple of scientific journals, I found that I could do a similar procedure with agrobacteria, which can use a disarmed tumor-inducing plasmid to inject a segment of tDNA from another binary plasmid into a plant genome (I'm still not 100% sure how that works or where in the genome it gets incorporated, but I know it works at least). I would like to try and put egfp into a plant and have it expressed at detectable levels. I've had people tell me it's not too difficult to do. If any of you have done it before, or if you know what is involved, could you possibly give me some pointers on how/where I can order the appropriate plasmids, and which strain of agrobacteria I can use? I found a couple of protocols, so I have a general idea of what I'm gonna do (floral dip), but I'm still a bit iffy on how to select the right plasmids and bacteria, and where exactly on the binary plasmid I should ligate my gene of interest. Any help is much appreciated :).

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[Sebastian S Cocioba]

Sorry forgot to add that agro inserts into the genome randomly and can do so multiple times. The binary vector has a t-dna region delineated with a left and right border motif. You will see articles citing circuits that read like LB-MCS-Pnos-kanR-Tnos-RB. Your gene of interest along with appropriate promoter and terminator goes in the mcs like any ecoli plasmid. The tdna region always has those borders and a preassembled circuit for expressing a selection marker typically an herbicide resistance gene. That way you can kill off any non-transformed tissue and regenerate from only the transformed cells. If you are doing a transformation experiment you may want to regenerate a few plants to account for multiple insertions which may or may not get silence in the next generation. Multiple inserts can be considered as viral load and it will get cut out altogether. To check for insert count, grow transgenic plant until it makes seeds, then plate seeds in vitro onto media containing the selection herbicide and allow to germinate. You'd want around 50 seeds Per plate. 

Once the seeds sprout, you'll notice some didnt germinate, some are happy and green, and if you are lucky, some are pale or white. If all germinated seeds are green, you have two or more inserts. You basically want a mendelian segregation of 75% resistant and 25% susceptible. At that point you know its just one insert.

 
Sent from my iPad

On Nov 27, 2014, at 6:03 AM, antomicblitz <aml...@ucsc.edu> wrote:

I'm currently an undergrad studying molecular biology, and I recently joined a community lab after taking biochemistry lab at my school. In my class, we transformed a colony of E. coli using egfp and I thought it was really cool, and I've kind of been obsessed with trying it with other organisms as well, so I wanted to see if maybe I could do the same thing with a plant. After spending a few days at the library and searching through a couple of scientific journals, I found that I could do a similar procedure with agrobacteria, which can use a disarmed tumor-inducing plasmid to inject a segment of tDNA from another binary plasmid into a plant genome (I'm still not 100% sure how that works or where in the genome it gets incorporated, but I know it works at least). I would like to try and put egfp into a plant and have it expressed at detectable levels. I've had people tell me it's not too difficult to do. If any of you have done it before, or if you know what is involved, could you possibly give me some pointers on how/where I can order the appropriate plasmids, and which strain of agrobacteria I can use? I found a couple of protocols, so I have a general idea of what I'm gonna do (floral dip), but I'm still a bit iffy on how to select the right plasmids and bacteria, and where exactly on the binary plasmid I should ligate my gene of interest. Any help is much appreciated :).

[antomicblitz]

Wow! Than you sebastian! That was some incredible information! I will try to do this next weekend. With any luck, I'll have some glowing plants 😊. I would love to get in touch with you and follow you as well, since I am just starting to learn this stuff and I think it's really fun :).

[Mega [Andreas Stuermer]]

Hi! AFAIK there already are pCambia Vectors which already contain EGFP unter a plant promoter. So ready to use for your floral dip. 

It may be best to try floral dip for you, because it yields quick results without needing tissue culture.

[Mega [Andreas Stuermer]]

http://www.markergene.com/Categories/plants.php 

e.g. this one 

M1593	pCambia1302 Plant Expression Vector		20 μg	$114.45	114.45
Vector contains kanamycin resistance gene for bacterial selection, hygromycin B resistance gene for plant selection and the mgfp5 reporter gene

[Mega [Andreas Stuermer]]

Ah I forgot to mention, you can also try a transient transformation. Just streak the leaves with the agobacterium solution, and they will get the gene inserted. However, the leaves get brown after x weeks, and die off. So the whole plant is not a transgene, it's a mosaic. Still nice to try with GFP. For some plants you cann add vanillin to agrobacterium to enhance the aggressivity (it acts as a wound signal).