User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/05/13

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χρόνος πέρασμα May 13th 2011

 * I searched for the genetic background of the Phaseolus vulgaris mutant R32-BS, allegedly an excellent candidate in which to test our system, and found several incoveniences with this organism:

**Apparently, the canadian group that created the mutant had already somehow characterize the gene responsible for the phenotype (increment of nudule number). The gene is called nts. The group also made two other mutants, one plant that produces inactive nodules and other that does not even nodulate. Mixing these plant variants produced some plants that had the wild-type phenotype, showing some sort of genetic complementation. ("Complementation of Nodulation Genes of Various Mutants in Common Bean (Phaseolus vulgaris L)", S. J. Park and B. R. Buttery, The Journal of Herecfity 1997:88(6)).

**The plant-rhizobia symbiosis is severly affected, as the nitrogen fixation activity is diminished, the nodule size reduces as the number of nodules increases, the ratio of active/inactive nodules is been dramatically reduced in this mutant (this means a greater proportion of the nodules aren't functioning properly); it has been hypothesized the reason for this is that the plant is incapable of delivering the necessary amount of photosynthates to all the nodules, delivering these limited compounds equitatively to all the nodules, and so making most (if not all) of the nodules non-functional as any recieves the proper photosynthate concentration. (DOI: 10.1111/j.1469-8137.1992.tb00088.x)

I propose we left aside the use of the mutant R32-BS. It was first proposed to use it in the project as it was inferred that as the number of nodules was greater, the nitrogen fixation should be greater and thus the hydrogen production. But as it has been said, the number of nodules is indeed bigger, but many of the nodules are not-operational; and they are unable to fix as much nitrogen as the wild-type plant. For the sake of our project, we must concentrate our efforts (and time) in other aspects of the project instead of dealing with the "unfittest" plant ever.

We explained them the project we intend to make. I estimated the number of plants we intent to use is ~>300, Esperanza told us that is the average number of plants they usually plant in an experiment (See Dropbox document "Uso de Invernaderos" in the folder "wetlab" for more info about the derivation of this number). They are willing to let us use their greenhouse as well as their cultive rooms. We expect to plant 5 Ph. vulgaris per plant pot, this gives us ~60 plant pots.
 * I, along with Paulina Alatriste, had a meeting with Esperanza Martínez and Ivonne Toledo to talk about their help in lending us greenhouse space.

At first, I had the idea that we should let grow the plants with our bacteria in a normal plant pot and when the time arrived (15 days post-inoculation), we should transplant our plants in the appropiate container in which they would reside to extract and storage the molecular hydrogen. We asked Esperanza to what level of stress this operation would put the plant and she told us that she wouldn't recommend us to do this as the plants might not handle this stressful procedure.

She told us that we could just try to use the assay they usually use to test for nitrogen fixation. The plant is separated from its roots. The roots (along with the nodules) remained alive up to 24 hours after separation from the rest of the plant; Esperanza and Ivonne told us the root behaves just as if it hadn't been separated at all for many hours. The root is put inside an special vial that contains a gelified solution that acts as the rest of the plant in providing the necessary nutrients to the root/nodules. The vial is sealed, all is left to do is to adapt the tip of the sealed vial in such a way that the air inside of it can be extracted with a seringe (just as the video posted on the team wall in which a toy car is powered with a fuel cell that uses hydrogen produced by algae), the air could be then assayed with a gas chromatographer to test for hydrogen production and nitrogen fixation. This experiment is almost extrapolable to the "R.etli-Ph.vulgaris symbiosis" system as the roots remain almost intact. We think this experiment should suffice in proving the succesful functioning of our genetic circuit inside the nodules. For the purpose of the project, or Proof-of-Principle, this experiment will be easy, practical and sufficient.

On tuesday, Ivonne and I will go see Fede's lab greenhouses to see which of them (Esperanza´s or Fede's) are better for our project. Fede has already agreed to lend us some of his greenhouses space.

Esperanza also told us that if we had concerns respecting the isolation of our plants (wheter the plants contaminate other experiments or our plants result to be contaminated with other rhizobia), we could use space in their cultivation rooms. We plan to test for hydrogen production just as the nodules are completely mature (this happens 12-15 days after the inoculation has been performed), at this stage, the plant are small enough to have them all in the cultivation rooms. Contamination inside these rooms is extremely rare, so this problem would be almost completely solved should we decide to this space.

Esperanza and Ivonne expect us to begin planting as soon as possible to train ourselves in the handle of plants. They seemed happy to help us in any difficulty we might encounter. We told Esperanza of our previous plans to use the mutant R32-BS, and she told us that if the information was correct, she agreed we shouldn't bother using it in our project, instead, she told us that another mutant exists that contrary to R32-BS, is very efficient in fixing nitrogen. She was also curious we chose to utilize Rhizobium etli CFN42, as it is almost as unfit as R32-BS, she recommended us to use instead the strains CIAT652 and Ch 24.10.


 * Finally, I helped other team members to finish revising the letter proposal to OilSands. I modified the parts concerning the methology that stated we were to integrate into R. etli's chromosome our constructions and the use of the Phaseolus vulgaris supernodulating mutant.