IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/09: Difference between revisions

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*Find out how to do a c-terminal GFP fusion for membrane proteins agrB and agrC
*Find out how to do a c-terminal GFP fusion for membrane proteins agrB and agrC
**Can we fuse GFP and still expect them to localize to the membrane?
**Can we fuse GFP and still expect them to localize to the membrane?
***Seems possible in theory for AgrB.  We got lucky because the signal peptide is not on the N terminus, and both the N and C terminus are intracellular.  http://www.jbc.org/cgi/content/full/277/38/34736#F1
***AgrB transmembrane topology (http://www.jbc.org/cgi/content/full/277/38/34736#F1).  The signal peptide is not on the N terminus, and both the N and C terminus are intracellular.   
***For AgrC, the N terminus is probably extracellular.  The C terminus is probably intracellular.  http://openurl.ingenta.com/content?genre=article&issn=0950-382X&volume=28&issue=3&spage=655&epage=662 (see figure 2 B).  The article doesn't say where the signal peptide is.
***AgrC transmembrane topology (http://openurl.ingenta.com/content?genre=article&issn=0950-382X&volume=28&issue=3&spage=655&epage=662 (see figure 2 B)), the N terminus is probably extracellular.  The C terminus is probably intracellular.  The article doesn't say where the signal peptide is.
**Should we do a antibody tag instead?
**Should we do a antibody tag instead?


Revision as of 02:15, 11 August 2008

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Result from Erythromycin test

Problem : colonies on each plate

  • Reason : Erythromycin has to be diluted in ethanol, not water
  • To be sure, took a bacillus colony with no resistance and plated it onto Ery 0.5

On Monday we will remake the Erythromycin plates, and replate everything with the proper controls. Unfortunately, we do not have a positive control for erythromycin resistance, but if things die (i.e. negative control works), we should be in business.

Plans for Monday

Lab Work

  • Be sure that the Spc50 plates are working, as the control for Spc50 grew and shouldn't have. We already know that things can die at Spc100 (the double crossover test from earlier this week) so it is unlikely that the stock is bad. We should test Spc50 again with regular bacillus and with E. coli at 50-100 ug/ml and hope that they die.
  • Finish all the agr PCRs, but especially the RBS.
  • Confirm that amyE insertion works, using the amylase/ery tests.
  • Confirm that with ECE153 inserted, we get detectable fluoresence in a microscope.
  • Confirm that ECE166 transformation works, using plasmid miniprepping.
    • Find the qiagen miniprep kit or adapt the protocol.
  • Confirm that when ECE166 is transformed, we get detectable fluoresence in a microscope.

Book/Computer Work

  • Find good promoters in vectors and order PCR primers to get them out
  • Find out how to assay for/make AIP
    • Find/Contact Jim Ajioka
  • Find out how to do a c-terminal GFP fusion for membrane proteins agrB and agrC

Longer Term Plans

  • Ligate the RBS into a BB vector so we can build a RBS/GFP construct.
  • Put the RBS/GFP construct into ECE166 and ECE 153.
  • Compare the 'out of the box' ECE153 with ECE153 with RBS/GFP biobrick inserts
  • Compare the 'out of the box' ECE166 with ECE166 with RBS/GFP biobrick inserts




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