contents

1. Introduction

2. How does fim switch works

2.1. The fim switch(wild-type)

2.1.1. Result of the FimB/FimE assay on the fim switch(wild-type)

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

FimB inverts the fim switch in [ON] state to [OFF] state and in [OFF] state to [ON] state with approximately equal probability. FimB enable prisoner coli to select its option (cooperation or defection) randomly (Fig.3-3-1-1.). We showed bidirectional inversion of fim switch by FimB. This is the first case in iGEM to show random inversion of a promoter.
	Fig.3-3-1-1.In the presence of FimB, promoter in fim switch can be inverted at random.
	On the other hand, FimE inverts the fim switch predominantly in the [ON] state to [OFF] state (McClain, 1991). For implementation of tit-for-tat strategy, the most successful strategy for Prisoner’s Dilemma, we used the FimE (Fig. 3-3-1-2.).
Fig.3-3-1-2.Prisoner A with tit-for-tat strategy
Our biggest achievement is that we have established a tripartite relationship between the three fim related parts, fim switch and FimB/FimE (Fig. 3-3-1-3).
	Fig.3-3-1-3.Overview of the fim related parts in iGEM

2. How does fim switch works

In the wild-type of E.coli K-12, two fim proteins invert the fim switch, which is a specific 314 bp DNA sequence containing a promoter, to modulate its own expressions (Abraham, 1985). The fim switch has two states, ON and OFF. The promoter in the fim switch directs transcription to the right when the fim switch is in [ON] state. In [OFF] state of the fim switch, on the other hand, the promoter directs transcription to the left.
	Fig.3-3-2-1.The relationship between the fim related genes.

The fim switch is inverted by two recombinases, FimB(wild-type) (BBa_K1632010) and FimE(wild-type) (BBa_K1632011). These proteins have distinct activities. FimB inverts the fim switch in [ON] state to [OFF] state direction and in [OFF] state to [ON] state direction with approximately equal probability. On the other hand, FimE inverts the fim switch predominantly in [ON] state to [OFF] state direction (McClain, 1991).

　　　

We designed two fim switches: the fim switch(wild-type) and the fim switch(Tokyo_Tech) which promoter can be exchanged with an arbitrary promoter.

　　　

In the first fim switch, the fim switch(wild-type), the sequence is derived from that of a wild-type. The fim switch(wild-type) we constructed are named fim switch[default ON](wild-type) (BBa_(BBa_K1632004) and fim switch[default OFF](wild-type) (BBa_K1632005). In the second fim switch, the fim switch(Tokyo_Tech), the promoter of the fim switch(wild-type) is replaced with J23119 promoter(BBa_J23119) and two restriction enzyme cut sites are added in each side of the promoter. Due to this addition of the restriction enzyme cut sites, we were able to replace the J23119 promoter in the fim swtich(Tokyo_Tech). For example, we replaced J23119 promoter with Lac promoter (BBa_K1632006). The fim switch(Tokyo_Tech) we constructed are named fim switch[default ON](Tokyo_Tech/J23119) (BBa_K1632000), fim switch[default OFF](Tokyo_Tech/J23119) (BBa_K1632000), and fim switch[default ON](Tokyo_Tech/B0010) (BBa_K1632006).

2.1 The fim switch(wild-type)

Fig. 3-3-2-2. The overview of the fim switch(wild-type)

We designed the fim switch(wild-type). The sequence of our fim switch(wild-type) is derived from that of a wild-type. The fim switch(wild-type) has a sigma 70 promoter which functions constitutively. We submitted two parts, one in the [ON] state (BBa_K1632004) and the other in [OFF] state (BBa_K1632005).

We confirmed the inversion of our fim switch(wild-type) by FimB and by FimE. Considering the experimental results, fimB(wild-type) inverts the fim switch(wild-type) in both [ON] state to [OFF] state and [OFF] state to [ON] state with approximately equal probability. Similarly FimE(wild-type) inverts the fim switch(wild-type) predominantly in [ON] state to [OFF] state. In order to confirm inversion more precisely, we also show the percentage of [ON] state with induction by arabinose and without induction. Also we show inversion in the level of DNA sequencing.

In summary, establishing a tripartite relationship between the fim switch and FimB/FimE is an unprecedented achievement in iGEM.

2.1.1. Result of the FimB/FimE assay on the fim switch(wild-type)

Fig.3-3-2-3. New plasmids we constructed to confirm the function of fim switch

The fim switch is inverted by two recombinases, FimB(wild-type) (BBa_K1632010) and FimE(wild-type) (BBa_K1632011). These proteins have distinct activities. FimB inverts the fim switch in both [ON] state to [OFF] state and [OFF] state to [ON] state direction with approximately equal probability. On the other hand, FimE inverts the fim switch predominantly in [ON] state to [OFF] state (McClain, 1991).

In order to assay the function of FimB(wild-type) and FimE(wild-type) for fim switch(wild-type), we added a GFP coding sequence on the downstream of the fim switch.The fim switch[default ON](wild-type)_gfp (BBa_K1632007) emitts fluorescence when expressed, while the fim switch [default OFF](wild-type)_gfp(BBa_K1632008) does not emit florescence when expressed. We also added p_BAD/araC (BBa_I0500) on the upstream of both fimB(wild-type) and fimE(wild-type). p_BAD/araC_fimB(wild-type) (BBa_K1632012) and p_BAD/araC_fimE(BBa_K1632013) can induce the expression of FimB(wild-type) or FimE(wild-type) in the presence of arabinose. We co-transformed a fim switch_gfp and a p_BAD/araC_fimB or _fimE in the E. coli DH5alpha strain(Fig. 3-3-2-3.). We measured the fluorescence intensity of the cells induced by different concentraions of arabinose. From the experimental results(Fig. 3-3-2-4), FimB(wild-type) inverted the fim switch[default ON](wild-type) from [ON] state to [OFF] state and the fim switch[defult OFF](wild-type) from [OFF] state to [ON] state, depending on the concentration of arabinose. FimE(wild-type) inverted the fim switch[default ON](wild-type) from [ON] state to [OFF] state but did not invert the fim switch[default OFF](wild-type) from [OFF] state to [ON] state, depending on the concentration of arabinose.

Fig.3-3-2-4. The intensity of fluroscence in cells measured using flowcytometer.

2.1.1.1 FimB dependent fim switch(wild-type) state assay

　　　

We newly constructed plasmid, PBAD/araC_fimB(wild-type) (BBa_K1632012) that produces FimB(wild-type). We also prepared two other new plasmids, (BBa_K1632007 and (BBa_K1632008(Fig. 3-5-1-2). (BBa_K1632012 enables arabinose-inducible expression of FimB(wild-type). In both [ON] state and [OFF] state, fim switch(wild-type) is placed upstream of GFP coding sequence.
	Fig.3-3-2-5.New plasmids we constructed to confirm the function of fim switch.
	First, we measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results (Fig. 3-3-2-6.), we confirmed that the fim switch (wild-type) is inverted from both [ON] state to [OFF] state and [OFF] state to [ON] state.
Fig. 3-3-2-6.The intensity of fluroscence in cells measured using flowcytometer
First, we measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results (Fig. 3-3-2-6.), we confirmed that the fim switch (wild-type) is inverted from both [ON] state to [OFF] state and [OFF] state to [ON] state. 　　　  Next, in order to confirm inversion more precisely, we also show the percentage of ON state (Fig. 3-3-2-7) and inversion in the level of DNA sequencing (Fig. 3-3-2-8). Fig.3-3-2-7 and Fig.3-3-2-8 clearly shows inversion from both [ON] state to [OFF] state and [OFF] state to [ON] state. E. coli harboring the [ON] fim switch plasmid glows in green fluorescence while E. coli harboring the [OFF] fim switch plasmid does not glow under ultra violet light. Cells transformed by the plasmid mixture extracted from the experiment formed two types of colonies: those with strong fluorescence and those with little background fluorescence. 　　　  Also, sequence complementarity in the specific region of the fim switch(wild-type) shows intended inversion of the fim switch from [ON] state to [OFF] state. Furthermore, the nearly equal number of the colonies between fluorescent and non-fluorescent suggest random inversion of the fim switch by FimB expression.
Fig. 3-3-2-7. Determination of percetage of ON state and colony formation using plasmid mixture extracted cell expressing FimB.	Fig. 3-3-2-8. DNA sequencing results of fim switch from [ON] state to [OFF] state

僕は今ここにいる。

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.

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

　　　　　　

HbiF

Gene Name	Year	College	Parts Number	Sequence Confirmation
HbiF	2012	Michigan	BBa_K88000	None

　　　　　　

fim switch

Gene Name	Year	College	Parts Number	Sequence Confirmation
fim switch inverted repeat left IRL natural	2012	Michigan	BBa_K1077000	Inconsistent
fim switch inverted repeat left IRR natural	2012	Michigan	BBa_K1077001	Inconsistent
J23100 fim switch b0034 GFP	2012	Michigan	BBa_K1077003	Inconsistent
J23100 fim switch b0034 GFP	2012	Michigan	BBa_K1077005	Inconsistent
J23100 fim switch b0034 amilCP ON orientation	2012	Michigan	BBa_K1077007	Inconsistent

　　　　　　

This system was made by 2013_Michigan.The characteristics of their fim switch is to switch the forced ON-to-OFF or OFF-to-ON by applying aTc or AHL.

Fig.3-3-1-1. The detail of their fim switch is shown below.

Fig.3-3-1-1. もう知らん

　　　　　　

The repression of tet repressor for tet promoter is weakened by the addition of aTc. Therefore, fimE (BBa_K137007), (which is on the) downstream of the tet promoter is expressed. As a result, fim switch is inverted from ON-to-OFF. On the other hand, lux promoter is activated by adding AHL. Therefore, hbiF(BBa_K880000) , (which is on the) downstream of lux promoter is expressed. The hbiF protein inverts fim switch in the OFF-to-ON direction. In other words, the hbiF protein has function completely opposite to the fimE protein.
    Two big problems are confirmed from the point of unbiased view.
    First, their fim switch and fimE did not work completely well because the result of default ON was only shown on their wiki and parts registry. In other words, the data of default OFF was not shown. Without the data of default OFF, the fimE protein work ON-to-OFF direction. Second, it is very difficult to judge working parts correctly because setting positive control and negative control was not enough in all experiments.
    It was impossible to verify the reproducibility because the nucleotide sequence was completely different between the distribution from HQ and the information shown on parts registry.
   

4. Reference

　　　　　　

1. 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.
2. 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 USA 82(17):5724-7
3. Ian C. Blomfield et al. (1997) Integration host factor stimulates both FimB- andFimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli. Molecular Microbiology 23(4), 705–717
4. 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 5. D L Gally et al. (1993) Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media. J Bacteriol. Oct; 175(19): 6186–6193.