Composite

Part:BBa_K3900053:Design

Designed by: Louisa Lehmann   Group: iGEM21_Bielefeld-CeBiTec   (2021-09-30)


pBad:Trz:dTer:ssTNT.R3:araBAD:PrompC:GFP:T1


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
    Illegal BamHI site found at 3725
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2102
    Illegal NgoMIV site found at 2224
    Illegal NgoMIV site found at 2354
    Illegal AgeI site found at 495
    Illegal AgeI site found at 3560
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4542
    Illegal SapI site found at 3542


Design of the signaling cascade

We chose to make the signaling cascade inducible to be able to compare the output of the cascade before and after expression. This also reduces stress during early growth phases. We chose the L-arabinose inducible pBAD promotor system, consisting of AraC, the regulator of the arabinose operon and the AraBAD promotor, which is regulated by AraC activity. After induction with L-arabinose, the promotor has a low to medium activity, reducing stress and formation of inclusion bodies. The AraBAD promotor controls the expression of the synthetic membrane-bound Trz receptor and the ssTNT receptor [1]. The sequence of the TNT receptor was modified to be cut out by HindIII to simplify exchange with our computationally designed receptors. However, the TNT receptor was missing a periplasmic localization peptide and was therefore not exported into the periplasmic space after expression, thus could not activate the signaling cascade. The OmpR-inducible OmpC promotor regulates the expression of the GFP, that marks the ending of the signaling cascade. OmpR occurs natively in E. coli and is therefore not required to be expressed by separately [2]. Since OmpC is activated by osmotic stress, it is important to adjust the concentrations of additives. In our experiments we used 0.2% (w/v; 0.013 M) arabinose for induction of expression without significant induction by changes in osmolarity.

References

  1. Looger, L. L., Dwyer, M. A., Smith, J. J. & Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185–190 (2003).
  2. https://www.uniprot.org/uniprot/P0AA16.