Difference between revisions of "Part:BBa K1981201"

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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.
 
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.
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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.
+
In AI-2 Response Device A, GFP expression is under the control of promoter, <i>plsr</i>. 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.
  
 
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===Usage and Biology===
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===1. Usage and Biology===
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This device is used to reflect AI-2 concentration in the nature or artificial environment. By co-culturing ‘AI-2 controllers’ with E.coli consisting of AI-2 Response Device A , we can know how the ‘controller cells’ manipulate the AI-2 level in the extracellular environment by simply measure the GFP expression level.
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This device is used to reflect AI-2 concentration in the nature or artificial environment. By co-culturing ‘AI-2 controllers’ with <i>E.coli</i> consisting of AI-2 Response Device A , we can know how the ‘controller cells’ manipulate the AI-2 level in the extracellular environment by simply measure the GFP expression level.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Usage and Biology===
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==1. Usage and Biology==
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This device is used to reflect AI-2 concentration in the nature or artificial environment. By co-culturing ‘AI-2 controllers’ with E.coli consisting of AI-2 Response Device A , we can know how the ‘controller cells’ manipulate the AI-2 level in the extracellular environment by simply measure the GFP expression level.
+
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.
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[[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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 +
In AI-2 Response Device A, GFP expression is under the control of promoter, <i>lsr</i>. 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.
 +
[[Image:T--NKU China--AI-2 Response Device A Map.png|400px|thumb|center|'''Figure 2:''' AI-2 Response Device A on plasmid pTrcHisB.]]
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==2. Characterization==
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 +
===2.1 Construction verification===
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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.
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[[Image:NKU China Device PCR Colony Verification.png|800px|thumb|center|'''Figure 3:''' Colony PCR Verification for AI-2 Response Device A.]]
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 +
 
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===2.2 Response ability to exogenously added AI-2===
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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.
 +
 
 +
[[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.]]
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===2.3 Co-culture with AI-2 Controllers===
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===2.3.1 Co-culture with AI-2 Consumers===
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AI-2 Consumers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 uptake (<i>lsrACDB</i>), phosphorylation (<i>lsrK</i>), and degradation (<i>lsrFG</i>), which can directly absorb and degrade AI-2 in the nature or artificial environment. When <i>E.coli</i> 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.
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[[Image:NKU China Device A with AI-2 Consumer.png|600px|thumb|center|'''Figure 5:''' GFP expression of AI-2 Response Device A when co-cultured with AI-2 Consumers.]]
  
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===Characterization===
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===2.3.2 Co-culture with AI-2 Suppliers===
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===Construction verification===
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AI-2 Suppliers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 production (<i>luxS</i>, <i>mtn</i>), which can directly supply and enrich the AI-2 molecular level in the nature or artificial environment. When <i>E.coli</i> 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.
  
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[[Image:AI-2 Response Device B Construction Map by NKU China.png|800px|thumb|center|'''Figure 10:''' Activity assay of each purified fraction of the cultivation with ECOL. Samples were re-buffered into H<sub>2</sub>O and the protein amount in each fraction has been adjusted. The measurements were done using the [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night. The first number indicates the percentage of used elution buffer, whereas the second number stands for the fraction number of this elution.]]
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[[Image:NKU China Device A with AI-2 Supplier NKU China.png|600px|thumb|center|'''Figure 6:''' GFP expression of AI-2 Response Device A when co-cultured with AI-2 Suppliers.]]

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


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.