Team:Tokyo Tech/Experiment/Overview of fim inversion system

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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

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.

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

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.

2.1. Wild type fim switch

Fig.3-3-2-1-1. ここに図の名前を打て

Fim switch (WT) is derived from wild type sequence. Fim switch (wild type) have sigma 70 promoter which is constitutively promoter. We submitted two parts in each state, default ON (BBa_K1632004) and OFF (BBa_K1632005) The inversion of fim switch (Wild-Type) by FimB/FimE was confirmed. From the assay, the FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction with approximately equal efficiencies. Similarly the FimE protein inverts fim switch predominantly in the ON-to-OFF direction. Creating tripartite relationship using FimE-FimB-Fimswitch, is an unprecedented achievement in iGEM.

2.1.1. The design of wild type fim switch

Fig.3-3-2-1-1. ここに図の名前を打て

Sigma 70 promoter is located in fim switch (WT).
    Switching is also influenced by at least three global regulator, leucine-responsive regulatory protein (Lrp), H-NS and integration host factor (IHF).
    Switching frequencies are regulated by both temperature media and that these effects appear to be independent.
    Though a common mechanism, the FimB protein and the FimE protein inverts the fim switch. Two fim protein invert the sequence between IRL and IRR. The invertible sequence is flanked by 9bp inverted repeat, and each repeat is in turn flanked by non-identical recombinase-binding elements (RBEs). RBEs is bound by fimB or fimE. Changing REBs makes FimE or FimB work strangely [論文].
   

詳細版 IHF LRP強調 RE

2.1.2. Result of FimB/FimE Assay to fimS(TT)

For the inverson of fim switch by fimB or fimE, on the downstream of fim switch, we added gfp, so that fim switch[ON]-gfp (BBa_K1632007) and fim switch [OFF]-gfp(BBa_K1632008) differ in the point of florescence. Next, on the upstream of fimB or fimE, we added pBAD/araC. pBAD/araC-fimB(BBa_K1632012) and pBAD/araC-fimE(BBa_K1632011) induced fimB or fimE in the presence of arabinose. We transformed fim switch-gfp and pBAD/araC-fim in the Ecoli DH5alfa strain. We measured the fluorescence intensity of the cells induced by arabinose.
    From the assay, the inversion of fim switch (WT) by two recombinase, fimB and fimE, was confirmed correctly in both default ON state and default OFF state depending on the concentration of arabinose.

2.1.2.1 FimS-FimB

In the experiments, we setted four arabinose concentration. 

2.1.2.2 FimS-FimE

アッセイから具体的な文章/データはもらう

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