IGEM:UNAM Genomics Mexico/2009/Notebook/iGEM 2011/2011/05/09
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χρόνος πέρασμα May 9th 2011
She provided us with the papers that talk about the plasmids we intend to use (pK18mobsacb, pRK404 and pBBRMCS5). Here is a brief summary of what she said: Rhizobium etli CFN 42 possesses six plasmids beside the chromosome. One of these is called pSin or pD; here reside most of the symbiotic-specific proteins. An operon regulated by the transcription factor NifA has been duplicated in this plasmid; this operon has the gene tha codifies for the nitrogenase, as well as several other proteins required for the proper performance of the nitrogenase.
Two R. etli CFN 42 mutant strains exist that are incapable of fixing nitrogen, but that nodulate perfectly. One of these has inactivated by a mutation the promoter of the operon that regulates the nitrogenase, this one does not fix nitrogen; the other consists of a point mutation that inactivates the nitrogenase. It has been observed that if only one copy of this operon has the mutation, nitrogen fixation activity halves; if both copies are mutated, nitrogen fixation activity is completely abolished. Maluye strongly recommend us to test the system in the wild-type bacteria, the single mutant and the double mutant, to see if hydrogen production happens to be directly affected by this step-by-step reduction of nitrogen fixation. Unfortunately, we can only dispose of two mutant versions, the one with the inactivated promoter and the one that has only one copy of the nitrogenase inactivated. Maluye will try to find the double mutant strain.
One important contribution Maluye said to me is that we might begin to elucidate an important biological question: Why does Rhizobium etli lack of a hydrogenase? Some find this fact intriguint, as some very closely related bacteria do have capture hydrogenases (Sinorhizobium meliloti and Rhizobium leguminosarum). Is it that the actual intrinsic biological system excludes the possibility of having a hydrogenase because it wouldn't be functionnal? Among other questions.
I had some doubts concerning the 5kb long sequence he selected for the constructions integration into R. etli chromosome. He chose that region because of the lack of experimental evidence of coding-regions inside it (although the region might be ideed transcribed, as almost all R. etli genome), as well as because it is near some actively expressed genes. The problem is that inside the sequence provided there is large inverted duplication that might result to be tricky to work with when we try to integrate our system. This duplication consist of two Insertion Sequences (IS) that are also found in another plasmid, making it even trickier; as the plasmid integration consist of sequence homology.
Miguel strongly suggests us not to try to do the chromosome integration for many reasons. One of these is the lack of time, as many researches have told us, only this particular step might take many months to achieve. Another point is the following: If we succeed in producing large amounts of hydrogen, then the project will turn out to be very useful; R. etli is one of the worst strains in the Rhizobia-World in many aspects, the region for chromosome integration so far considered is unique of R. etli, so the system wouldn't prove to be completely modular (meaning that some modifications should be taken in order to introduce the genetic circuit to other rhizobia) and to find another shared region in the chromosome is not as simply as it sounds.
Miguel also told me that there is another R.etli mutant strain that we could utilize. It has mutated two genes, RpoH1 and RpoH2, both polymerase stress responsive sigma factors. It has been shown that these two genes inactivated somehow indirectly affect the nitrogen fixation pathway, completely abolishing it.
She will be the one that will provide us with the Phaseolus vulgaris variation that has been mutated and produces many more nodules that average. This mutant is called R32, was brought from Canada. She agreed to spare some of her very small stock of seeds, we have to start growing them as soon as possible to obtain even more seeds. R32 has not been yet fully characterized, but the most obvious phenotype is that the plant is extremely small, yellow and ... as I saw them, almost dying.
Approximately only half of the seeds planted succeed to grow and once the “adulthood” is reached, the plant produces a mean of 2 pods with 5 seeds each. So, for each seed we plant we expect to obtain 5 seeds. It does not seem an appropiate candidate to test our system.
I have told Geo I will send her an approximation of the number of seeds we would like her to lend us. She will help us with the plantation. The plants don't need lots of care... we just plant them and irrigate them twice or three times per week.