Composite

Part:BBa_K1981201

Designed by: Fankang Meng   Group: iGEM16_NKU_China   (2016-10-14)
Revision as of 02:20, 15 October 2016 by MengFankang Nankai (Talk | contribs)


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 succeesflly 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, 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.

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 show that deicve can result in different GFP expression in responding to different AI-2 concentration .

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.
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