Device

Part:BBa_K1412600

Designed by: Zehua Chen   Group: iGEM14_XMU-China   (2014-09-08)


Endow the CL-1(E.coli engineered strain) the ability of chemotaxis and quorum sensing

What it is

The composite part enables the chemotaxis of the engineering CL-1 be regulated by AHL and AiiA, which promotes quorum sensing and oscillation.

What it does

The luxI promoter drives production of the luxI, aiiA, and CheZ genes. LuxI enzymatically produces a small molecule AHL, which can diffuse out of the cell membrane and into neighboring cells, activating the luxI promoter. AiiA negatively feedback regulates the circuit by acting as an effective protease for AHL. At the beginning, the small colony of individual cells cannot produce enough inducer to activate expression of the luxI promoter. However, once the population reaches a critical density, there is a “burst” of transcription of the luxI promoters, resulting in increasing levels of LuxI, AiiA, and CheZ (and the bacteria will move outward). As AiiA accumulates, it begins to degrade AHL, and after a sufficient time, the promoters return to their inactivated state. The production of AiiA is then attenuated, which permits another round of AHL accumulation and another burst of the promoters. The growth-ring formation circuit.png

How to use it in your project

The emergence of quorum sensing and oscillation will cause the periodic expression of CheZ, which will lead to the cyclical growth of colony radius.Then we can use it as a biological timer.

Experimental data

Bacteria rings formed by CL-1 with oscillation circuit

Bacteria rings.jpgWild-type E.coli(CL-M).png

Experiments show that bacteria could just form several rings in 48 hours. Afterwards, no bacteria ring formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (right) which are quiet different from wild-type (left). We make sure that chemotaxis is reprogrammed successfully but not as expected.

Cultivating CL-1 in Plate with Cm and Tet(halve the concentration of LB)

The grown curve of CL-1 with oscillation(1).png

We make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve, the curve show that the rate is stable.

Cultivating CL-1 in Plate with Cm and Tet

The grown curve of CL-1 with oscillation(2).png

We make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve, the curve show that the rate is stable.

protocol

1. Transformation

2. Extract plasmids

3. Digestion

4. DNA gel electrophoresis

5. Gel Extraction

6. Ligation

7. Transformation

8. Extract plasmids

9. Digestion

10. DNA gel electrophoresis

11. Transform the plasmid into CL-1



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1821
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101
    Illegal BsaI.rc site found at 1970
    Illegal BsaI.rc site found at 3073


Reference

[1] [http://www.ncbi.nlm.nih.gov/pubmed/21998392/ Sequential Establishment of Stripe Patterns in an Expanding Cell Population Chenli Liu et al.Science 334, 238 (2011);DOI: 10.1126/science.1209042]

[2] [http://www.ncbi.nlm.nih.gov/pubmed/20090747/ A synchronized quorum of genetic clocks Tal Danino et al. Nature. 2010 Jan 21;463(7279):326-30. doi: 10.1038/nature08753]

More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]

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