Device

Part:BBa_K2205016

Designed by: Lais Takiguchi   Group: iGEM17_Newcastle   (2017-09-21)

Description

The part BBa_K2205016 was designed as Reporter unit of the Sensynova multicellular framework for biosensors development (Figure 1).

Framework generic.jpg

This part was made to test the modularity of the system by replacing the sfGFP reporter module of the Sensynova platform design with the chromoprotein variants aeBlue (BBa_ K1033929).

The aeBlue protein is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equine. It was first extracted and characterized by Shkrob et al. 2005 under the name aeCP597 and codon optimised for E. coli by Bioneer Corp. The protein has an absorption maximum at 597nm and a deep blue colour visible to the naked eye. The protein aeBlue has significant sequence homologies with proteins in the GFP family. The coding sequence for this protein was originally submitted to the registry as BBa_K1033916 by the 2012 Uppsala iGEM team.

The part BBa_ K1033929 is under the control of our connector 2, part BBa_K2205013, meaning it is normally repressed except in the presence of connector 2 (C12 – RHL).

Framework Blue Variant Pic.jpg

Characterisation

Initial testing of this chromoprotein reporter variant was conducted by inoculating 1ml of LB containing the antibiotic Chloramphenicol with a colony from each colour proven by colony PCR and sequencing data to be correct and grown at 37° for 2 hours as well as a control of wildtype DH5α.


Cultures were then plated onto a LB agar + Chloramphenicol plate which were then innoculated in four locations with 10μl of connector 2 (C12 - Rhl) and grown overnight resulting in the picture below.

Framework Blue Variant.jpg

In order to test this chromoprotein reporter variant into the Sensynova framework, cultures of IPTG detector, processor unit and reporter module were inoculated and grown overnight in LB+chloramphenicol (12,5ng/μl).

The cultures were then diluted at OD600: 0,1 and mixed together to obtain co-cultures with ratio 1:1:1 and 1:1:13. Some samples were supplemented with 1mM IPTG to induce the expression of quorum sensing molecules and eventually achieve the chromoprotein visualisation (Figure 3).

Blue_pellets2.jpg

Figure 3: Pellets collected after overnight co-cultures of IPTG + processor + Blue Chromoprotein reporter in ratios 1:1:1 and 1:1:13, with and without 1mM IPTG.

References

Shkrob, M., Yanushevich, Y., Chudakov, D., Gurskaya, N., Labas, Y., Poponov, S., Mudrik, N., Lukyanov, S. and Lukyanov, K. (2005). Far-red fluorescent proteins evolved from a blue chromoprotein fromActinia equina. Biochemical Journal, 392(3), pp.649-654.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 776


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