Difference between revisions of "Part:BBa K1981202"
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<partinfo>BBa_K1981202 short</partinfo>
 
<partinfo>BBa_K1981202 short</partinfo>
  
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Device B long description
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This composite part consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a LsrR coding sequence BBa_K091001, two double terminators BBa_B0015.
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In AI-2 Response Device B, GFP expression is under the control of promoter, plsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. In AI-2 Response Device A, 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 Device B, 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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===Initial activity tests of purified fractions===
 
  
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A cultivation of ECOL has been done and the fractions of the purification were analyzed further on protein content and re-buffered subsequently into deionized H<sub>2</sub>O. To determine the protein content afterwards because of loss of proteins through re-buffering, another [http://2012.igem.org/Team:Bielefeld-Germany/Amsterdam/Labjournal#Tuesday_October_17th/ protein concentration measurement] has been done. The re-buffered fractions have been incubated with 0.4 mM CuCl<sub>2</sub> to gain higher activity of the laccases, because they are copper-dependent. Standard activity tests were done with all ECOL fractions with adjusted protein content for comparison. The experimental setup included the ECOL fractions, Britton-Robinson buffer (pH 5) and 0.1 mM ABTS. Measurements were done at 25 °C. Resulting, one fraction showed very high activity in comparison to the other fractions (see Fig. 10). This fraction, fraction 50% 2, oxidized up to 23 µM ABTS after 5 hours. The first number of the sample indicates the percentage of used elution buffer, whereas the second number stands for the fraction number of this elution. This fraction was set as containing 90 % ECOL laccase of the whole protein content. Therefore a ECOL concentration of 63,9 µg mL<sup>-1</sup> was gained. This fraction was analyzed further on pH optimum, temperature dependency and ABTS saturation.
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===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(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.  
  
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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 B Construction.png|800px|thumb|center|'''Figure 1:''' Colony PCR Verification for AI-2 Response Device B.]]
  
  
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===Characterization===
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===2.2 Response ability to exogenously added AI-2===
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AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.
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[[Image:NKU China exogenously Added AI-2 On DeviceB NKU China.png|800px|thumb|center|'''Figure 1:''' GFP expression of AI-2 Response Device B when add exogenous AI-2.]]

Revision as of 23:09, 14 October 2016


Autoinducer-2 Response Device B

This composite part consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a LsrR coding sequence BBa_K091001, two double terminators BBa_B0015. In AI-2 Response Device B, GFP expression is under the control of promoter, plsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. In AI-2 Response Device A, 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 1996


Usage and Biology

Activity Analysis of ECOL

Characterization

2.1 Construction verification

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

Figure 1: Colony PCR Verification for AI-2 Response Device B.


2.2 Response ability to exogenously added AI-2

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

Figure 1: GFP expression of AI-2 Response Device B when add exogenous AI-2.