User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/07/25

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χρόνος πέρασμα July 25th 2011

ABSTRACT

  • Transcriptional terminator B0015 characterization design.

  • In order to characterize the transcriptional terminator we're planning to use, B0015 [1], I'm making the constructions that will be needed, we are to characterize it in Rhizobium etli just as it had been done in Escherichia coli [2].
  • I have found several bioparts that contain distinct fluorescent proteins that will serve us to make the constructs. The general layout of the construction should be as follows: A promoter, an RBS, CDS of a flourescent protein, the terminator to be characterize (B0015), an RBS, another distinct CDS of a fluorescent protein and finally another terminator. The point of this is to see how often the second FP is observed, as one wouldn't expect to see it as the first terminator should halt the transcription machinery. The ratio of these two FP will help to see how efficient is the terminator.
  • The bioparts I've found are: I20260 [3], this serves as the first part of the construction, it has a constitutive promoter and a GFP; E0430 [4], this serves as the second part of the construction, it has a YFP; J04450 [5], this serves as the first part of the construction, it has a LacI regulated promoter and a RFP; and finally E0422 [6], serves as the second part of the construction, it has a CFP.
Fluorescent Protein Excitation wavelength Emission wavelength
GFP (I20260) 501 nm 511 nm
RFP (J04450) 584 nm 607 nm
YFP (E0430) 514 nm 527 nm
CFP (E0422) 439 nm 476 nm


  • As the devise that measures the fluorescent intensities works using narrow windows in the electromagnetic spectrum, it should be preferable to use the two most distant FP in the construction to avoid problems trying to distinguish one flourescence from another. Having the RFP with the CFP proves to be the best option. Besides, having an inducible promoter instead of a constitutive one should prove very useful as it could be pretty easy to measure basal expression of both FP and to perform the appropriate corrections to the measurement. Anyway, I will try to make as many variants of the FP as possible to enrich the terminator characterization.
  • As of today, three of these four bioparts are at our disposition: E0430 (transformed last week), J04450 (transformed last week) and I20160 (transformed by Gustavo Ruíz and Melissa Molho). Part E0422 will be provided by our advisor Enrique Paz.
  • Today I left incubating the bacteria carrying the plasmids with the bioparts E0430 and J04450 in order to let them ready for tomorrow plasmid extraction. 2 test tubes per biopart were left with 5 ml of LB with 5 μl of ampicillin at a concentration of 100 μg/ml.



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