Part:BBa_K1067001:Design
Twin-arginine translocation reporter
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1225
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1225
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1225
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1225
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 149
Design Notes
To visualize that GFP SF actually is exported, we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). As we initially found, if you just grow cells in LB and induce the GFP SF/RFP expression, there will be too much GFP SF in the cytoplasm to get a good resolution between cytoplasm and periplasm. That is why it’s important to devise a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; when expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF into the cytoplasm.
After optimizing the procedure we made a final version which can be found in the [http://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm DTU-Denmark 2013 wiki methods page]. This also includes how to use background subtraction to get a better resolution.
References
Skoog, Karl, et al. "Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli." Journal of bacteriology 194.3 (2012): 584-586.