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Team:TU Darmstadt/Protocols/Activity assay
From 2012.igem.org
Contents |
Activity Assays
ELISA Reader
LB-Agar-Tributyrin
- inoculate the plate at 5 points with DH5α containing 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 BBa_K808030 (RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
- check every day for lysis area
LB-Agar-Bromothymol 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 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
