Composite

Part:BBa_K1981201

Designed by: Fankang Meng   Group: iGEM16_NKU_China   (2016-10-14)


Autoinducer-2 Response Device A

This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. In AI-2 Response Device A, GFP expression is under the control of promoter, plsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. The expression of GFP can directly response to the AI-2 level in the environment, which is an alternative way to reflect the AI-2 concentration in the nature or artificial environment.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 898


1. Usage and Biology

This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. We firstly isolated promoter lsr from E.coli MG1655. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we successfully constrcuted plsr+ GFP +double terminator by using homologous recombination technology.

Figure 1: Schematic overview of the AI-2 Response Device A.

In AI-2 Response Device A, GFP expression is under the control of promoter, lsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. The expression of GFP can directly response to the AI-2 level in the environment, which is an alternative way to reflect the AI-2 concentration in the nature or artificial environment.

Figure 2: AI-2 Response Device A on plasmid pTrcHisB.

2. Characterization

2.1 Construction verification

AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249bp), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115bp). The total length of AI-2 Response Device A is 1111bp.

Figure 3: Colony PCR Verification for AI-2 Response Device A.


2.2 Response ability to exogenously added AI-2

We fisrtly tested whether AI-2 Response Device A can respond to different AI-2 concentration. We directly added exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that deicve can respond to different AI-2 concentration resulting in different GFP expression.

Figure 4: GFP expression of AI-2 Response Device A when add exogenous AI-2.

2.3 Co-culture with AI-2 Controllers

2.3.1 Co-culture with AI-2 Consumers

AI-2 Consumers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), which can directly absorb and degrade AI-2 in the nature or artificial environment. When E.coli consisting of AI-2 Response Device A are co-cultured with AI-2 Consumers, the GFP expression of AI-2 Response Device A is directly decreased compared to control group.

Figure 5: GFP expression of AI-2 Response Device A when co-cultured with AI-2 Consumers.

2.3.2 Co-culture with AI-2 Suppliers

AI-2 Suppliers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 production (luxS, mtn), which can directly supply and enrich the AI-2 molecular level in the nature or artificial environment. When E.coli consisting of AI-2 Response Device A are co-cultured with AI-2 Suppliers, the GFP expression of AI-2 Response Device A is directly increased compared to control group.

Figure 6: GFP expression of AI-2 Response Device A when co-cultured with AI-2 Suppliers.
[edit]
Categories
Parameters
None