Composite

Part:BBa_K4115040

Designed by: Kaijun Wang   Group: iGEM22_ShanghaiTech_China   (2022-09-30)
Revision as of 14:17, 11 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], Figure 1. Thus we can detect whether LuxI is functional by observing the fluorescence intensity under UV light.

Figure 1: Lux QS system path.






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 and gain Figure 2.

Figure 2: 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.
























We put the gel beads of the experimental group and the control group on a confocal microscope for imaging observation, and found that there was a significant difference in fluorescence intensity between the two groups (Figure 3-4). Thus, the Lux system was proved to be functional between E. coli.

Figure 3: 10X Confocal image of the control group's gel beads.
Figure 4: 10X Confocal image of the experimental group's gel beads.


























Quantitative analysis

Experiment approach

1. Centrifuge the overnight E. coli culture transformed with BBa_K4115040(express LuxI consititutively) at 12000 rpm for 1 min. Take out the supernatants for use.
2. Dilute the overnight E. coli culture that transformed with BBa_K4115039 to OD600~0.1.
3. Three groups are set: 1) Blank: diluted cultures in step2, autoinducer is not supplied to blank group 2) 10-7 M 3OC6 HSL: 1:1000 add the 10-3 M autoinducer into the diluted cultures, the final autoinducer concentration is 10-7 M 3) Supernatant: centrifuge the diluted cultures at 3500 rpm for 10 min, resuspend the cells with the supernatant in step1. Culture all these three groups at 37℃ with shaking at 220rpm for 3h.
4. After three hours, add 200ul to each well of the black bottom 96-well plate for fluorescence intensity testing. Use an excitation wavelength of 485nm and an emission wavelength of 535nm.
5. Add 200ul to each well of the transparent 96-well plate for OD600 measurement.

Results

We process the raw data by dividing the fluorescence intensity by the OD600 and get Figure 2. Thus, we can conclude the response intensity of LuxR to 3O6C HSL produced by BBa_K4115040.

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 the E. coli culture.

























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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