Project

Part:BBa_K494006

Designed by: Haissi Cui, Tilman Flock, Sebastian Gude, Christoph Hartlmüller, Florian Praetorius, Jan Schüürmann, Tobi Wauer, Philipp Wortmann   Group: iGEM10_TU_Munich   (2010-10-26)

Exemplary insert for BBa_K494001 (Trp-Term and Trp-Sig)

In general we want to provide a new, scalable principle of gene regulation based on termination and antitermination which can be further developed, tested and optimizted by everybody. To enable that in the easiest way possible we decided to not just add the switches we designed but instead the material needed for evaluation. Therefore we focus on providing the parts needed for verification and testing of new individual switches.


This is a ready to use part consisting of our screening backbone BBa_K494001 and a Trp-terminator in front of mCherry for testing reasons. Following mCherry a short DNA sequence is encoded (the signal) which is transcriped into a short transmitter RNA which binds to a certain region of the Trp-terminator leading to antitermination. This signal is IPTG inducible, so in theory the terminator can be switched from "off" to "on" by an input.
This part belongs to the screening system for evaluation of individual switches constructed on the principle of bioLOGICS, in this case BBaK494004 may serve as a reference to compare results with those presented by the TU Munich 2010 iGEM Team. Together with BBa_K494005 (Terminator only) and BBa_K494002 (positive control for mCherry expression) it is a setup to evaluate termination and antitermination efficiency of the Trp-Terminator.

In theory upon induction with IPTG the signal will be transcripted into transmitter RNA which binds to the Trp-terminator, leading to antitermination and therefore allowing transcription of mCherry which can be detected as a rise of red fluorescence. Other switching elements and terminators can be tested in a similiar way based on the K494001 backbone and K494006 may serve as a reference then.

The Trp-Terminator (which does not exist as an individual BioBrick since there are enough thoroughly tested Terminators yet in the registry) is based on an attenuator stem loop in front of the trp-operon of E. coli. The corresponding signal was constructed as decribed in the TU Munich iGEM 2010 wiki.


Emission spectra of induced and uninduced BBa_K494006 ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None