Difference between revisions of "Part:BBa K1981202"

Revision as of 03:21, 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, 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 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.

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 ==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 <i>E.coli MG1655</i>. 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.
+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.
 [[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.]]
-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.
+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.
 [[Image:T--NKU China--AI-2 Response Device B Map.png|400px|thumb|center|'''Figure 2:''' AI-2 Response Device B on plasmid pTrcHisB.]]