Difference between revisions of "Part:BBa K1981202"
Line 27: Line 27:
 
===Characterization===
 
===Characterization===
 
===2.1 Construction verification===
 
===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.  
+
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.  
  
 
[[Image:NKU China Device B Construction.png|800px|thumb|center|'''Figure 1:''' Colony PCR Verification for AI-2 Response Device B.]]
 
[[Image:NKU China Device B Construction.png|800px|thumb|center|'''Figure 1:''' Colony PCR Verification for AI-2 Response Device B.]]
Line 33: Line 33:
  
 
===2.2 Response ability to exogenously added AI-2===
 
===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 LsrR coding sequence BBa_K091001(954bp), two double terminators BBa_B0015(115). The total length of AI-2 Response Device A is 2209bp.
 
  
−
[[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.]]
+
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 respond to different AI-2 concentration resulting in different GFP expression
 +
[[Image:NKU China exogenously Added AI-2 On Device B NKU China.png|800px|thumb|center|'''Figure 1:''' GFP expression of AI-2 Response Device B when adding exogenous AI-2.]]

Revision as of 01:24, 15 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. 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, 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 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.

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 show that deicve can respond to different AI-2 concentration resulting in different GFP expression

File:NKU China exogenously Added AI-2 On Device B NKU China.png
Figure 1: GFP expression of AI-2 Response Device B when adding exogenous AI-2.