Team:TU Darmstadt/Protocols/Activity assay

From 2012.igem.org

(Difference between revisions)
Adrian E (Talk | contribs)
(Created page with "<html> <link rel="stylesheet" href="https://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/css&action=raw&ctype=text/css" type="text/css" /> <div id="TUD"> <div id="...")
Newer edit →

Revision as of 15:16, 16 September 2012

Contents

Activity Assay

ELISA Reader

LB-Agar-Tributyrin

  • inoculate the plate at 5 points with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032](L-arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA) and incubate at 37°C
  • controll group is from the 6th point of same bacterial strain but carrying only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030] (RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
  • check every day for lysis area

LB-Agar-Bromthymol blue

  • stick in six sterile PET particles
  • inoculate the PET particles with DH5α containing pSB1C3-whole-construct and incubate at 37°C
  • check every day for colour change (pH decrease when PET gets digested)
  • controll group is on LB-Agar-Bromthymol blue without arabinose

DYT-medium

  • inoculate 50 mL DYT-medium-CAM with bacteria containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
  • incubate at 37°C to an OD600 = 0.6
  • incubate on ice for 15 min before inducing
  • prepare sterile test tubes
  • fill the tubes in the way shown in table 1
Component tst tube 1 test tube 2 test tube 3 test tube 4
DYT-medium x x x x
PET particle x x x
bacteria x x x
induced x x x
  • check every day the colour of the DYT-medium and take a picture
  • digestion of PET particles leads to a decreasing pH and will be shown by a yellow color
Retrieved from "http://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay"