Difference between revisions of "Part:BBa K1981201"
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==1. Usage and Biology== | ==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 <i>lsr</i> from <i>E.coli</i> 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. | + | 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 <i>lsr</i> from <i>E.coli</i> MG1655. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we successfully constrcuted <i>plsr</i>+ <i>GFP</i> +double terminator by using homologous recombination technology. |
[[Image:AI-2 Response Device AConstruction Map by NKU China.png|900px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 Response Device A.]] | [[Image:AI-2 Response Device AConstruction Map by NKU China.png|900px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 Response Device A.]] | ||
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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. | 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. | ||
| − | [[Image: | + | [[Image:T--NKU China--Proof-15.png|500px|thumb|center|'''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 Co-culture with AI-2 Controllers=== | ||
Latest revision as of 02:31, 20 October 2016
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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.
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.
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.
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.
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.
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.
