Device

Part:BBa_K2205018

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


Sensynova Framework (Connector 2B + spisPink (Complete) - rCell)

Description

The part BBa_K2205018 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 spisPink (BBa_K1033925).

The spisPink protein is a pink chromoprotein extracted from the coral Stylophora pistillata. It was first extracted and characterized by Alieva et al. under the name spisCP (GenBank: ABB17971.1) and codon optimized for E. coli by Genscript. The protein has an absorption maximum at 560 nm giving it a pink colour visible to the naked eye. The strong colour is readily observed in both LB or on agar plates after less than 24 hours of incubation.

The part BBa_K1033925 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 Pink 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 Pink 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).

Pink_pellets2.jpg

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

References

[1] Alieva, Naila O., et al. "Diversity and evolution of coral fluorescent proteins." PLoS One 3.7 (2008): e2680.

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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