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 does 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 does 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)

アッセイから具体的な文章/データはもらう

2.2. Tokyo_Tech fim switch

The inversion of fim switch (TT) by FimB/FimE was not confirmed correctly. From the assay, the FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction correctly. However the FimE protein didn’t inverts fim switch predominantly in the ON-to-OFF direction. In the assay, the FimE protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein.

2.2.1. The design of wild type fim switch

We designed another fim switch with standardized interchangeable promoter, fim switch (TT). Only one difference between wild type and TokyoTech is to change sigma 70 promoter to J23119 promoter and insert two restriction enzyme sites in each front (SalIand BamHI) and back (BglII and MluI). By insertion of restriction enzymes, fim switch (TokyoTech) have standardized interchangeble change promoter. Except for insertion of restriction enzyme sites, basically, the design of TokyoTech fim switch is similar with Wild Type fim switch.
    The inversion of fim switch () by FimB/FimE was not confirmed correctly. From the assay, the FimB protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction correctly. However the FimE protein didn’t inverts fim switch predominantly in the ON-to-OFF direction. In the assay, the FimE protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein.
   

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

3. Comparison other team’s fim switch

　　　　　　

Our big achievement have created tripartite relationship, Fim switch-FimB/FimE.
    In this chapter, the novelty of fim switch-FimB/FimE is shown by comparison of past iGEM teams’ fim parts. In the past Jamboree, some teams submitted fim genes. However the information of fim genes are so complicated that it is necessary to clarify the situation.

Fig.A. ここに図の名前を打て

　　　　　　

Fig. A shows the summary of past iGEM team’s fim parts. These submitted parts which show some data, is divided to three groups as shown in theigure bellow.

1. FimB(2013_Toronto)
⇒3.1.
2. FimS(2006_Caltech)-FimE(2006_Caltech)
⇒3.2.
3. FimS(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)
⇒3.3.

　　　　　　

From the result of each group, each one is evaluated as shown as below.

　　　　　　

1. FimB(2013_Toronto) ⇒No date about inversion
2. FimS(2006_Caltech)-FimE(2006_Caltech)
⇒No date about inversion
3. FimS(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)
⇒Not enough date about inversion
From these evaluations, we achieved to create tripartite relationship, Fim switch-FimB/FimE.

Fig.南蛮文化. ここに図の名前を打て

3.1. FimB(2013_Toronto)

Fig.A. ここに図の名前を打て

　　　　　　

In the past jamboree, 2013_Toronto only submitted FimB(BBa_K137007) and showed some data. However, their data clearly did not relate to the inversion of fim switch. Their assay is to search difference of some parameters like OD600 in FimB (+) and FimB (-). So the result of 2013_Toronto did not show any data of the inversion of the fim switch.

3.2. Fim switch(2006_Caltech)-FimE(2006_Caltech)

Fig.A. ここに図の名前を打て

　　　　　　

FimE

Gene Name	Year	College	Parts Number	Sequence Confirmation
FimE	2008	Caltech	BBa_K137007	None

　　　　　　

fim switch

Gene Name	Year	College	Parts Number	Sequence Confirmation
FimE IRR	2008	Caltech	BBa_K137008	None
FimE IRL	2008	Caltech	BBa_K137010	None
GFP fimE switch with 150 bp tetR promoter	2008	Caltech	BBa_K137057	None
GFP fimE switch with 250 bp tetR promoter	2008	Caltech	BBa_K137058	None
GFP fimE switch with 350 bp tetR promoter	2008	Caltech	BBa_K137059	None
GFP fimE switch with 450 bp tetR promoter	2008	Caltech	BBa_K137060	None
GFP fimE switch with 550 bp tetR promoter	2008	Caltech	BBa_K137061	None
GFP fimE switch with 650 bp tetR promoter	2008	Caltech	BBa_K137062	None

　　　　　　

In the past jamboree, 2006_Caltech only submitted FimE(BBa_K137007) and showed some data. Assay protocol is here).

Fig.A. 2006_Caltech

　　　　　　

They wanted to determine the range of length which FimE will act on and to determine how the length affects fimE activities. Thus they tested how the length of the recombination site affects FimE’s ability to detect the two flanking binding sites. So GFP fluorescence / OD600 was observed in each sample in which the length of the inversion site differs.
   However their data cannot be trusted completely with respect to inversion of fim switch. There are several reasons. First of all, they did not set positive controls and negative controls. Secondly, they didn’t write down information of plasmids used in the experiments (backbone, gene).
   The nucleotide sequence of 2006_Caltech fimE and wild type fim E did not match. So, we designed FimE (BBa_K1632011) whose nucleotide sequence completely matchs that of the wild type fimE. Comparing these two parts, two differences were confirmed. First, fimE of 2008_Caltech lacks the nucleotide sequence of N-terminal 15 residues. Second, fimE of 2008_Caltech differs from the nucleotide sequence of 2015_TokyoTech by C-terminal 2 residues as shown below.

　　　　　　

2008_Caltech:5’-...-TAA-TAA-Suffix-3’
2015_TokyoTech : 5’-...-GTT-TGA-Suffix-3'

3.3. Fim switch(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)

Fig.3-3-2-1. タイムアウト

　　　　　　

FimE

Gene Name	Year	College	Parts Number	Sequence Confirmation
FimE	2008	Caltech	BBa_K137007	None

　　　　　　

   

4. Reference

　　　　　　

DNA反転(プロトコール作成時に使用)
Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4
FimE FimBの塩基配列(プライマー作成に使用)
Klemm P. (1986) Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J. 5(6):1389-93.
FimB/Eの役割
McClain MS et al. (1991) Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli. J Bacteriol 173(17):5308-14.
FimSwitch Design
Ian C. Blomfield et al. (1997) Integration host factor stimulates both FimB- andFimE-mediated site-specific DNA inversion that controlsphase variation of type 1 fimbriae expression in Escherichia coli. Molecular Microbiology 23(4), 705–717
John M. Abraham et al. (1985) An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli. Proc Natl Acad Sci U S A 82(17):5724-7
Matthew P. McCusker et al. (2008) DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries
determine the directionality of the fim genetic switch in Escherichia coli K-12. Molecular Microbiology 67(1): 171–187