Difference between revisions of "Part:BBa K1981202"
 
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<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, <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. In this device, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response compared to AI-2 response device A.
  
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1981202 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1981202 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1981202 parameters</partinfo>
 
<partinfo>BBa_K1981202 parameters</partinfo>
 
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==1. Usage and Biology==
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This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a LsrR coding squence BBa_K091001,  a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. We firstly isolated promoter lsr and lsrR gene from <i>E.coli MG1655</i>. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we succeesflly constrcuted TT+ <i>lsrR</i> + <i>plsr</i> + <i>GFP</i> + double terminator by using Homologous recombination technology.
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[[Image:AI-2 Response Device B Construction Map by NKU China.png|900px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 Response Device B.]]
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In AI-2 Response Device B, GFP expression is under the control of promoter, <i>lsr</i>. When phospho-AI-2 binds <i>LsrR</i>, 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 this device, additional LsrR expression enables additional repression of target genes for tighter regulation and delayed response compared to AI-2 response device A.
  
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===Usage and Biology===
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[[Image:T--NKU China--AI-2 Response Device B Map.png|400px|thumb|center|'''Figure 2:''' AI-2 Response Device B on plasmid pTrcHisB.]]
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==Activity Analysis of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863005 ECOL]==
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==2. Characterization==
  
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===Initial activity tests of purified fractions===
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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 LsrR coding sequence BBa_K091001(954bp), two double terminators BBa_B0015(115). The total length of AI-2 Response Device A is 2209bp.
  
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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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[[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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[[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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===2.2 Response ability to exogenously added AI-2===
  
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===Characterization===
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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.
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[[Image:NKU China exogenously Added AI-2 On DeviceB NKU China.png|500px|thumb|center|'''Figure 1:''' GFP expression of AI-2 Response Device B when adding exogenous AI-2.]]

Latest revision as of 03:03, 20 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, 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. In this device, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response compared to AI-2 response device A.

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


1. Usage and Biology

This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a LsrR coding squence BBa_K091001, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. We firstly isolated promoter lsr and lsrR gene from E.coli MG1655. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we succeesflly constrcuted TT+ lsrR + plsr + GFP + double terminator by using Homologous recombination technology.

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

In AI-2 Response Device B, 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. In this device, additional LsrR expression enables additional repression of target genes for tighter regulation and delayed response compared to AI-2 response device A.

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

2. 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 LsrR coding sequence BBa_K091001(954bp), two double terminators BBa_B0015(115). The total length of AI-2 Response Device A is 2209bp.

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


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