Difference between revisions of "Part:BBa K1981202"

Revision as of 01:27, 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

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

Revision as of 01:26, 15 October 2016 (view source) MengFankang Nankai (Talk \| contribs) ← Older edit		Revision as of 01:27, 15 October 2016 (view source) MengFankang Nankai (Talk \| contribs) Newer edit →
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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 show 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 show that deicve can respond to different AI-2 concentration resulting in different GFP expression
−	[[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 adding exogenous AI-2.]]	+	[[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.]]