Composite

Part:BBa_K4115040

Designed by: Kaijun Wang   Group: iGEM22_ShanghaiTech_China   (2022-09-30)
Revision as of 12:39, 4 October 2022 by Yurj (Talk | contribs)


J23101-B0034-LuxI-B0015

J23101-B0034-LuxI-B0015
Function Sythase
Use in E. coli
RFC standard RFC 10, RFC 10 compatible
Backbone pUC57
Submitted by ShanghaiTech_China

This composite part is used to produce 3OC6 HSL by LacI (BBa_C0161)[1] under a constitutive promoter J23101(BBa_J23101) and send it to the receiver protein LuxR (BBa_C0062)in the other E. coli.When the LuxR receives the signal, it will regulate and express the downstream report gene sfGFP [2], thus we can detect whether LuxI is functional by observing the fluorescence intensity under UV light.

Proof of function

We encapsulated E. coli with this composite part and E. coli with LuxR and sfGFP into alginate gel beads. The E. coli with LuxI secreted the 3OC6 HSL signal molecule, and the E. coli with LuxR received the signal and regulate the expression of downstream sfGFP, making it express. So we can detect green fluorescence in the latter E. coli under the irradiation of ultraviolet light.

Figure 1: sfGFP is expressed under the regulation of LuxR between E. coli in a single gel bead. Two parallel groups are given and the above two are control groups.
























Quantitative analysis

We set three groups,one is the blank group, where only E. coli with LuxR is added, the second is the group where 10-7M 3OC6 HSL is added, and the third one is the group where the supernatant of LuxI is added. We conclude the data by dividing the fluorescence intensity by the OD600 and get Figure 2.

Figure 2: LuxI supernatant induces LuxR to respond and express sfGFP.The data is in contract with 10-7M 3OC6 HSL added group and the blank group is also shown.

























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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

[1] Daer R, Barrett CM, Melendez EL, Wu J, Tekel SJ, Xu J, Dennison B, Muller R, Haynes KA. Characterization of diverse homoserine lactone synthases in Escherichia coli. PLoS One. 2018 Aug 23;13(8):e0202294. doi: 10.1371/journal.pone.0202294. PMID: 30138364; PMCID: PMC6107141.

[2] Kylilis, N., Tuza, Z.A., Stan, GB. et al. Tools for engineering coordinated system behaviour in synthetic microbial consortia. Nat Commun 9, 2677 (2018). https://doi.org/10.1038/s41467-018-05046-2

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