Latest revision as of 03:52, 19 September 2015

Overview of fim inversion system

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_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, P_BAD/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

For more information, see experiment page.

2.1.1.2 FimE dependent fim switch(wild-type) state assay

To confirm the function of fim switch(wild-type) in the presence of FimE(wild-type), we constructed Biobrick parts, (BBa_K1632013 as Fig. 3-3-2-9. (BBa_K1632013 enables arabinose-inducible expression of the FimE (wild-type). In (BBa_K1632007 and (BBa_K1632008, either the fim switch [default ON] or the fim switch [default OFF] is placed upstream of the GFP coding sequence.
	Fig.3-3-2-9.New plasmids we constructed to confirm the function of fim switch.
	Our purpose is to confirm that FimE (wild-type) inverts the fim switch (wild-type) from [ON] state to [OFF] state predominantly. First of all, we measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results (Fig. 3-3-2-10), we confirmed that our fim switch(wild-type) is inverted from [ON] state to [OFF] state predominatly.
Fig. 3-3-2-10.The intensity of fluroscence in cells measured using flowcytometer
Next, in order to confirm inversion more precisely, we also show the percentage of ON state (Fig. 3-3-2-11) and inversion in the level of DNA sequencing (Fig. 3-3-2-12). Fig. 3-3-2-11 shows inversion from [ON] state to [OFF] state predominantly. E. coli harboring the [ON] fim switch(wild-type) plasmid glows in green fluorescence while E. coli harboring the [OFF] fim switch(wild-type) 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 switch shows intended inversion of the fim switch(wild-type) from [ON] state to [OFF] state. Considering the percentage of [ON] state, FimE(wild-type) inverts the fim switch predominantly in [ON] state to [OFF] state.

Fig. 3-3-2-11. Determination of percetage of ON state and colony formation using plasmid mixture extracted cell expressing FimE.	Fig. 3-3-2-12. DNA sequencing results of fim switch.

For more information, see experiment page.

2.2. The fim switch(Tokyo_Tech)


Fig. 3-3-2-13. The overview of fim switch(Tokyo_Tech)

We designed a fim switch, which promoter can be exchanged with an arbitrary promoter, the fim switch(Tokyo_Tech) (Fig. 3-3-2-13).

We could not confirm the inversion of the fim switch(Tokyo_Tech) by FimE(wild-type). From the experimental results, FimB(wild-type) inverts the fim switch in both [ON] state to [OFF] state and [OFF] state to [ON] state as expected. However, FimE(wild-type) unexpectedly did not invert the fim switch(Tokyo_Tech) predominantly in [ON] state to [OFF] state. Instead, FimE(wild-type) inverted the fim switch(Tokyo_Tech) in both [ON] state to [OFF] state and in [OFF] state to [ON] state. In other words, FimE(wild-type) worked in the same way as FimB(wild-type).

Basically, the design of the fim switch(Tokyo_Tech) is similar to the fim switch(wild-type). The only difference is that we inserted restriction cut enzyme sites to fim switch(Tokyo_Tech). In detail, in fim switch(Tokyo_Tech), the sigma 70 promoter is exchanged to the J23119 promoter(BBa_J23119) and there are two restriction enzyme cut sites inserted each in the front (SalI and BamHI) and in the back (BglII and MluI) of the promoter. Due to the insertion of the restriction enzyme cut sites, fim switch(Tokyo_Tech) has a promoter which can we exchanged with an arbitrary promoter. We actually changed J23119 promoter in the fim switch(Tokyo_Tech) to Lac promoter(BBa_K1632006) as shown in the Fig. 3-3-2-14.


Fig. 3-3-2-14. The flow of changing the promoter in fim switch(Tokyo_Tech)

2.2.1. Results of the FimB/FimE assay on the fim switch(Tokyo_Tech)

As with assays of fim swtich(wild-type), in order to assay the function of FimB(wild-type) and FimE(wild-type) for fim switch(Tokyo_Tech), we added a GFP coding sequence on the downstream of the fim switch(Tokyo_Tech). The fim switch[default ON](Tokyo_Tech/J23119)_gfp (BBa_K1632003) emitts fluorescence when expressed, while the fim switch [default OFF](Tokyo_Tech/J23119)_gfp(BBa_K1632004) 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 (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(Tokyo_Tech)_gfp and a p_BAD/araC_fimB or _fimE in the E. coli DH5alpha strain. We measured the fluorescence intensity of the cells induced by different concentraions of arabinose. We could not confirm the inversion of the fim switch(Tokyo_Tech) by FimE(wild-type). From the experimental results, FimB(wild-type) inverts the fim switch(Tokyo_Tech) in both [ON] state to [OFF] state and [OFF] state to [ON] state as expected. However, FimE(wild-type) unexpectedly did not invert the fim switch(Tokyo_Tech) predominantly in [ON] state to [OFF] state. Instead, FimE(wild-type) inverted the fim switch(Tokyo_Tech) in both [ON] state to [OFF] state and [OFF] state to [ON] state. In other words, FimE(wild-type) worked in the same way as FimB(wild-type).

For more information, see the parts registry page(FimB_dependent_fim_switch_state_assay).

3. Comparison other team’s fim switch

Our biggest achievement is that we have established a tripartite relationship between the three fim related parts, the fim switch and FimB/FimE.

In this chapter, the novelty of constructing and assaying all three fim related parts (the fim switch, FimB, and FimE) is shown by comparison to the past iGEM teams’ fim related parts. Some teams have submitted fim related parts. However the information of the fim related parts are so complicated that we felt that it is necessary to clarify the situation.

These submitted parts which show some data, are divided into three groups as shown in the figure below (Fig. 3-3-3-1.)


Fig. 3-3-3-1. All past iGEM teams’ fim related parts.

From the results of each group, we evaluated each group’s data as shown below.
From these evaluations, we concluded that our establishment of a tripartite relationship between the fim switch and FimB/FimE, is an unprecedented achievement in iGEM.


Fig. 3-3-3-2. Summary of the fim related parts in iGEM.

3.1. fimB (2013 Toronto) : No data about inversion


Fig. 3-3-3-3. Overview of the fim related parts in iGEM.

FimB

Gene Name	Year	College	Parts Number	Sequence Confirmation
FimB	2013	Toronto	BBa_K1019000	None

Fig. 3-3-3-4. Lists of submitted fim parts.

2013_Toronto submitted fimB (BBa_K1019000) and showed some data. However, their data clearly did not relate to the inversion of fim switch. Their experimental result is to search the difference of certain parameters like OD600, in the condition of FimB (+) and FimB (-). Therefore, it can be concluded that the results of 2013_Toronto did not show any data referring to the inversion of the fim switch.

For detailed information, see Team:Toronto

3.2. fim switch(2008_Caltech)-fimE(2008_Caltech) : No data about inversion


Fig. 3-3-3-5. Overview of the fim related parts in iGEM.

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

Fig.3-3-3-6.Lists of submitted fim parts.


Fig. 3-3-3-7. The experimental result of 2008_Caltech

2008_Caltech only submitted fimE (BBa_K137007) and showed data about inversion (Fig. 3-3-3-7.). 2008_Caltech wanted to determine the range of length in which fimE (BBa_K137007) will function and to determine how the length affects the activities of FimE. Thus they tested how the length of the recombination site affects FimE’s ability to detect the two flanking binding sites. Therefore, 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. Because of the lack of controls, we cannot confirm that FimE inverts the fim switch in [ON] state to [OFF] state predominantly. Secondly, they didn’t write down the information of plasmids used in the experiments. The nucleotide sequence of 2008_Caltech’s fimE (BBa_K137007) and FimE(wild type) which was obtained from Ecogene did not match. Therefore, we designed FimE(wild-type) (BBa_K1632010) ) whose nucleotide sequence completely matchs that of the wild type’s 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 FimE(wild-type) by C-terminal 2 residues as shown in Fig. 3-3-3-8.


Fig. 3-3-3-8. The difference of nucleotide sequence between BBa_K137007) and BBa_K1632011) .

For detailed information, see 2008_Caltech wiki)

3.3. fim switch(2013_Michigan)-fimE(2008_Caltech)-hbiF(2012_Michigan) : Not enough data about inversion


Fig. 3-3-3-9. Overview of the fim related parts in iGEM.

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

Fig. 3-3-3-10.Lists of submitted fim parts.


Fig. 3-3-3-11. Overview of the fim related parts in iGEM.

The characteristic of the part (BBa_K1077002) ) constructed by Michigan, is that it can be inverted in [ON] state to [OFF] state direction or in [OFF] state to [ON] state done-sidedly with induction of aTc or AHL. The repression of the tet promoter by the tet repressor is weakened by the addition of aTc. Therefore, fimE (BBa_K137007) ), which is on the downstream of the tet promoter is expressed by inducing aTc. As a result, the fim switch is inverted from [ON] state to [OFF] state. In other words, the target gene which is on the downstream of the fim switch, is not expressed.

On the other hand, by the addition of AHL, lux promoter is activated. Therefore, hbiF (BBa_K880000) ), which is on the downstream of the lux promoter, is expressed. HbiF inverts the fim switch in [OFF] state to [ON] state. As a result, the target gene which is on the downstream of the fim switch, is expressed.

There are two immense issues to their system, judging from their experimental results. First of all, we could not conclude that their fim switch and fimE is functioning correctly, because the results were only shown for the fim switch[default ON] in their wiki and parts registry. In other words, the data of fim switch[default OFF] was not shown. Without the data of fim switch[default OFF], we can’t conclude that FimE (BBa_K137007) ) inverts the fim switch in [ON] state to [OFF] state predominantly.

Second of all, the results of Team Michigan, lack reproducibility because they have failed to set Positive controls and Negative controls in all of their experiments.

It was impossible to verify the reproducibility because the nucleotide sequence of this part (BBa_K1077002) ) was completely different between the distribution from the HQ and the information shown on the parts registry.

For detailed information, see 2013_Michigan wiki).

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.

@@ Line 25: / Line 25: @@
        <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
        <h3 class="link"><a href="#How">2. How does <i>fim</i>&nbsp;switch works</a></h3>
-        <h3 class="link2"><a href="#Wild">2.1. Wild type <i>fim</i>&nbsp;switch</a></h3>
+        <h3 class="link2"><a href="#Wild">2.1. The <i>fim</i> switch(wild-type)</a></h3>
-        <h3 class="link3"><a href="#tdesign">2.1.1. The design of wild type <i>fim</i>&nbsp;switch </a></h3>
+        <h3 class="link2"><a href="#Tokyofim">2.2. The <i>fim</i> switch(Tokyo_Tech)</a></h3>
-        <h3 class="link3"><a href="#wFimB/FimE">2.1.2. Result of FimB/FimE Assay to fimS(TT)</a></h3>
+         <h3 class="link3"><a href="#wdesign">2.2.1. Results of the FimB/FimE assay on the <i>fim</i> switch(Tokyo_Tech)</a></h3>
-        <h3 class="link3"><a href="#AHL">2.1.3. AHL dependent FimE Assay to fimS(TT)</a></h3>
+       <h3 class="link"><a href="#Comparison">3. Comparison of other teams’ <i>fim</i> related parts</a></h3>
-        <h3 class="link2"><a href="#Tokyofim">2.2. Tokyo_Tech <i>fim</i>&nbsp;switch</a></h3>
+        <h3 class="link2"><a href="#T31">3.1. FimB(2013_Toronto)</a></h3>
-         <h3 class="link3"><a href="#wdesign">2.2.1. The design of Tokyo_Tech <i>fim</i>&nbsp;switch </a></h3>
+        <h3 class="link2"><a href="#32">3.2. <i>fim</i> switch(2006_Caltech)-fimE(2006_Caltech)</a></h3>
-        <h3 class="link3"><a href="#tFimB/FimE">2.2.2. Result of FimB/FimE Assay to fimS(WT)</a></h3>
+        <h3 class="link2"><a href="#33">3.3. <i>fim</i> switch(2013_Michigan)-fimE(2006_Caltech)-hbiF(2012_Michigan)</a></h3>
-       <h3 class="link"><a href="#Comparison">3. Comparison other team’s <i>fim</i> switch</a></h3>
-        <h3 class="link2"><a href="#Toronto">3.1. FimB(2013_Toronto)</a></h3>
-        <h3 class="link2"><a href="#Caltech">3.2. <i>Fim</i>&nbsp;switch(2006_Caltech)-FimE(2006_Caltech)</a></h3>
-        <h3 class="link2"><a href="#Michigan">3.3. <i>Fim</i>&nbsp;switch(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)</a></h3>
        <h3 class="link"><a href="#Reference">4. Reference</a></h3>
        <br>
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        <td width="470px">
 　　　 <p class="text">FimB inverts the <i>fim</i> 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 <i>fim</i> switch by FimB. This is the first case in iGEM to show random inversion of a promoter. </p>
-　　　 <p class="text">FimB inverts the <i>fim</i> 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 <i>fim</i> switch by FimB. This is the first case in iGEM to show random inversion of a promoter.  </p>
        </td>
        <td width="470px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a4/Tokyo_Tech_fim_over_view1.png" width="400px"/>
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        <td width="940px">&nbsp;</td>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-1-1.</strong>In the presence of FimB , promoter in <i>fim</i> switch can be inverted at random.</h4>
+       <h4 align="center" class="fig"><strong>Fig.3-3-1-1.</strong>In the presence of FimB, promoter in <i>fim</i> switch can be inverted at random.</h4>
        <td>
        </tr>
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 　　　 <p class="text">On the other hand, FimE inverts the <i>fim</i> 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.).</p></tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-1-1.</strong>図の名前を変更In the presence of FimB , promoter in <i>fim</i> switch can be inverted at random.</h4></td>
+       <h4 align="center" class="fig"><strong>Fig.3-3-1-2.</strong>Prisoner A with tit-for-tat strategy</h4></td></tr>
+      <tr>
+      <td width="470px">
+　　　 <p class="text">Our biggest achievement is that we have established a tripartite relationship between the three <i>fim</i> related parts, <i>fim</i> switch and FimB/FimE (Fig. 3-3-1-3).</p>
+      </td>
+      <td width="470px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8b/Tokyo_Tech_fim_over_view3.png" width="400px"/>
+      </td>
+      </tr>
+      <tr>
+      <td width="940px">&nbsp;</td>
+      <td width="940px">
+      <h4 align="center" class="fig"><strong>Fig.3-3-1-3.</strong>Overview of the <i>fim</i> related parts in iGEM</h4>
+      <td>
+      </tr>
        </table>
+          <h2 id="How" class="smalltitle">2. How does <i>fim</i> switch works</h2>
+    <table width="940 px" border="0px">
+      <tr>
+      <td width="470px">
+　　　 <p class="text">In the wild-type of E.coli K-12, two fim proteins invert the <i>fim</i> switch, which is a specific 314 bp DNA sequence containing a promoter, to modulate its own expressions (Abraham, 1985).  The <i>fim</i> switch has two states, ON and OFF.  The promoter in the <i>fim</i> switch directs transcription to the right when the <i>fim</i> switch is in [ON] state.  In [OFF] state of the <i>fim</i> switch, on the other hand, the promoter directs transcription to the left.  </p></td>
+      <td width="470px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/17/Tokyo_Tech_fim_over_view4.png" width="400px"/>
+      </td>
+      </tr>
+      <tr>
+      <td width="940px">&nbsp;</td>
+      <td width="940px">
+      <h4 align="center" class="fig"><strong>Fig.3-3-2-1.</strong>The relationship between the <i>fim</i> related genes.</h4>
+      <td>
+      </tr>
+     </table>
+           <p class="text">The <i>fim</i> switch is inverted by two recombinases, FimB(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632010">BBa_K1632010</a>) and FimE(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632011">BBa_K1632011</a>). These proteins have distinct activities. FimB inverts the <i>fim</i> 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 <i>fim</i> switch predominantly in [ON] state to [OFF] state direction (McClain, 1991). </p>
+　　　 <p class="text">We designed two <i>fim</i> switches: the <i>fim</i> switch(wild-type) and the <i>fim</i> switch(Tokyo_Tech) which promoter can be exchanged with an arbitrary promoter.</p>
+　　　 <p class="text">In the first <i>fim</i> switch, the <i>fim</i> switch(wild-type), the sequence is derived from that of a wild-type. The <i>fim</i> switch(wild-type) we constructed are named <i>fim</i> switch[default ON](wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632004">BBa_K1632004</a>) and <i>fim</i> switch[default OFF](wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632005">BBa_K1632005</a>).
+In the second <i>fim</i> switch, the <i>fim</i> switch(Tokyo_Tech), the promoter of the <i>fim</i> 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 <i>fim</i> swtich(Tokyo_Tech).  For example, we replaced J23119 promoter with Lac promoter (<a href="http://parts.igem.org/Part:BBa_K1632006">BBa_K1632006</a>).  The <i>fim</i> switch(Tokyo_Tech) we constructed are named <i>fim</i> switch[default ON](Tokyo_Tech/J23119) (<a href="http://parts.igem.org/Part:BBa_K1632000">BBa_K1632000</a>), <i>fim</i> switch[default OFF](Tokyo_Tech/J23119) (<a href="http://parts.igem.org/Part:BBa_K1632000">BBa_K1632000</a>), and <i>fim</i> switch[default ON](Tokyo_Tech/B0010) (<a href="http://parts.igem.org/Part:BBa_K1632006">BBa_K1632006</a>).</p>
+              <h3 id="Wild" class="sub5">2.1 The <i>fim</i> switch(wild-type)</h3>
-          <h2 id="How" class="smalltitle">2. How does <i>fim</i> switch works</h2>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8b/Tokyo_Tech_fim_over_view3.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/2/20/Tokyo_Tech_fim_over_view5.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-2-1.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-2-2.</strong>&nbsp;The overview of the <i>fim</i> switch(wild-type)</h4>
        <td>
        </tr>
        </table>
-　　　　　　<p class="text">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).
+                    <p class="text3">We designed the <i>fim</i> switch(wild-type). The sequence of our <i>fim</i> switch(wild-type) is derived from that of a wild-type.  The <i>fim</i> switch(wild-type) has a sigma 70 promoter which functions constitutively.  We submitted two parts, one in the [ON] state (<a href="http://parts.igem.org/Part:BBa_K1632004">BBa_K1632004</a>) and the other in [OFF] state (<a href="http://parts.igem.org/Part:BBa_K1632005">BBa_K1632005</a>).</p>
-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).
+                    <p class="text3">We confirmed the inversion of our <i>fim</i> switch(wild-type) by FimB and by FimE.  Considering the experimental results, fimB(wild-type) inverts the <i>fim</i> 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 <i>fim</i> 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.</p>
-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.
+                    <p class="text3">In summary, establishing a tripartite relationship between the <i>fim</i> switch and FimB/FimE is an unprecedented achievement in iGEM. </p>
-</p><br>
+               <h3 id="tdesign" class="sub6">2.1.1. Result of the FimB/FimE assay on the <i>fim</i> switch(wild-type)</h3>
-              <h3 id="Wild" class="sub5">2.1. Wild type <i>fim</i> switch</h3>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8b/Tokyo_Tech_fim_over_view3.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/6/6f/Tokyo_Tech_fim_over_view6.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-2-1-1.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig.3-3-2-3.</strong>&nbsp;New plasmids we constructed to confirm the function of <i>fim</i> switch</h4>
        <td>
        </tr>
        </table>
-                     <p class="text3">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)
+                     <p class="text3">The <i>fim</i> switch is inverted by two recombinases, FimB(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632010">BBa_K1632010</a>) and FimE(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632011">BBa_K1632011</a>). These proteins have distinct activities. FimB inverts the <i>fim</i> 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 <i>fim</i> switch predominantly in [ON] state to [OFF] state (McClain, 1991). </p>
-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.
+                    <p class="text3">In order to assay the function of FimB(wild-type) and FimE(wild-type) for <i>fim</i> switch(wild-type), we added a GFP coding sequence on the downstream of the <i>fim</i> switch.The <i>fim</i> switch[default ON](wild-type)_gfp (<a href="http://parts.igem.org/Part:BBa_K1632007">BBa_K1632007</a>) emitts fluorescence when expressed, while the <i>fim</i> switch [default OFF](wild-type)_gfp(<a href="http://parts.igem.org/Part:BBa_K1632008">BBa_K1632008</a>) does not emit florescence when expressed. We also added p<sub>BAD/<i>araC</i></sub> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) on the upstream of both fimB(wild-type) and fimE(wild-type). p<sub>BAD/<i>araC</i></sub>_<i>fimB</i>(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632012">BBa_K1632012</a>) and p<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(<a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a>) can induce the expression of FimB(wild-type) or FimE(wild-type) in the presence of arabinose. We co-transformed a <i>fim</i> switch_gfp and a p<sub>BAD/<i>araC</i></sub>_<i>fimB</i> or _<i>fimE</i> in the <i>E. coli</i> 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 <i>fim</i> switch[default ON](wild-type) from [ON] state to [OFF] state and the <i>fim</i> switch[defult OFF](wild-type) from [OFF] state to [ON] state, depending on the concentration of arabinose.  FimE(wild-type) inverted the <i>fim</i> switch[default ON](wild-type) from [ON] state to [OFF] state but did not invert the <i>fim</i> switch[default OFF](wild-type) from [OFF] state to [ON] state, depending on the concentration of arabinose.
 </p>
-               <h3 id="tdesign" class="sub6">2.1.1. The design of wild type <i>fim</i>&nbsp;switch</h3>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/38/Tokyo_Tech_fim_over_view7.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-2-1-1.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig.3-3-2-4.</strong>&nbsp;The intensity of fluroscence in cells measured using flowcytometer.</h4>
        <td>
        </tr>
        </table>
-                    <p class="text4">Sigma 70 promoter is located in fim switch (WT).<br>&nbsp;&nbsp;&nbsp;
+<p class="text4"><strong>2.1.1.1 FimB dependent <i>fim</i> switch(wild-type) state assay</strong></p>
-Switching is also influenced by at least three global regulator, leucine-responsive regulatory protein (Lrp), H-NS and integration host factor (IHF).<br>&nbsp;&nbsp;&nbsp;
+<table width="800 px" border="0px">
-Switching frequencies are regulated by both temperature media and that these effects appear to be independent.<br>&nbsp;&nbsp;&nbsp;
+      <tr>
-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].<br>&nbsp;&nbsp;&nbsp;
+      <td width="400px">
-</p>
+　　　 <p class="text">We newly constructed plasmid, P<sub>BAD/<i>araC</i></sub>_<i>fimB</i>(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632012">BBa_K1632012</a>) that produces FimB(wild-type).  We also prepared two other new plasmids, (<a href="http://parts.igem.org/Part:BBa_K1632007">BBa_K1632007</a> and (<a href="http://parts.igem.org/Part:BBa_K1632008">BBa_K1632008</a>(Fig. 3-5-1-2).  (<a href="http://parts.igem.org/Part:BBa_K1632012">BBa_K1632012</a> enables arabinose-inducible expression of FimB(wild-type).  In both [ON] state and [OFF] state, <i>fim</i> switch(wild-type) is placed upstream of GFP coding sequence. </p>
-詳細版
+      </td>
-IHF LRP強調
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8e/Tokyo_Tech_fimB_summary1.png" width="350px"/>
-RE
+      </td>
-               <h3 id="wFimB/FimE" class="sub6">2.1.2. Result of FimB/FimE Assay to fimS(TT)</h3>
+      </tr>
-                    <p class="text4">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.<br>&nbsp;&nbsp;&nbsp;
+      <tr>
-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.</p>
+      <td width="400px">&nbsp;</td>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig.3-3-2-5.</strong>New plasmids we constructed to confirm the function of </i>fim</i> switch.</h4>
+      <td>
+      </tr>
+      <tr>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/4/4e/Tokyo_Tech_fim_over_view8.png" width="200px"/></div></td>
+      <td width="400px">
+　　　 <p class="text">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 <i>fim</i> switch (wild-type) is inverted from both [ON] state to [OFF] state and [OFF] state to [ON] state. </p></tr>
+　　　<tr>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-6.</strong>The intensity of fluroscence in cells measured using flowcytometer</h4></td><td width="400px">&nbsp;</td>
+</tr><tr>
+      <td width="400px" colspan="2">
+　　　 <p class="text4">&nbsp;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 <i>fim</i> switch (wild-type) is inverted from both [ON] state to [OFF] state and [OFF] state to [ON] state. </p>
+　　　 <p class="text4">&nbsp;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.  <i>E. coli</i> harboring the [ON] <i>fim</i> switch plasmid glows in green fluorescence while <i>E. coli</i> harboring the [OFF] <i>fim</i> 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.</p>
+　　　 <p class="text4">&nbsp;Also, sequence complementarity in the specific region of the <i>fim</i> switch(wild-type) shows intended inversion of the <i>fim</i> 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 <i>fim</i> switch by FimB expression.</p></td>
+<tr>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/3e/Tokyo_Tech_fimB_result1.png" width="350px"/></div></td>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/9/9d/Tokyo_Tech_fimB_result2.png" width="350px"/></div></td>
+<tr>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-7.</strong>  Determination of percetage of ON state and colony formation using plasmid mixture extracted cell expressing FimB.</h4>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-8.</strong>  DNA sequencing results of <i>fim</i> switch from [ON] state to [OFF] state</h4>
+</tr>
+</table>
+<p class="text4">For more information, see <a href="https://2015.igem.org/Team:Tokyo_Tech/Experiment/FimB_dependent_fim_switch_state_assay">experiment page.</a></p>
+<p class="text4"><strong>2.1.1.2 FimE dependent <i>fim</i> switch(wild-type) state assay</strong></p>
+<table width="800 px" border="0px">
+      <tr>
+      <td width="400px">
+　　　 <p class="text">To confirm the function of <i>fim</i> switch(wild-type) in the presence of FimE(wild-type), we constructed Biobrick parts, (<a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a> as Fig. 3-3-2-9.  (<a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a> enables arabinose-inducible expression of the FimE (wild-type).  In (<a href="http://parts.igem.org/Part:BBa_K1632007">BBa_K1632007</a> and (<a href="http://parts.igem.org/Part:BBa_K1632008">BBa_K1632008</a>, either the <i>fim</i> switch [default ON] or the <i>fim</i> switch [default OFF] is placed upstream of the GFP coding sequence. </p>
+      </td>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8a/Tokyo_Tech_arabinosefimEsummary.png" width="350px"/>
+      </td>
+      </tr>
+      <tr>
+      <td width="400px">&nbsp;</td>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig.3-3-2-9.</strong>New plasmids we constructed to confirm the function of </i>fim</i> switch.</h4>
+      <td>
+      </tr>
+      <tr>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f4/Tokyo_Tech_fim_over_view9.png" width="200px"/></div></td>
+      <td width="400px">
+　　　 <p class="text">Our purpose is to confirm that FimE (wild-type) inverts the <i>fim</i> switch (wild-type) from [ON] state to [OFF] state predominantly. First of all, we measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results (Fig. 3-3-2-10), we confirmed that our <i>fim</i> switch(wild-type) is inverted from [ON] state to [OFF] state predominatly.</p></tr>
+　　　<tr>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-10.</strong>The intensity of fluroscence in cells measured using flowcytometer</h4></td><td width="400px">&nbsp;</td>
+</tr><tr>
+      <td width="400px" colspan="2">
+　　　 <p class="text4">&nbsp;Next, in order to confirm inversion more precisely, we also show the percentage of ON state (Fig. 3-3-2-11) and inversion in the level of DNA sequencing (Fig. 3-3-2-12).  Fig. 3-3-2-11 shows inversion from [ON] state to [OFF] state predominantly.  <i>E. coli</i> harboring the [ON] <i>fim</i> switch(wild-type) plasmid glows in green fluorescence while <i>E. coli</i> harboring the [OFF] <i>fim</i> switch(wild-type) 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 switch shows intended inversion of the <i>fim</i> switch(wild-type) from [ON] state to [OFF] state. Considering the percentage of [ON] state, FimE(wild-type) inverts the <i>fim</i> switch predominantly in [ON] state to [OFF] state.</p>
+　　　 <p class="text4">&nbsp;</p></td>
+<tr>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/7/70/Tokyo_Tech_fimE_5555555.png" width="350px"/></div></td>
+      <td width="400px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/19/Tokyo_Tech_arabinosefimE_result2.png" width="350px"/></div></td>
+<tr>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-11.</strong>  Determination of percetage of ON state and colony formation using plasmid mixture extracted cell expressing FimE.</h4>
+      <td width="400px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-2-12.</strong> DNA sequencing results of <i>fim</i> switch.</h4>
+</tr>
+</table>
+<p class="text4">For more information, see <a href="https://2015.igem.org/Team:Tokyo_Tech/Experiment/FimE_dependent_fim_switch_state_assay">experiment page.</a></p>
-<p class="text4"><strong>2.1.2.1 FimS-FimB</strong></p>
-In the experiments, we setted four arabinose concentration.
-<p class="text4"><strong>2.1.2.2 FimS-FimE</strong></p>
-アッセイから具体的な文章/データはもらう
-               <h3 id="AHL" class="sub6">2.1.3. AHL dependent FimE Assay to fimS(TT)</h3>
-                    <p class="text4">アッセイから具体的な文章/データはもらう</p>
-              <h3 id="Tokyofim" class="sub5">2.2. Tokyo_Tech <i>fim</i>&nbsp;switch</h3>
+              <h3 id="Tokyofim" class="sub5">2.2. The <i>fim</i> switch(Tokyo_Tech)</h3>
-                     <p class="text">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.</p>
+<table width="940 px" border="0px">
-               <h3 id="wdesign" class="sub6">2.2.1. The design of wild type <i>fim</i>&nbsp;switch</h3>
+      <tr>
-                    <p class="text4">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.<br>&nbsp;&nbsp;&nbsp;
+      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/5/54/Tokyo_Tech_fim_over_view10.png" width="500px"/>
-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.<br>&nbsp;&nbsp;&nbsp;</p>
+      </td>
-               <h3 id="tFimB/FimE" class="sub6">2.2.2. Result of FimB/FimE Assay to fimS(TT)</h3>
+      </tr>
-                    <p class="text4"></p>
+      <tr>
+      <td width="940px">
+      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-3-2-13.</strong>&nbsp; The overview of <i>fim</i> switch(Tokyo_Tech)</h4>
+      <td>
+      </tr>
+      </table>
+                    <p class="text2">We designed a <i>fim</i> switch, which promoter can be exchanged with an arbitrary promoter, the <i>fim</i>  switch(Tokyo_Tech) (Fig. 3-3-2-13). </p>
+                     <p class="text2">We could not confirm the inversion of the <i>fim</i> switch(Tokyo_Tech) by FimE(wild-type).  From the experimental results, FimB(wild-type) inverts the <i>fim</i> switch in both [ON] state to [OFF] state and [OFF] state to [ON] state as expected.  However, FimE(wild-type) unexpectedly did not invert the <i>fim</i> switch(Tokyo_Tech) predominantly in [ON] state to [OFF] state.  Instead, FimE(wild-type) inverted the <i>fim</i> switch(Tokyo_Tech) in both [ON] state to [OFF] state and in [OFF] state to [ON] state. In other words, FimE(wild-type) worked in the same way as FimB(wild-type).</p>
+                    <p class="text2">Basically, the design of the <i>fim</i> switch(Tokyo_Tech) is similar to the <i>fim</i> switch(wild-type). The only difference is that we inserted restriction cut enzyme sites to <i>fim</i> switch(Tokyo_Tech). In detail, in <i>fim</i> switch(Tokyo_Tech), the sigma 70 promoter is exchanged to the J23119 promoter(<a href="http://parts.igem.org/Part:BBa_J23119">BBa_J23119</a>) and there are two restriction enzyme cut sites inserted each in the front (SalI and BamHI) and in the back (BglII and MluI) of the promoter. Due to the insertion of the restriction enzyme cut sites, <i>fim</i> switch(Tokyo_Tech) has a promoter which can we exchanged with an arbitrary promoter. We actually changed J23119 promoter in the <i>fim</i> switch(Tokyo_Tech) to Lac promoter(<a href="http://parts.igem.org/Part:BBa_K1632006">BBa_K1632006</a>) as shown in the Fig. 3-3-2-14.
+</p>
+<table width="940 px" border="0px">
+      <tr>
+      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/thumb/5/5c/Tokyo_Tech_fim_over_view11.png/683px-Tokyo_Tech_fim_over_view11.png" width="500px"/>
+      </td>
+      </tr>
+      <tr>
+      <td width="940px">
+      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-3-2-14.</strong>&nbsp;The flow of changing the promoter in <i>fim</i> switch(Tokyo_Tech)</h4>
+      <td>
+      </tr>
+      </table>
+               <h3 id="tFimB/FimE" class="sub6">2.2.1. Results of the FimB/FimE assay on the <i>fim</i> switch(Tokyo_Tech)</h3>
+                    <p class="text3">As with assays of <i>fim</i> swtich(wild-type), in order to assay the function of FimB(wild-type) and FimE(wild-type) for <i>fim</i> switch(Tokyo_Tech), we added a GFP coding sequence on the downstream of the <i>fim</i> switch(Tokyo_Tech). The <i>fim</i> switch[default ON](Tokyo_Tech/J23119)_gfp (<a href="http://parts.igem.org/Part:BBa_K1632003">BBa_K1632003</a>) emitts fluorescence when expressed, while the <i>fim</i> switch [default OFF](Tokyo_Tech/J23119)_gfp(<a href="http://parts.igem.org/Part:BBa_K1632004">BBa_K1632004</a>) does not emit florescence when expressed. We also added p<sub>BAD/<i>araC</i></sub>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) on the upstream of both FimB(wild-type) and FimE(wild-type). p<sub>BAD/<i>araC</i></sub>_<i>fimB</i> (<a href="http://parts.igem.org/Part:BBa_K1632012">BBa_K1632012</a>) and p<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(<a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a>) can induce the expression of FimB(wild-type) or FimE(wild-type) in the presence of arabinose. We co-transformed a <i>fim</i> switch(Tokyo_Tech)_gfp and a p<sub>BAD/<i>araC</i></sub>_<i>fimB</i> or _<i>fimE</i> in the <i>E. coli</i> DH5alpha strain. We measured the fluorescence intensity of the cells induced by different concentraions of arabinose.
+We could not confirm the inversion of the <i>fim</i> switch(Tokyo_Tech) by FimE(wild-type). From the experimental results, FimB(wild-type) inverts the <i>fim</i> switch(Tokyo_Tech) in both [ON] state to [OFF] state  and [OFF] state to [ON] state as expected. However, FimE(wild-type) unexpectedly did not invert the <i>fim</i> switch(Tokyo_Tech) predominantly in [ON] state to [OFF] state. Instead, FimE(wild-type) inverted the <i>fim</i> switch(Tokyo_Tech) in both [ON] state to [OFF] state and [OFF] state to [ON] state. In other words, FimE(wild-type) worked in the same way as FimB(wild-type).</p>
+                    <p class="text3">For more information, see the parts registry page(<a href="https://2015.igem.org/Team:Tokyo_Tech/Experiment/FimB_dependent_fim_switch_state_assay">FimB_dependent_fim_switch_state_assay</a>).</p>
            <h2 id="Comparison" class="smalltitle">3. Comparison other team’s <i>fim</i> switch</a></h2>
-　　　　　　<p class="text">Our big achievement have created tripartite relationship, Fim switch-FimB/FimE.<br>&nbsp;&nbsp;&nbsp;
+　　　　　　<p class="text">Our biggest achievement is that we have established a tripartite relationship between the three <i>fim</i> related parts, the <i>fim</i> switch and FimB/FimE.</p>
-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.</p>
+　　　　　　<p class="text">In this chapter, the novelty of constructing and assaying all three <i>fim</i> related parts (the <i>fim</i> switch, FimB, and FimE) is shown by comparison to the past iGEM teams’ <i>fim</i> related parts.  Some teams have submitted <i>fim</i> related parts.  However the information of the <i>fim</i> related parts are so complicated that we felt that it is necessary to clarify the situation. </p>
+　　　　　　<p class="text">These submitted parts which show some data, are divided into three groups as shown in the figure below (Fig. 3-3-3-1.)</p>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/2/20/Tokyo_Tech_fim_over_view5.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f0/Tokyo_Tech_fim_over_view12.png" width="600px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.A.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-1.</strong>&nbsp;All past iGEM teams’ <i>fim</i> related parts.</h4>
        <td>
        </tr>
        </table>
-　　　　　　<p class="text">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.</p>
+　　　　　　<p class="text">From the results of each group, we evaluated each group’s data as shown below. <br>
-                    <p class="text4">
+From these evaluations, we concluded that our establishment of a tripartite relationship between the <i>fim</i> switch and FimB/FimE, is an unprecedented achievement in iGEM.</p><br>
-. FimB(2013_Toronto)<br>
-	⇒3.1.<br>
-. FimS(2006_Caltech)-FimE(2006_Caltech)<br>
-	⇒3.2.<br>
-. FimS(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)<br>
-	⇒3.3.<br>
-</p>
-　　　　　　    <p class="text2">From the result of each group, each one is evaluated as shown as below.</p>
-　　　　　　    <p class="text2">1. FimB(2013_Toronto)
-	⇒No date about inversion<br>
-. FimS(2006_Caltech)-FimE(2006_Caltech)<br>
-	⇒No date about inversion<br>
-. FimS(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)<br>
-	⇒Not enough date about inversion<br>
-From these evaluations, we achieved to create tripartite relationship, Fim switch-FimB/FimE.<br>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a4/Tokyo_Tech_fim_over_view1.png" />
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/0c/Tokyo_Tech_fim_over_view13.png" width="600px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.南蛮文化.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-2.</strong>&nbsp;Summary of the <i>fim</i> related parts in iGEM.</h4>
        <td>
        </tr>
        </table>
-              <h3 id="Toronto" class="sub5">3.1. FimB(2013_Toronto)</h3>
+               <h3 id="31" class="sub5">3.1. fimB (2013 Toronto) : No data about inversion</h3>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/2/20/Tokyo_Tech_fim_over_view5.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/0c/Tokyo_Tech_fim_over_view13.png" width="600px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.A.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-3.</strong>&nbsp;Overview of the <i>fim</i> related parts in iGEM.</h4>
        <td>
        </tr>
        </table>
-　　　　　　    <p class="text2">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.</p>
+　　　　　　    <p class="text2"><strong>FimB</strong></p>
-              <h3 id="Caltech" class="sub5">3.2. <i>Fim</i>&nbsp;switch(2006_Caltech)-FimE(2006_Caltech)</h3>
+                 <table width="90%" border="2" style="border-collapse: collapse;">
+                   <tbody>
+                      <tr height="20px">
+                       <th width="20%">Gene Name</th>
+                       <th width="10%">Year</th>
+                       <th width="10%">College</th>
+                       <th width="20%">Parts Number</th>
+                       <th width="30%">Sequence Confirmation</th>
+                      </tr>
+                      <tr height="20px">
+                       <td width="20%">FimB</td>
+                       <td width="10%">2013</td>
+                       <td width="10%">Toronto</td>
+                       <td width="20%"><a href="http://parts.igem.org/Part:BBa_K1019000">BBa_K1019000</a></td>
+                       <td width="30%">None</td>
+                       </tr>
+                      </tbody></table><br>
+      <h4 align="center" class="fig"><strong>Fig. 3-3-3-4.</strong> Lists of submitted <i>fim</i> parts.</h4>
+   <p class="text">2013_Toronto submitted fimB (<a href="http://parts.igem.org/Part:BBa_K1019000">BBa_K1019000</a>) and showed some data. However, their data clearly did not relate to the inversion of <i>fim</i> switch. Their experimental result is to search the difference of certain parameters like OD600, in the condition of FimB (+) and FimB (-). Therefore, it can be concluded that the results of 2013_Toronto did not show any data referring to the inversion of the <i>fim</i> switch. </p>
+   <p class="text">For detailed information, see <a href="https://2013.igem.org/Team:Toronto">Team:Toronto</a></p>
+               <h3 id="32" class="sub5">3.2. <i>fim</i> switch(2008_Caltech)-fimE(2008_Caltech) : No data about inversion</h3>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/6/6f/Tokyo_Tech_fim_over_view6.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/af/Tokyo_Tech_fim_over_view14.png" width="600px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.A.</strong>&nbsp;ここに図の名前を打て</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-5.</strong>&nbsp;Overview of the <i>fim</i> related parts in iGEM.</h4>
        <td>
        </tr>
@@ Line 232: / Line 343: @@
                         <td width="30%">None</td>
                         </tr>
-                       </tbody></table><br>
+                       </tbody></table>
 　　　　　　    <p class="text2"><strong><i>fim</i> switch</strong></p>
                   <table width="90%" border="2" style="border-collapse: collapse;"><h3>
@@ Line 300: / Line 421: @@
                         </tr>
                        </tbody></table></h3>
-　　　　　　    <p class="text2">In the past jamboree, 2006_Caltech only submitted FimE(BBa_K137007) and showed some data. Assay protocol is  <a href="https://2008.igem.org/Team:Caltech/Protocols/FimE">here</a>).</p>
+      <h4 align="center" class="fig"><strong>Fig.3-3-3-6.</strong>Lists of submitted <i>fim</i> parts.</h4>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/38/Tokyo_Tech_fim_over_view7.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/thumb/8/84/Tokyo_Tech_fim_over_view15.png/800px-Tokyo_Tech_fim_over_view15.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.A.</strong>&nbsp;2006_Caltech</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-7.</strong>&nbsp; The experimental result of 2008_Caltech</h4>
        <td>
        </tr>
        </table>
-　　　　　　    <p class="text2">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. <br>&nbsp;&nbsp;&nbsp;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).
+　　　　　　    <p class="text2">2008_Caltech only submitted fimE (<a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) and showed data about inversion (Fig. 3-3-3-7.). 2008_Caltech wanted to determine the range of length in which fimE (<a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) will function and to determine how the length affects the activities of FimE. Thus they tested how the length of the recombination site affects FimE’s ability to detect the two flanking binding sites. Therefore, GFP fluorescence / OD600 was observed in each sample in which the length of the inversion site differs. </p>
-<br>&nbsp;&nbsp;&nbsp;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.<p>
+　　　　　　    <p class="text2">However their data cannot be trusted completely with respect to inversion of <i>fim</i> switch. There are several reasons. First of all, they did not set Positive controls and Negative controls. Because of the lack of controls, we cannot confirm that FimE inverts the <i>fim</i> switch in [ON] state to [OFF] state predominantly. Secondly, they didn’t write down the information of plasmids used in the experiments.
-　　　　　　    <p class="text4">2008_Caltech:5’-...-TAA-TAA-Suffix-3’<br>
+The nucleotide sequence of 2008_Caltech’s fimE (<a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) and FimE(wild type) which was obtained from <a href="http://www.ecogene.org/old/geneinfo.php?eg_id=EG10312) ">Ecogene</a> did not match. Therefore, we designed FimE(wild-type) (<a href="http://parts.igem.org/Part:BBa_K1632010">BBa_K1632010</a>) ) whose nucleotide sequence completely matchs that of the wild type’s 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 FimE(wild-type) by C-terminal 2 residues as shown in Fig. 3-3-3-8.
-_TokyoTech	: 5’-...-GTT-TGA-Suffix-3'<br></p>
+</p>
-              <h3 id="Michigan" class="sub5">3.3. <i>Fim</i>&nbsp;switch(2013_Michigan)-FimE(2006_Caltech)-HbiF(2012_Michigan)</h3>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/4/4e/Tokyo_Tech_fim_over_view8.png" width="480px"/>
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/thumb/3/35/Tokyo_Tech_fim_over_view16.png/800px-Tokyo_Tech_fim_over_view16.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-2-1.</strong>&nbsp;タイムアウト</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-8.</strong>&nbsp;The difference of nucleotide sequence between <a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) and <a href="http://parts.igem.org/Part:BBa_K1632011">BBa_K1632011</a>) .</h4>
+      <td>
+      </tr>
+      </table>
+<p class="text2">For detailed information, see <a href="https://2008.igem.org/Team:Caltech">2008_Caltech wiki</a>) </p>
+              <h3 id="33" class="sub5">3.3. <i>fim</i> switch(2013_Michigan)-fimE(2008_Caltech)-hbiF(2012_Michigan) : Not enough data about inversion</h3>
+    <table width="940 px" border="0px">
+      <tr>
+      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/ff/Tokyo_Tech_fim_over_view17.png" width="480px"/>
+      </td>
+      </tr>
+      <tr>
+      <td width="940px">
+      <h4 align="center" class="fig"><strong>Fig. 3-3-3-9.</strong> Overview of the <i>fim</i> related parts in iGEM.</h4>
        <td>
        </tr>
@@ Line 415: / Line 551: @@
                         </tr>
                        </tbody></table>
-　　　　　　    <p class="text2">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.</p>
+      <h4 align="center" class="fig"><strong>Fig. 3-3-3-10.</strong>Lists of submitted <i>fim</i> parts.</h4>
      <table width="940 px" border="0px">
        <tr>
-       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f4/Tokyo_Tech_fim_over_view9.png" />
+       <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/e/ee/Tokyo_Tech_fim_over_view18.png" width="480px"/>
        </td>
        </tr>
        <tr>
        <td width="940px">
-       <h4 align="center" class="fig"><strong>Fig.3-3-1-1.</strong>&nbsp;The detail of their fim switch is shown below.</h4>
+       <h4 align="center" class="fig"><strong>Fig. 3-3-3-11.</strong> Overview of the <i>fim</i> related parts in iGEM.</h4>
        <td>
        </tr>
        </table>
-    <table width="940 px" border="0px">
+<p class="text2">The characteristic of the part (<a href="http://parts.igem.org/Part:BBa_K1077002">BBa_K1077002</a>) ) constructed by Michigan, is that it can be inverted in [ON] state to [OFF] state direction or in [OFF] state to [ON] state done-sidedly with induction of aTc or AHL. The repression of the tet promoter by the tet repressor is weakened by the addition of aTc. Therefore, fimE (<a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) ), which is on the downstream of the tet promoter is expressed by inducing aTc. As a result, the <i>fim</i> switch is inverted from [ON] state to [OFF] state. In other words, the target gene which is on the downstream of the <i>fim</i> switch, is not expressed. </p>
-      <tr>
+<p class="text2">On the other hand, by the addition of AHL, lux promoter is activated. Therefore, hbiF (<a href="http://parts.igem.org/Part:BBa_K880000">BBa_K880000</a>) ), which is on the downstream of the lux promoter, is expressed. HbiF inverts the <i>fim</i> switch in [OFF] state to [ON] state. As a result, the target gene which is on the downstream of the <i>fim</i> switch, is expressed. </p>
-      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/5/54/Tokyo_Tech_fim_over_view10.png" width="600px" />
+ <p class="text2">There are two immense issues to their system, judging from their experimental results.
-      </td>
+First of all, we could not conclude that their <i>fim</i> switch and fimE is functioning correctly, because the results were only shown for the <i>fim</i> switch[default ON] in their wiki and parts registry. In other words, the data of <i>fim</i> switch[default OFF] was not shown. Without the data of <i>fim</i> switch[default OFF], we can’t conclude that FimE (<a href="http://parts.igem.org/Part:BBa_K137007">BBa_K137007</a>) ) inverts the <i>fim</i> switch in [ON] state to [OFF] state predominantly. </p>
-      </tr>
+  <p class="text2">Second of all, the results of Team Michigan, lack reproducibility because they have failed to set Positive controls and Negative controls in all of their experiments.</p>
-      <tr>
+ <p class="text2"> It was impossible to verify the reproducibility because the nucleotide sequence of this part (<a href="http://parts.igem.org/Part:BBa_K1077002">BBa_K1077002</a>) ) was completely different between the distribution from the HQ and the information shown on the parts registry.</p>
-      <td width="940px">
+ <p class="text2"> For detailed information, see <a href="https://2013.igem.org/Team:Michigan">2013_Michigan wiki</a>).</p>　<br>
-      <h4 align="center" class="fig"><strong>Fig.3-3-1-1.</strong>&nbsp;もう知らん</h4>
-      <td>
-      </tr>
-      </table>
-　　　　　　    <p class="text2">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.<br>&nbsp;&nbsp;&nbsp;
-Two big problems are confirmed from the point of unbiased view.<br>&nbsp;&nbsp;&nbsp;
-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.<br>&nbsp;&nbsp;&nbsp;
-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.<br>&nbsp;&nbsp;&nbsp;
-</p><br>

Difference between revisions of "Team:Tokyo Tech/Experiment/Overview of fim inversion system"

Latest revision as of 03:52, 19 September 2015

Overview of fim inversion system

contents

1. Introduction

Fig.3-3-1-1.In the presence of FimB, promoter in fim switch can be inverted at random.

Fig.3-3-1-2.Prisoner A with tit-for-tat strategy

Fig.3-3-1-3.Overview of the fim related parts in iGEM

2. How does fim switch works

Fig.3-3-2-1.The relationship between the fim related genes.

2.1 The fim switch(wild-type)

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

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

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

Fig.3-3-2-5.New plasmids we constructed to confirm the function of fim switch.

Fig. 3-3-2-6.The intensity of fluroscence in cells measured using flowcytometer

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

Fig.3-3-2-9.New plasmids we constructed to confirm the function of fim switch.

Fig. 3-3-2-10.The intensity of fluroscence in cells measured using flowcytometer

Fig. 3-3-2-11. Determination of percetage of ON state and colony formation using plasmid mixture extracted cell expressing FimE.

Fig. 3-3-2-12. DNA sequencing results of fim switch.

2.2. The fim switch(Tokyo_Tech)

Fig. 3-3-2-13. The overview of fim switch(Tokyo_Tech)

Fig. 3-3-2-14. The flow of changing the promoter in fim switch(Tokyo_Tech)

2.2.1. Results of the FimB/FimE assay on the fim switch(Tokyo_Tech)

3. Comparison other team’s fim switch

Fig. 3-3-3-1. All past iGEM teams’ fim related parts.

Fig. 3-3-3-2. Summary of the fim related parts in iGEM.

3.1. fimB (2013 Toronto) : No data about inversion

Fig. 3-3-3-3. Overview of the fim related parts in iGEM.

Fig. 3-3-3-4. Lists of submitted fim parts.

3.2. fim switch(2008_Caltech)-fimE(2008_Caltech) : No data about inversion

Fig. 3-3-3-5. Overview of the fim related parts in iGEM.

Gene Name

Fig.3-3-3-6.Lists of submitted fim parts.

Fig. 3-3-3-7. The experimental result of 2008_Caltech

Fig. 3-3-3-8. The difference of nucleotide sequence between BBa_K137007) and BBa_K1632011) .

3.3. fim switch(2013_Michigan)-fimE(2008_Caltech)-hbiF(2012_Michigan) : Not enough data about inversion

Fig. 3-3-3-9. Overview of the fim related parts in iGEM.

Fig. 3-3-3-10.Lists of submitted fim parts.

Fig. 3-3-3-11. Overview of the fim related parts in iGEM.

4. Reference