Team:Tokyo Tech/Experiment/Overview of fim inversion system
Overview of fim inversion system
We introduce fim system
contents
1. Introduction
2. How to fim switch works
2.1. Wild type fim switch
2.1.1. The design of wild type fim switch
2.1.2. Result of FimB/FimE Assay to fimS(TT)
2.1.3. AHL dependent FimE Assay to fimS(TT)
2.2. Tokyo_Tech fim switch
2.2.1. The design of Tokyo_Tech fim switch
2.2.2. Result of FimB/FimE Assay to fimS(WT)
3. Comparison other team’s fim switch
3.1. FimB(2013_Toronto)
3.2. Fim switch(2006_Caltech)-FimE(2006_Caltech)
3.3. Fim switch(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)
4. Reference
1. Introduction
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Fig.3-3-1-1. ここに図の名前を打て |
In order to make the E. coli decide for themselves, we needed a part that can switch back and forth at random. We decided that the fim switch can do this job. Our big achievement have created a tripartite relationship, fim switch-FimB/FimE.
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Fig.3-3-1-2. ここに図の名前を打て |
In the wild-type of E.coli K-12, two Fim proteins(fimbriae) invert the Fim switch, a specific 314 bp DNA sequence containing a promoter, to modulate its own expressions [1]. Fim switch has two states, ON and OFF. In the ON state, the promoter in (the fim switch transcripts to the right. On the other hand, in the OFF, promoter in fim switch transcripts to the left. In the following sentence, “ON” and “OFF" represent fim switch [default ON] and fim switch[default OFF].
Fim switch is inverted by two recombinases, FimB and FimE. These proteins have distinct activities. The FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction with approximately equal efficiencies. On the other hand, the FimE protein inverts fim switch predominantly in the ON-to-OFF direction [2]. FimB and FimE gene was amplified by PCR. These PCR primers were designed by [2].
2. How to fim switch works
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Fig.3-3-2-1. ここに図の名前を打て |
We designed two fim switchs: a wild-type one and a one with standardized interchangeable promoter. The first fim switch is derived from wild type sequence. We named the wild type’s fim switch as fim switch[default ON](WT) and fim switch[default OFF](WT). (The second fim switch has been added four restriction sequences and a replaceable promoter the fim swtich(WT).) We designed the second fim switch by adding four restriction sequences and a replaceable promoter to the fim swtich(WT). We will name this artificial fim switch as fim switch[default ON](TT: promoter’s name) and fim switch[default OFF](TT: promoter’s name). we made fim switch[default ON](TT: J23119) and fim switch[default ON](TT: Lac). From the FLA assay, the FimB protein and FimE protein from chromosome didn’t affect the inversion of plasmid. The detail data is on the assay page.
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Fig.3-3-3-2. ここに図の名前を打て |
2.1. Wild type fim switch
2.1.1. The design of wild type fim switch
2.1.2. Result of FimB/FimE Assay to fimS(TT)
2.1.3. AHL dependent FimE Assay to fimS(TT)
2.2. Tokyo_Tech fim switch
2.2.1. The design of wild type fim switch
2.2.2. Result of FimB/FimE Assay to fimS(TT)
3. Comparison other team’s fim switch
3.1. FimB(2013_Toronto)
3.2. Fim switch(2006_Caltech)-FimE(2006_Caltech)
3.3. Fim switch(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)
4. Reference