Difference between revisions of "Team:OUC-China/Results"

Latest revision as of 02:29, 18 October 2018

Team OUC-China: Main

1. THE RESULTS OF FIRST SYSTEM: MINITOE
2. THE RESULTS OF SECOND SYSTEM: MINITOE FAMILY
3. THE RESULT OF THIRD SYSTEM: MINITOE POLYCISTRON
4. THE RESULT OF FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM

Results

To help you enjoy our result in a more convenient and smooth way, here are some key concepts in our discussion.
1) miniToe system
2) miniToe family system (The miniToe family refers to the whole second system below)
3) miniToe polycistron system (The miniToe polycistron refers to the whole third system below)
4) miniToe based motility detection system

The first system (miniToe system) is the basics of other three systems. As description we introduced above, on the level of DNA, we design a miniToe part and construct it to circuits. On the level of RNA, we focus on miniToe structure. In the miniToe family system, we focus on Csy4 hairpin which is a part of miniToe part. In fact, the Csy4 hairpin is the miniToe target region. A cis-repressive RNA (crRNA) served as a translation suppressor by pairing with RBS is critical part of miniToe structure.

Also, the miniToe system is a kind of translation activator. In miniToe family, the capabilities of different Csy4 mutants reflect their activation efficiency to turn on translation. So the fluorescence intensity reflect activation efficiency which is a comprehensive ability including the capabilities of recognition and cleavage.

1. The results of first system: miniToe

1.1 Plasmid construction

First, we use an inducible promoter Ptac to regulate the expression of Csy4 (pCsy4). Without the inducer isopropyl-β-d-thiogalactoside (IPTG), no Csy4 is produced. Otherwise, Csy4 can produce. As for another plasmid pRepoter, the superfolder green fluorescent protein (sfGFP) is the reporter gene to reflect output of our system under miniToe regulation, the expression of this gene is driven by a constitutive promoter named J23119 from Anderson family. The Csy4 hairpin is inserted between RBS and cis-repressive RNA region.

Fig.1-1 The two plasmids of miniToe system. The pCsy4 is constructed for the expression of Csy4. The pReporter contains miniToe part.

1.2 The measurement of growth rates

After circuit construction to get two plasmids pCsy4 and pReporter, we transformed them into E. coli DH5 Alpha and got the recombinant strain with miniToe system successfully. For the sake of functional test, 5 different groups are set, the control group E. coli DH5 Alpha, the pCsy4 only group, the pReporter only group, the pCsy4&pReporter with IPTG group and the pCsy4&pReporter without IPTG group. As preliminary experiment, the growth rate measurements are essential. The curve below demonstrates all the groups have almost the same tendency of OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of recombinant strain. The metabolic stress by two plasmids is not harmful to the recombinant strains.

Fig.1-2 Growth curve of strains we used in experiments. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)

1.3 Proof of function

The microplate reader is used to test the intensity of superfolder green fluorescent protein (sfGFP) which is changed over time. The aim is to prove that miniToe system can control the downstream gene expression during the whole cultivation period.

The following chart shows the dynamic curve measured by microplate reader every two hours. The yellow line refers to the test group which is recombinant strain (with the whole miniToe system) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity in recombinant strain (with the whole miniToe system) without IPTG (0mM). The green line refers to another control group which only has pReporter without the pCsy4 in strain. The results help us to prove two problems in miniToe system.

Fig.1-3 The intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups. The yellow line refers to a test group with IPTG (0.125mM). The blue line refers to a group without IPTG (0mM). The green line refers to a control group only with pReporter. Error bars represent standard deviation of three biological replicates. (Measured by microplate reader)

The first problem is whether miniToe structure can fold exactly on the level of RNA. In order to deal with the problem, the prediction of secondary structure is needed by using mfold and RNAfold. The result of prediction shows miniToe structure can fold correctly after transcription.

Fig.1-4 The structure prediction of full-length transcript of this circuit as well as miniToe structure. The miniToe structure is on the left of picture and the full-length transcript of this circuit is on the right of picture. The red frame indicates the places of miniToe structure in the full-length transcript of this circuit.

As the result showed in Fig1-3, a control group (the green line) is relatively stable during the whole process comparing with other another control group (IPTG=0) and test group(IPTG=0.125mM). It means the miniToe structure without Csy4 folds well on the level of RNA and also keep OFF state so the changes of fluorescence intensity cannot be detected.

The second problem that need to be proved is whether miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig1-3, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with the IPTG and the blue line is a control group without IPTG. It is not difficult to find that the fluorescence intensity of control group (the blue line) is always lower than test group (the yellow line). These data strongly support that the increased expression of the target gene sfGFP is indeed due to cleavage of Csy4 site that exposed the RBS to restore translation. It means miniToe system can work successfully.

We also tested miniToe system by flow cytometric. In Fig.1-5, it's easy to distinguish the two groups (blue & white) and the test group (+IPTG) has the obvious increase compared with the control group (-IPTG). The result shows the same conclusions mentioned before.

Fig.1-5 Flow cytometric measurement of fluorescence of sfGFP. Histograms show distribution of fluorescence in samples with test group with IPTG (green) and control group without IPTG (white). Crosscolumn number shows fold increase of sfGFP fluorescence. The test group is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). And the control group is a recombinant strain (with the whole miniToe system including two plasmids) without IPTG (0 mM).

1.4 Discussion

Combining the biology and math, we discuss the dynamics of sfGFP in the Fig.1-3. In order to explain in detail, Fig.1-6 presents the dynamics of all species in the miniToe system.

Fig.1-6 The dynamics of all species in the miniToe system

In the Fig.1-3, the red line which represents the dynamics of sfGFP which increases in the beginning and then drop down to a stable level. Because the capability of Csy4's cleavage is stronger. And the capability of mRNA's production [cis-repressive RNA-RBS-mRNA _gfp] is relate weaker which results in the decline of [mRNA_gfp] after 10 hours. Before we add IPTG to induce the Ptac, the [cis-repressive RNA-RBS-mRNA _gfp] is accumulated controlled by a constitutive promoter. After we add IPTG, the initial concentration of [cis-repressive RNA-RBS-mRNA _gfp] plays an important role in the production of sfGFP during the first 10-hour. Even though the rate of cleavage is faster than the production of [cis-repressive RNA-RBS-mRNA _gfp], the concentration of mRNA keeps increasing. But once the original [cis-repressive RNA-RBS-mRNA _gfp] is consumed, the [mRNA_gfp] stop increasing and drop down to a stable level. So the balance of the product rate and decay rate can keep.

See more details in model! Click here !

1.5 collaborations

Fig.1-7 The result from other four teams which proved our conclusions. Error bars represent standard deviation of four biological replicates.

We also collaborated with other 4 teams, and they helped us in proving our results by wet experiments in their labs. Thank you! Click here to see more details!

2. The results of second system: miniToe family

2.1 Plasmid construction

After building the ODE model, we use it to simulate the dynamics of sfGFP. Comparing with the experimental data, it fits perfectly, which indicates that the model is reliable about first system. By analyzing the sensitivity of the GFP level in the system to cleavage rate by model, it is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. It means we may change the expression level of sfGFP if we employ different mutants of Csy4 proteins.

There are two ways in second system which can help to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe mutants. There are all the plasmids we used in Fig.2-1 and Fig.2-2.

Fig.2-1 The plasmids of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4 and we also use it in first system. The pCsy4-Q104A is a plasmid which contains Csy4-Q104A. The pCsy4-Y176F is a plasmid which contains Csy4-Y176F. The pCsy4-F155A is a plasmid which contains Csy4- F155A. The pCsy4-H29A is a plasmid which contains Csy4- H29A.

Fig.2-2 The plasmids of miniToe mutants. The pReporter is a plasmid which contains miniToe-WT and we also use it in first system. The pReporter-1 is a plasmid which contains miniToe-1. The pReporter-2 is a plasmid which contains miniToe-2. The pReporter-3 is a plasmid which contains miniToe-3. The pReporter-4 is a plasmid which contains miniToe-4. The pReporter-5 is a plasmid which contains miniToe-5.

After plasmid construction, we proved the functions of Csy4 mutants by wet experiments first.

2.2.1 Proof of functions about Csy4 mutants

In this part, three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. Our expectation is that by using new Csy4 mutants, the expression level of sfGFP vary with Csy4s' capabilities. It means that miniToe family members present various expression of target genes.

Prediction

Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. The models help us go further this year!

Fig.2-3 The predication: the fluorescence intensities by different Csy4 mutants along with time

We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.

2.2.2 The result by Microscope

First, we tested the capabilities of five Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. In Fig.2-4, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s' capabilities of cleavage decrease one by one. The Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.

Fig.2-4-1 The expression of sfGFP by strain-Csy4.

Fig.2-4-2 The expression of sfGFP by strain-Csy4-Q104A.

Fig.2-4-3 The expression of sfGFP by strain-Csy4-Y176F.

Fig.2-4-4 The expression of sfGFP by strain-Csy4-F155A.

Fig.2-4-5 The expression of sfGFP by strain-Csy4-H29A.

Fig.2-4 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows E. coli without fluorescence excitation. The images on the right represent situation when fluorescence excitation.

2.2.3 The result by flow cytometer

The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.2-5, and their intensities of fluorescence are from strong to weak.

Fig.2-5 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.

Fig.2-6 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low which prove that they have different capabilities of cleavage.

2.2.4 The result by microplate reader

Besides all the works before, we also need to know more information about the Csy4 mutants in entire cultivation period. Even though we known that our Csy4 mutants have differentiated expression level in ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectations.

So we tested five test stains individually (strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A) by microplate reader every two hours. The green lines in all the images represents strain-miniToe-only group keep stable. It means the miniToe structure fold well and lock the process of translation without Csy4. And the five test groups show different characteristics. In Fig.2-7-A, the group strain-Csy4 shows the same result with the first system. The switch turns off without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the Csy4-WT has strongest capabilities (Fig.2-7-F). In the Fig.2-7-B, the tendency of fluorescence intensities by Csy4-Q104A is similar with Csy4-WT. And the expression level is lower than Csy4-WT. The Csy4-Y176F’s capabilities ranks the third. What is special is Csy4-H29A. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.2-7-E. In summary, we put all the test groups together in Fig.2-7-F, the picture shows prediction by model match the result perfectly in Fig.2-8.

Fig.2-7 The fluorescence intensities of sfGFP by microplate reader. A. strain-Csy4. B. strain-Csy4-Q104A. C. strain-Csy4-Y176F. D. strain-Csy4-F155A. E. strain-Csy4-H29A. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A.

Fig.2-8 The comparison about model and result by microplate reader.

By all the experiments mentioned before, we proved that Csy4 mutants work as expectations successfully. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A. And the original sequences of Csy4 part has been submitted by other iGEM teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.

2.3 Proof of functions about hairpin mutants

We design a new cis-regulatory RNA element named miniToe in first system. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function. At the same time, the Csy4 site is a kind of hairpin (wild type).

In order to meet our goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. After testing Csy4 mutants, we insert the new hairpin mutants in the miniToe. So the miniToe (contains hairpin-WT) is change to five mutants named miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5. For the sake of convenience, we named miniToe part from first system a new name in second system, miniToe-WT.

By flow cytometry measurement, we rank them by their capabilities.The recombination strains for test both have same pCsy4 but different plasmids contain different miniToe parts in the following. The recombination strains to test the functions of miniToe mutants are strain-miniToe (pCsy4&pReporter), strain-miniToe-1 (pCsy4 &pReporter-1), strain-miniToe-2 (pCsy4&pReporter-2), strain-miniToe-3 (pCsy4&pReporter-3), strain-miniToe-4 (pCsy4&pReporter-4), strain-miniToe-5 (pCsy4&pReporter-5). The results are listed in the order: miniToe-WT>miniToe-5>miniToe-1>miniToe-4>miniToe-2>miniToe-3.

Fig.2-9 The fluorescence intensities of sfGFP about hairpin mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-miniToe (Black), strain-miniToe-5 (Red), strain-miniToe-1 (Green), strain-miniToe-4 (Blue), strain-miniToe-2 (Cyan), strain-miniToe-3 (Yellow). Crosscolumn number shows fold increase of sfGFP fluorescence.

Fig.2-10 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The six test groups present different fluorescence intensities from high to low which prove that they have different capabilities.

2.4 Proof of functions about miniToe family

In Design page , we found it is possible to use one system to meet diverse aims which means by using our miniToe system, people can create more flexible gene circuits with different expression level.

With the help of our model, 4 Csy4 mutants and 5 hairpin mutants are selected. We tested each mutant and got positive data supporting model prediction. Then we set up a function verification experiment with 5*6 combinations of Csy4 and hairpin including wild types. So the 30 combinations are the candidates for miniToe family. And we should test all of them to select the positive members finally.

And we tested our system by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.2-11. We rank them by the heat map and then select the groups from different expression levels. As you can see, in the heat map, the expression levels of some groups are almost the same. So we just give up some combinations and then select the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.2-12. The user-friendly system meets the flexible needs in study about regulating different levels of expression.

Fig.2-11 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.

Fig.2-12 The members of miniToe family.

3. The result of third system: miniToe polycistron

3.1 The purpose of experiment

The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.
1) First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
2) Then we selected some miniToe parts and inserted them between, before and behind sfGFP and mCherry. For example, for bi-cistron, then three miniToe parts will be inserted. for three genes in polycistron, then four miniToe parts will be inserted, and so on.

This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more miniToe polycistron based on miniToe family.

Fig.3-1 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system.

3.2 Proof of functions

The result by microplate reader has been shown in the Fig.3-2. After culturing for 10 hours, the rate of fluorescence intensities by sfGFP/mCherry was changed by miniToe family. The group A is a control group whose circuits have no miniToe part. The ratio of fluorescence intensities by sfGFP/mCherry is about 6.81 which means the gene near the promoter has much higher expression than the gene far from promoter in a normal polycistron. The test group-polycistron A has been changed by miniToe system because the ratio of fluorescence intensities decrease to 4.38. To our surprise, the test group-polycistron B shows the significant change whose rate is about 2.82. It means the ratio of gene expression can be regulated by miniToe family. In the future, the miniToe family create more possibilities in regulating the ratio of gene expression.

Fig.3-2 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates.

4. The result of fourth system: miniToe based Motility detection system

4.1 The purpose of designing the experiment

As is shown in the first system miniToe, we created a new method to regulate the downstream gene expression. Furthermore, we proved that our system can be enlarged and then we created miniToe family system based on the mutation of miniToe part and Csy4 mutants. As and translation regulation tool, MiniToe can also be used in application scenario of molecular mechanism research. Sometimes scientists may puzzle with the functions of certain gene or protein without in-depth study. One general method to study them is knock-out or knock-in methods. In this way, organisms show some phenotypic change related to particular gene. However, if we want to know better about the functions of the gene, we may need more tool to change gene expression at different levels.

By using our system, the motility of E. coli can be regulated. We transformed the miniToe system into E. coli whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque. ΔmotA strains (the motA-deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.

So we did a lot of wet lab works to test minToe system by applying it to the detection of E. coli motility. We construct plasmid by putting the motA behind miniToe part. So the target gene motA can be regulated by miniToe system.

Fig.4-1 The process of motility detection system

4.2 Proof of functions

Five groups are set, a test group and four control groups. And the results shown below proved that our system can work as expectation.

Fig.4-2 The control groups A and B including positive group and negative group. Plates were inoculated with E. coli RP437 (A1, A2, A3) that have motility and they can move arbitrarily in the plates. The plates on right are ΔmotA strains(the motA-deletion strain) (B1, B2, B3), E. coli RP6666, which have no motility so the strains stay on the center. We have three biological replicates in this experiment.

Fig.4-3 The test group C. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe part).Without the motA, the E. coli cannot move. And the Csy4 have no big influence on strain compared with the ΔmotA strain. The little round of papers indicates the places of inducer IPTG (Isopropyl β D thiogalactopy ranoside). We have three biological replicates in the experiment.

Fig.4-4 The test group D. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe part but no Csy4. The circuit is on the control of miniToe part and its downstream gene motA can be regulated without Csy4. So MotA can be produced. We have three biological replicates in the experiment.

Fig.4-5 The test group E. The strain we culture in plates is miniToe-motA with Csy4. The strain have the miniToe part and Csy4 which means motA can be regulated by miniToe. In the picture, the E. coli move everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene motA come into play. The E. coli can move everywhere in the plate. We have three biological replicates in the experiment.

Fig.4-6 The migration dimensions. The ratio of migration area /whole plate. This chart is made by numerical integration

As we can see, test group strains can move everywhere in the plate and the control groups strains can not move.The test group work as expectation compared to the control groups. But there is no time for us to test more miniToe mutants and Csy4 mutants in miniToe family. We want to realize the function of regulation by using different miniToe family members in the future. So we still have a lot of work to do.

protocol

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+					<div class="sticky">
+<div class="topleft2">
+<br /><br /><br />
+<ul class="side-nav" style="width:150px">
+  <li><a href="#tips1"id="thu">1. THE RESULTS OF FIRST SYSTEM: MINITOE</a></li>
+  <li><a href="#tips2"id="thu">2. THE RESULTS OF SECOND SYSTEM: MINITOE FAMILY</a></li>
+  <li><a href="#tips3"id="thu">3. THE RESULT OF THIRD SYSTEM: MINITOE POLYCISTRON</a></li>
+  <li><a href="#tips4"id="thu">4. THE RESULT OF FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM</a></li>
+</ul>
+</div>
+</div>
+								<div class="nb">
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 								<!-- Features -->
 									<section class="box features">
-										<h2 class="major"><span>Results</span></h2>
+										<h2 class="major"><font size="7"><span>Results</span></font></h2>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/29/T--OUC-China--project-_overview-upupup.jpg" width="800"></div>
-<p>
+<p><font size="4">
-<h3>The result of first system</h3>
+<br />To help you enjoy our result in a more convenient and smooth way, here are some key concepts in our discussion.
-<br /><h4 ><font size="3">Plasmid construction</font></h4>
+<br />
-First, we use an inducible promoter Ptac to regulate the expression of Csy4. Without the IPTG, the circuit of Csy4 closes at the same time. On the contrary, Csy4 enzyme produce and help regulate the expression of downstream genes of miniToe structure in another plasmid as a part of our first system. Also, we use the promoter J23199 from Anderson family which is a constitutive promoter to regulate the reporter circuit. So if our system works well, we will get some curves for fluorescence intensity as our expectation.
+) miniToe system<br />
+) miniToe family system (The miniToe family refers to the whole second system below)<br />
+) miniToe polycistron system (The miniToe polycistron refers to the whole third system below)<br />
+) miniToe based motility detection system<br /><br />
+The first system (miniToe system) is the basics of other three systems. As description we introduced above, on the level of <strong>DNA</strong>, we design a <strong>miniToe part</strong> and construct it to circuits. On the level of <strong>RNA</strong>, we focus on <strong>miniToe structure</strong>. In the miniToe family system, we focus on Csy4 <strong>hairpin</strong> which is a part of <strong>miniToe part</strong>. In fact, the Csy4 hairpin is the miniToe target region. <strong>A cis-repressive RNA (crRNA)</strong> served as a translation suppressor by pairing with RBS is critical part of miniToe structure.
+ <br /><br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/7/72/T--OUC-China--Pic.jpg" width="650" >
+</div>
+<br />
+<br />
+Also, the miniToe system is a kind of <strong>translation activator</strong>. In miniToe family, the <strong>capabilities</strong> of different Csy4 mutants reflect their <strong>activation efficiency</strong> to turn on translation. So the fluorescence intensity reflect <strong>activation efficiency</strong> which is a comprehensive ability including the capabilities of recognition and cleavage.<br />
+<br />	</font size="4">
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/ff/T--OUC-China--project-_overview.png "  width="800"></div>
+<br /><br />
+<a id="tips1"></a><h3><font size="6">1. The results of first system: miniToe</font></h3>
+<br /><h4 ><font size="5">1.1 Plasmid construction</font></h4><br />
+First, we use an inducible promoter P<i>tac </i> to regulate the expression of Csy4 (pCsy4). Without the inducer isopropyl-β-d-thiogalactoside (IPTG), no Csy4 is produced. Otherwise, Csy4 can produce. As for another plasmid pRepoter, the superfolder green fluorescent protein (sfGFP) is the reporter gene to reflect output of our system under miniToe regulation, the expression of this gene is driven by a constitutive promoter named J23119 from Anderson family. The Csy4 hairpin is inserted between RBS and cis-repressive RNA region.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/6/68/T--OUC-China--res1.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.1-1 The two plasmids of miniToe test system.</p></div>
+  <div align="center"><p >Fig.1-1 The two plasmids of miniToe system. The pCsy4 is constructed for the expression of Csy4. The pReporter contains miniToe part.</p></div>
-<br /><h4 ><font size="3">Selective Medium Assay</font></h4>
+<br /><h4 ><font size="5">1.2 The  measurement of growth rates</font></h4><br />
-After circuit construction to get the two plasmids we mentioned before, we transformed both of them into E.coli DH5 Alpha and got the miniToe testing strain successfully. Because the promoter Tac has high leakage in LB medium so we culture our recombinant strain in M9 medium. We measured the growth rate of both our engineered strain and the negative control as preliminary experiment. As a result, the curve well demonstrates that the strain with our system has almost some OD600 with the negative control strain during the entire cultivation period. It means that our system has no big negative influence on the growth of strain. The metabolic stress by two plasmids is not harm to our recombinant strain.
+After circuit construction to get two plasmids pCsy4 and pReporter, we transformed them into <i>E. coli</i> DH5 Alpha and got the recombinant strain with miniToe system successfully. For the sake of functional test, 5 different groups are set, the control group <i>E. coli</i> DH5 Alpha, the pCsy4 only group, the pReporter only group, the pCsy4&pReporter with IPTG group and the pCsy4&pReporter without IPTG group. As preliminary experiment, the growth rate measurements are essential. The curve below demonstrates all the groups have almost the same tendency of OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of recombinant strain. The metabolic stress by two plasmids is not harmful to the recombinant strains.
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/9/9f/T--OUC-China--res2.png" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/d/d3/T--OUC-China--JCod1.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.1-2 Growth curve of strains of our recombinant strain (with the whole miniToe system including two plasmids) and negative control groups. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)</p></div>
+  <div align="center"><p >Fig.1-2 Growth curve of strains we used in experiments. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)</p></div>
-  <h4 ><font size="3">Proof of function</font></h4>
+  <h4 ><font size="5">1.3 Proof of function</font></h4><br />
-We use microplate reader to test the fluorescence intensity of sfGFP which is changed over time. getting data to support our idea directly that our system can control the downstream gene expression during the whole cultivation period.
+The microplate reader is used to test the intensity of superfolder green fluorescent protein (sfGFP) which is changed over time. The aim is to prove that miniToe system can control the downstream gene expression during the whole cultivation period. <br />
-<br />The following chart shows the dynamic curve measured by microplate reader. We test our system every two hours. The yellow line is the symbol of test group which is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity by a recombinant strain(with the whole miniToe system including two plasmids) without IPTG (0mM). The green line is also a control group in our system, it shows the fluorescence intensity of sfGFP by a strain with only one kind of plasmids (pReporter) which only has miniToe structure without the Csy4. The result by this curve help us to prove two functions in miniToe system.					<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ed/T--OUC-China--res3.png" height="400"> </div>
+<br>The following chart shows the dynamic curve measured by microplate reader every two hours. The yellow line refers to the test group which is recombinant strain (with the whole miniToe system) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity in recombinant strain (with the whole miniToe system) without IPTG (0mM). The green line refers to another control group which only has pReporter without the pCsy4 in strain. The results help us to prove two problems in miniToe system.					<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ed/T--OUC-China--res3.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.1-3 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains we tested in the chart. The yellow line is a test group with IPTG (0.125mM). The blue line is a control group without IPTG (0mM). The green line is a control group with only one plasmid (pReporter). </p></div>
+  <div align="center"><p >Fig.1-3 The intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups. The yellow line refers to a test group with IPTG (0.125mM). The blue line refers to a group without IPTG (0mM). The green line refers to a control group only with pReporter. Error bars represent standard deviation of three biological replicates. (Measured by microplate reader) </p></div>
-  <br />The first problem is whether our miniToe structure fold exactly. So first we predict our secondary structure by using mfold(http://unafold.rna.albany.edu/?q=mfold) and RNAfold(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). We predict the whole structure of our circuit and structure of miniToe to see if our structure can fold directly on the level of RNA. By the result of prediction, we just find our structure can fold directly after transcription.
+  <br />The first problem is whether miniToe structure can fold exactly on the level of RNA. In order to deal with the problem, the prediction of secondary structure is needed by using <a href='http://unafold.rna.albany.edu/?q=mfold'>mfold</a>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/5c/T--OUC-China--design2-2.png" height="400"> </div>
+  and <a href='http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi'>RNAfold</a>. The result of prediction shows miniToe structure can fold correctly after transcription.   <br />	<br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/5c/T--OUC-China--design2-2.png" height="800"> </div>
 <br />
-  <div align="center"><p >Fig.1-4 The structure prediction of the whole circuit and miniToe. The structure of miniToe is on the right of picture and the structure of whole circuit is on the left of picture. The red frame indicates the miniToe structure in the whole circuit.  </p></div>
+  <div align="center"><p >Fig.1-4 The structure prediction of full-length transcript of this circuit as well as miniToe structure. The miniToe structure is on the left of picture and the full-length transcript of this circuit is on the right of picture. The red frame indicates the places of miniToe structure in the full-length transcript of this circuit.  </p></div>
- But in fact, we also need to prove that our miniToe can fold directly in reality by experiments. As the result showed in Fig1-3, one of the control group (the green line) is relatively stable during the whole process comparing with two other strains. This means the miniToe without Csy4 folds well in secondary structure on the level of RNA and also keep the OFF state so we can’t detect the changes of fluorescence intensity by sfGFP because the translation of sfGFP is closed.		<br />
+As the result showed in Fig1-3, a control group (the green line) is relatively stable during the whole process comparing with other another control group (IPTG=0) and test group(IPTG=0.125mM). It means the miniToe structure without Csy4 folds well on the level of RNA and also keep OFF state so the changes of fluorescence intensity cannot be detected.	<br /><br />
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ed/T--OUC-China--res3.png" height="400"> </div>
-<br />
- <div align="center"><p >Fig.1-3 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains we tested in the chart. The yellow line is a test group with IPTG (0.125mM). The blue line is a control group without IPTG (0mM). The green line is a control group with only one plasmid (pReporter). </p></div>
+The second problem that need to be proved is whether miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig1-3, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with the IPTG and the blue line is a control group without IPTG. It is not difficult to find that the fluorescence intensity of control group (the blue line) is always lower than test group (the yellow line). These data strongly support that the increased expression of the target gene sfGFP is indeed due to cleavage of Csy4 site that exposed the RBS to restore translation. It means miniToe system can work successfully.
-The second problem we need to prove is that whether our miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig1-3 we can find that there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with the IPTG and the blue line is a control group without IPTG. It is not difficult to find that the fluorescence intensity of control group (the blue line) is always lower than test group (the yellow line). This means our system can work successfully.
-<br />
-At the same time, we find the control group without IPTG (the blue line) has leakage compared with other two group. Because the control group with only one plasmid (the green line) has the stable and low expression of sfGFP, the leakage may result from the Inductive promoter Ptac. Even though the control group has leakage of sfGFP, we can prove the function of our system successfully. But in the future, we may have more time to find a better promoter which is also suitable for our system.
 <br />
-We also test our system by Flow cytometric, the blue group showed in the Fig.1-5 is the test group when the white group is a control group. It’s easy to distinguish the two groups and the test group has the obvious increase compare to the control group. The result shows the same conclusions we mentioned before.
+<br>We also tested miniToe system by flow cytometric. In Fig.1-5, it's easy to distinguish the two groups (blue & white) and the test group (+IPTG) has the obvious increase compared with the control group (-IPTG). The result shows the same conclusions mentioned before.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/0/0b/T--OUC-China--res5.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.1-5 Flow cytometric measurement of fluorescence of sfGFP. Histograms show distribution of fluorescence in samples with test group with IPTG (green) and control group without IPTG(white). Crosscolumn number shows fold increase of sfGFP fluorescence. The strain we use in test group is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). And the control group is a recombinant strain (with the whole miniToe system including two plasmids) without IPTG (0 mM).  </p></div>
+  <div align="center"><p >Fig.1-5 Flow cytometric measurement of fluorescence of sfGFP. Histograms show distribution of fluorescence in samples with test group with IPTG (green) and control group without IPTG (white). Crosscolumn number shows fold increase of sfGFP fluorescence. The test group is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). And the control group is a recombinant strain (with the whole miniToe system including two plasmids) without IPTG (0 mM).  </p></div>
- 另一个问题，曲线为什么会下降？
-<br /><h4 ><font size="3">Discussion</font></h4>
+<br /><h4 ><font size="5">1.4 Discussion</font></h4><br />
-Combining the biology and math, we discuss the dynamics of GFP in the Fig.1-3 now. In order to explain in detail, we present the dynamics of all species in the miniToe system in Fig.1-6.
+Combining the biology and math, we discuss the dynamics of sfGFP in the Fig.1-3. In order to explain in detail, Fig.1-6 presents the dynamics of all species in the miniToe system.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/2/2e/T--OUC-China--res6.png" height="400"> </div>
 <br />
   <div align="center"><p >Fig.1-6 The dynamics of all species in the miniToe system </p></div>
-In the Fig.1-3, the red line which represents the dynamics of GFP which increases in the beginning and then drop down to a stable level. The reason is that the capability of Csy4’s cleavage is stronger. And the capability of mRNA’s production()  is relate weaker which results in the decline of  after 10 hours. Before we add IPTG to induce the Ptac, the   is accumulated because it is under controlled by a constitutive promoter. After we add IPTG, the initial concentration of  plays an important role in the production of GFP during the first 10-hour. Even though the rate of cleavage is faster than the production of , the concentration of mRNA keeps increasing. But once the original  is consumed, the  stop increasing and drop down to a stable level. So the balance of the product rate and decay rate can kept. This is the reason why the level of sfGFP keep stable finally in Fig1-3.
+In the Fig.1-3, the red line which represents the dynamics of sfGFP which increases in the beginning and then drop down to a stable level. Because the capability of Csy4's cleavage is stronger. And the capability of mRNA's production [cis-repressive RNA-RBS-mRNA <sub>gfp</sub>] is relate weaker which results in the decline of [mRNA<sub>gfp</sub>] after 10 hours. Before we add IPTG to induce the P<i>tac</i>, the [cis-repressive RNA-RBS-mRNA <sub>gfp</sub>] is accumulated controlled by a constitutive promoter. After we add IPTG, the initial concentration of [cis-repressive RNA-RBS-mRNA <sub>gfp</sub>] plays an important role in the production of sfGFP during the first 10-hour. Even though the rate of cleavage is faster than the production of [cis-repressive RNA-RBS-mRNA <sub>gfp</sub>], the concentration of mRNA keeps increasing. But once the original [cis-repressive RNA-RBS-mRNA <sub>gfp</sub>] is consumed, the [mRNA<sub>gfp</sub>] stop increasing and drop down to a stable level. So the balance of the product rate and decay rate can keep.
-<br />See more details in model! Click here!
+<br><br />See more details in model! Click <a href='https://2018.igem.org/Team:OUC-China/miniToe'> here </a>!
-<br />(https://2018.igem.org/Team:OUC-China/miniToe)
+<br /><br /><h4 ><font size="5">1.5 collaborations</font></h4>
-<br /><br /><br />collaboration：结果对比正确/SDU/SK
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/4c/T--OUC-China--res72.jpg" height="700"> </div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e4/T--OUC-China--res7.png" height="250"> </div>
 <br />
-  <div align="center"><p >Fig.1-7 The result from other four teams which have proved our conclusions. </p></div>
+  <div align="center"><p >Fig.1-7 The result from other four teams which proved our conclusions. Error bars represent standard deviation of four biological replicates. </p></div>
-<br />We also have collaborations with other 4 teams, and they help us in proving our results by experiments in their labs. Thank you! See more details here!   (https://2018.igem.org/Team:OUC-China/Collaborations)
+<br />We also collaborated with other 4 teams, and they helped us in proving our results by wet experiments in their labs. Thank you! Click <a href='https://2018.igem.org/Team:OUC-China/Collaborations'> here </a> to see more details!
-							</p>
+</p>
 <p>
-<h3>The result of second system</h3>
+<a id="tips2"></a><h3><font size="6">2. The results of second system: miniToe family</font></h3>
-<br /><h4 ><font size="3">Plasmid construction</font></h4>
+<br /><h4 ><font size="5">2.1 Plasmid construction</font></h4>
-In our second system, we find it possible to achieve our aim by using the mutants of Csy4 and mutants of miniToe structure. There are two ways in our second system which can help us to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe muntants.
+<br />After building the ODE model, we use it to simulate the dynamics of sfGFP. Comparing with the experimental data, it fits perfectly, which indicates that the model is reliable about first system. By analyzing the sensitivity of the GFP level in the system to cleavage rate by model, it is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. It means we may change the expression level of sfGFP if we employ different mutants of Csy4 proteins. <br /><br />
-<br />By point mutantion and synthesis by company, we have obtained all the circuits below.
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/4f/T--OUC-China--res21.png" width="800"> </div>
+There are two ways in second system which can help to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe mutants. There are all the plasmids we used in Fig.2-1 and Fig.2-2. <br /><br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/4f/T--OUC-China--res21.png" width="900"> </div>
 <br />
-  <div align="center"><p >Fig.2-1 The circuits of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4 and we also use it in first system. The pCsy4-Q104A is a plasmid which contains Csy4-Q104A. The pCsy4-Y176F is a plasmid which contains Csy4-Y176F. The pCsy4-F155A is a plasmid which contains Csy4- F155A. The pCsy4-H29A is a plasmid which contains Csy4- H29A.</p></div>
+  <div align="center"><p >Fig.2-1 The plasmids of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4 and we also use it in first system. The pCsy4-Q104A is a plasmid which contains Csy4-Q104A. The pCsy4-Y176F is a plasmid which contains Csy4-Y176F. The pCsy4-F155A is a plasmid which contains Csy4- F155A. The pCsy4-H29A is a plasmid which contains Csy4- H29A.</p></div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--OUC-China--res22.png" width="800"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--OUC-China--res22.png" width="900"> </div>
+<br />
+ <div align="center"><p >Fig.2-2 The plasmids of miniToe mutants. The pReporter is a plasmid which contains miniToe-WT and we also use it in first system. The pReporter-1 is a plasmid which contains miniToe-1. The pReporter-2 is a plasmid which contains miniToe-2. The pReporter-3 is a plasmid which contains miniToe-3. The pReporter-4 is a plasmid which contains miniToe-4. The pReporter-5 is a plasmid which contains miniToe-5.</p></div>
+<div align="center"><p ><img src="https://static.igem.org/mediawiki/2018/d/d3/T--OUC-China--pCsy4.jpg" height="500"> </p></div>
+<div align="center"><p ><img src="https://static.igem.org/mediawiki/2018/5/51/T--OUC-China--pRep.jpg" height="500"> </p></div>
+<br />After plasmid construction, we proved the functions of Csy4 mutants by wet experiments first.<br />
+<br /><h4 ><font size="5">2.2.1 Proof of functions about Csy4 mutants</font></h4><br />
+In this part, three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. Our expectation is that by using new Csy4 mutants, the expression level of sfGFP vary with Csy4s' capabilities. It means that miniToe family members present various expression of target genes.
 <br />
- <div align="center"><p >Fig.2-2 The circuits of miniToe mutants. The pReporter is a plasmid which contains miniToe-WT and we also use it in first system. The pReporter-1 is a plasmid which contains miniToe-1. The pReporter-2 is a plasmid which contains miniToe-2. The pReporter-3 is a plasmid which contains miniToe-3. The pReporter-4 is a plasmid which contains miniToe-4. The pReporter-5 is a plasmid which contains miniToe-5.</p></div>
+<br /><h4 ><font size="5">Prediction</font></h4><br />
-<br />After plasmid construction, we prove the functions of Csy4 mutants first.
+Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. The models help us go further this year!
-<br /><h4 ><font size="3">Proof of functions about Csy4 mutants</font></h4>
-In this part, we have three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. At the same time, we focus on the capacities of Csy4’s cleavage (including all the mutants we design). Our expectation is that by using our new Csy4 mutants, the fluorescence intensities of sfGFP can vary upon the rates of Csy4s’ cleavage which means that the result presents various expression of target genes.
-<br />(图：此处可以有一张多酶预测图)
-<br /><h4 ><font size="3">Prediction</font></h4>
-	Before the experiments, we have proved our ideas by model. The predication below shows the possibilities of different expression levels by different Csy4 mutants. So the model help us to know our improvement deeply this year!
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/ca/T--OUC-China--res23.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.2-3 The predication: The fluorescence intensities by different Csy4 mutants along with time</p></div>
+  <div align="center"><p >Fig.2-3 The predication: the fluorescence intensities by different Csy4 mutants along with time </p></div>
-<br /><h4 ><font size="3">The result by Microscope</font></h4>
+<br />We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter. <br /><br />
-<br />First, we have tested five different Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. We have cultured them in the solid medium in plates until the bacterial colonies can be observed by naked eyes. At that time, the sfGFP have been accumulated so we can see the fluorescence by microscope. Because the five Csy4s have different capabilities of cleavage, we want to see different intensities of fluorescent by eyes. As we can see in Fig.2-4, we have cultured the five different strains for same time which both have the same miniToe circuit but have totally diffenent Csy4 mutants. From top to bottom in Fig.2-4, there are fluorescence imagines by fluorescent microscope which indicate Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A in sequence. We can observe visible distinctions in these imagines. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s’ capabilities of cleavage decrease one by one. So the imagines indicate the Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/7/7f/T--OUC-China--plasmid.jpg" height="200"> </div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/a/a2/T--OUC-China--res24.png" width="400"> </div>
-<br />
+<br /><h4 ><font size="5">2.2.2 The result by Microscope</font></h4>
-  <div align="center"><p >Fig.2-4 The fluorescence imagines by fluorescent microscope. From top to bottom, the imagines shows the expression of sfGFP by Csy4-WT&miniToe, Csy4-Q104A&miniToe, Csy4-Y176F&miniToe, Csy4-F155A&miniToe and Csy4-H29A&miniToe in sequence. The plotting scale is on the right corner of each imagine.
+<br />First, we tested the capabilities of five Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. In Fig.2-4, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s' capabilities of cleavage decrease one by one. The Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.<br /><br />
-</p></div>
-<br /><h4 ><font size="3">The result by flow cytometer</font></h4>
-<br /> The qualitative experiment is not enough to analyze the Csy4s. So we test our system by flow cytometer when we have cultured them for ten hours in M9 medium. The expression of five groups’ sfGFP are showed in Fig.2-5, and they are Csy4-WT&miniToe, Csy4-Q104A&miniToe, Csy4-Y176F&miniToe, Csy4-F155A&miniToe and Csy4-H29A&miniToe. We find that 5 groups’ fluorescence intensities have a obvious order from Csy4-WT to Csy4-H29A which means the capabilities of cleavage decrease one by one. The strongest one is Csy4-WT. The second one is Csy4-Q104A. The third one is Csy4-Y176F. The fourth one is Csy4-F155A and the Csy4-H29A is the weakest. (此处无对照) As the Fig.2-5 shown, the relative expression level can be measured by flow cytometer at the same time.
+<div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/28/T--OUC-China--JCYWT.png" width="800"> </div>
+<div align="center"><p > Fig.2-4-1 The expression of sfGFP by strain-Csy4.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/1/19/T--OUC-China--JCYQ2.png" width="800"> </div>
+  <div align="center"><p > Fig.2-4-2 The expression of sfGFP by strain-Csy4-Q104A.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/bb/T--OUC-China--JCYY.png" width="800"> </div>
+ <div align="center"><p > Fig.2-4-3 The expression of sfGFP by strain-Csy4-Y176F.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/88/T--OUC-China--JCYF.png" width="800"> </div>
+ <div align="center"><p > Fig.2-4-4 The expression of sfGFP by strain-Csy4-F155A.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/fd/T--OUC-China--JCYH.png" width="800"> </div>
+ <div align="center"><p > Fig.2-4-5 The expression of sfGFP by strain-Csy4-H29A.</p></div>
+<div align="center"><p >Fig.2-4 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows <i>E. coli</i> without fluorescence excitation. The images on the right represent situation when fluorescence excitation.</p></div>
+</div>
+<br /><h4 ><font size="5">2.2.3 The result by flow cytometer</font></h4>
+<br /> The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.2-5, and their intensities of fluorescence are from strong to weak.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e8/T--OUC-China--fig2-5z.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.2-5 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with Csy4-WT&miniToe (Blank), Csy4-Q104A&miniToe (Orange), Csy4-Y176F&miniToe (Red), Csy4-F155A&miniToe (Blue), Csy4-H29A&miniToe (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.</p></div>
+  <div align="center"><p >Fig.2-5 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.</p></div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/26/T--OUC-China--res26.png" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/d/da/T--OUC-China--JCPE.png" height="400"> </div>
 <br />
   <div align="center"><p >Fig.2-6 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low which prove that they have different capabilities of cleavage.  </p></div>
-<br /><h4 ><font size="3">The result by microplate reader</font></h4>
+<br /><h4 ><font size="5">2.2.4 The result by microplate reader</font></h4>
-<br />Besides all the works we have done before, we also need to know more information about the Csy4s we design. Even though we have known that our Csy4 mutants have differentiated expression level after ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectation.
+<br />Besides all the works before, we also need to know more information about the Csy4 mutants in entire cultivation period. Even though we known that our Csy4 mutants have differentiated expression level in ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectations.
-<br />So we tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the imagines represent the control group, “miniToe only” group and the green lines keep stable which means the miniToe structure can close the expression of downstream genes. And the test groups show different characteristics. As we can see in Fig.2-7-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without IPTG works (As the blue line shows). And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT (Fig.2-7-F). In the Fig.2-7-B, the tendency of increase of fluorescence intensities by Csy4-Q104A is almost same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What is special is Csy4-H29A. We have mentioned Csy4-H29A before. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.2-7-E. （此处缺一个解释）In summary, we put all the test groups together in Fig.2-7-F, the picture shows our prediction by model match the result perfectly in Fig.2-8.
+<br><br />So we tested five test stains individually (strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A) by microplate reader every two hours. The green lines in all the images represents strain-miniToe-only group keep stable. It means the miniToe structure fold well and lock the process of translation without Csy4. And the five test groups show different characteristics. In Fig.2-7-A, the group strain-Csy4 shows the same result with the first system. The switch turns off without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the Csy4-WT has strongest capabilities (Fig.2-7-F). In the Fig.2-7-B, the tendency of fluorescence intensities by Csy4-Q104A is similar with Csy4-WT. And the expression level is lower than Csy4-WT. The Csy4-Y176F’s capabilities ranks the third. What is special is Csy4-H29A. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.2-7-E. In summary, we put all the test groups together in Fig.2-7-F, the picture shows prediction by model match the result perfectly in Fig.2-8.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/5/56/T--OUC-China--res27.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.2-7 The fluorescence intensities of sfGFP by microplate reader. A. Csy4-WT&miniToe. B. Csy4-Q104A&miniToe. C. Csy4-Y176F&miniToe. D. Csy4-F155A&miniToe. E. Csy4-H29A&miniToe. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants.  </p></div>
+  <div align="center"><p >Fig.2-7 The fluorescence intensities of sfGFP by microplate reader. A. strain-Csy4. B. strain-Csy4-Q104A. C. strain-Csy4-Y176F. D. strain-Csy4-F155A. E. strain-Csy4-H29A. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A. </p></div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/ba/T--OUC-China--res28.png" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/ba/T--OUC-China--res28.png" height="300"> </div>
 <br />
   <div align="center"><p >Fig.2-8 The comparison about model and result by microplate reader.
 </p></div>
-By all the experiments mentioned before, we have proved that our Csy4 mutants work as expectation successfully. And the original part Csy4 has been submitted by other teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.
+By all the experiments mentioned before, we proved that Csy4 mutants work as expectations successfully. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A. And the original sequences of Csy4 part has been submitted by other iGEM teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.	<br />
+<br /><h4 ><font size="5">2.3 Proof of functions about hairpin mutants</font></h4><br />
-<br /><h4 ><font size="3">Proof of functions about hairpin mutants</font></h4>
+We design a new cis-regulatory RNA element named miniToe in first system. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function. At the same time, the Csy4 site is a kind of hairpin (wild type).
-In order to meet this goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. After testing Csy4 mutants, we have tested another way that may help us to create more possibilities. We also proved that we can get some different hairpin mutants by changing the sequences of hairpin-WT.
+<br />
-<br />We also have redesigned 5 hairpin mutants and tested them by flow cytometry and rank them by their capacities. Finally we just found that the rank of them is miniToe WT>miniToe 5>miniToe 1>miniToe 4>miniToe 2>miniToe 3.
+<br />In order to meet our goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. After testing Csy4 mutants, we insert the new hairpin mutants in the miniToe. So the miniToe (contains hairpin-WT) is change to five mutants named miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5. For the sake of convenience, we named miniToe part from first system a new name in second system, miniToe-WT.
+<br>
+<br />
+By flow cytometry measurement, we rank them by their capabilities.The recombination strains for test both have same pCsy4 but different plasmids contain different miniToe parts in the following. The recombination strains to test the functions of miniToe mutants are strain-miniToe (pCsy4&pReporter), strain-miniToe-1 (pCsy4 &pReporter-1), strain-miniToe-2 (pCsy4&pReporter-2), strain-miniToe-3 (pCsy4&pReporter-3), strain-miniToe-4 (pCsy4&pReporter-4), strain-miniToe-5 (pCsy4&pReporter-5). The results are listed in the order: miniToe-WT>miniToe-5>miniToe-1>miniToe-4>miniToe-2>miniToe-3.
+<br /><br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/d/df/T--OUC-China--plasmid1.jpg" height="200"> </div>
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/7/73/T--OUC-China--res29.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.2-9 The fluorescence intensities of sfGFP about hairpin mutants by flow cytometer. Histograms show distribution of fluorescence in samples with Csy4-WT&miniToe-WT (Blank), Csy4-WT&miniToe 5 (Red), Csy4-WT&miniToe 1 (Green), Csy4-WT&miniToe 4 (Blue), Csy4-WT&miniToe 2 (Cyan), Csy4-WT&miniToe 3 (Yellow). Crosscolumn number shows fold increase of sfGFP fluorescence.
+  <div align="center"><p >Fig.2-9 The fluorescence intensities of sfGFP about hairpin mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-miniToe (Black), strain-miniToe-5 (Red), strain-miniToe-1 (Green), strain-miniToe-4 (Blue), strain-miniToe-2 (Cyan), strain-miniToe-3 (Yellow). Crosscolumn number shows fold increase of sfGFP fluorescence.
 </p></div>
-	<div align="center"><img src="https://static.igem.org/mediawiki/2018/9/97/T--OUC-China--res210.png" height="400"> </div>
+	<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/3c/T--OUC-China--JCHE.png" height="400"> </div>
 <br />
   <div align="center"><p >Fig.2-10 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The six test groups present different fluorescence intensities from high to low which prove that they have different capabilities.
@@ Line 253: / Line 371: @@
-<br /><h4 ><font size="3">Proof of functions about miniToe family</font></h4>
+<br /><h4 ><font size="5">2.4 Proof of functions about miniToe family</font></h4><br />
-As we talk about before in DESIGN page, we found it is possible to use one system to meet diverse aims which means by using our miniToe system, people can create more flexible gene circuit with different expression level.
+In <a href='https://2018.igem.org/Team:OUC-China/Design'> Design page </a>, we found it is possible to use one system to meet diverse aims which means by using our miniToe system, people can create more flexible gene circuits with different expression level.
 <br />
-In order to meet this goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. And we have proved that all of them are the good materials to form a bigger group. So we just combine all the mutants together. The combinations of different Csy4 mutants and hairpin mutants which compose a small library give us more possibilities in use. We named those combinations miniToe family which is the second system of our project.
+<br> With the help of our model, 4 Csy4 mutants and 5 hairpin mutants are selected. We tested each mutant and got positive data supporting model prediction. Then we set up a function verification experiment with 5*6 combinations of Csy4 and hairpin including wild types. So the 30 combinations are the candidates for miniToe family. And we should test all of them to select the positive members finally.
 <br />
-And we have tested our system by flow cytometry. All the 30 groups’ intensities of fluorescence are shown in Fig.2-11. We rank them by the heat map and then select the groups from different expression levels. As you can see, in this heat map, some groups have the similar expression levels. So we just give up some combinations and then select the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.2-12.
+<br>And we tested our system by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.2-11. We rank them by the heat map and then select the groups from different expression levels. As you can see, in the heat map, the expression levels of some groups are almost the same. So we just give up some combinations and then select the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.2-12. The user-friendly system meets the flexible needs in study about regulating different levels of expression. <br /><div align="center"><img src="https://static.igem.org/mediawiki/2018/2/28/T--OUC-China--res211.png" height="700"> </div>
-The user-friendly system meet the flexible needs in study which can help user’s need about different levels of expression. <br /><div align="center"><img src="https://static.igem.org/mediawiki/2018/2/28/T--OUC-China--res211.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.2-11 The heat map generated from flow cytometry data reflecting 30 groups’ intensities of fluorescence by sfGFP
+  <div align="center"><p >Fig.2-11 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.
 </p></div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/6b/T--OUC-China--res212.png" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/6b/T--OUC-China--res212.png" height="300"> </div>
 <br />
   <div align="center"><p >Fig.2-12 The members of miniToe family.
@@ Line 272: / Line 389: @@
 <p>
-<h3>The result of the third system—— miniToe polycistron </h3>
+<a id="tips3"></a><h3><font size="6">3. The result of third system: miniToe polycistron </font> </h3>
-<br /><h4 ><font size="3">The setting of test groups and control groups</font></h4>
+<br /><h4 ><font size="5">3.1 The purpose of experiment</font></h4><br />
-The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe structure into circuits, more than one gene can be regulated. So in this system, we focus on the ratio of gene expression. We tested polycistron system by two target genes, sfGFP and mCherry.
+The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins. <br />
-						<br />
+)	First, sfGFP and mCherry is used as a test system in bi-cistron circuit. <br />
-Three kinds of groups have been set. One is the bicistron circuit without miniToe structures. In order to make sure our miniToe structure folded as expectation, we have created the recombinant strain (control group) which only has the circuit constructed by miniToe without Csy4. The test group both have miniToe polycistron and Csy4.
+)	Then we selected some miniToe parts and inserted them between, before and behind sfGFP and mCherry. For example, for bi-cistron, then three miniToe parts will be inserted. for three genes in polycistron, then four miniToe parts will be inserted, and so on.
 <br />
-This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will tested more polycistron based on miniToe family.
+<br>This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more miniToe polycistron based on miniToe family.
+<div align="center"><img src=" https://static.igem.org/mediawiki/2018/a/a6/T--OUC-China--res31.jpg" height="300"> </div>
-<br /><h4 ><font size="3">Proof of functions</font></h4>
+ <div align="center"><p >Fig.3-1 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system. </p></div>
-The result by microplate reader has been shown in the Fig.3-1.
-<div align="center"><img src="1" height="400"> </div>
+<br /><h4 ><font size="5">3.2 Proof of functions</font></h4>
+<br>The result by microplate reader has been shown in the Fig.3-2. After culturing for 10 hours, the rate of fluorescence intensities by sfGFP/mCherry was changed by miniToe family. The group A is a control group whose circuits have no miniToe part. The ratio of fluorescence intensities by sfGFP/mCherry is about 6.81 which means the gene near the promoter has much higher expression than the gene far from promoter in a normal polycistron. The test group-polycistron A has been changed by miniToe system because the ratio of fluorescence intensities decrease to 4.38. To our surprise, the test group-polycistron B shows the significant change whose rate is about 2.82. It means the ratio of gene expression can be regulated by miniToe family. In the future, the miniToe family create more possibilities in regulating the ratio of gene expression.
+<br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/42/T--OUC-China--res322.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.3-1</p></div>
+  <div align="center"><p >Fig.3-2 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates. </p></div>
 							</p>
 <p>
-<h3>The result of miniToe Motility detection system</h3>
+<a id="tips4"></a><h3><font size="6">4. The result of fourth system: miniToe based Motility detection system</font></h3>
-<br /><h4 ><font size="3">Proof of functions</font></h4>
+<br /><h4 ><font size="5">4.1 The purpose of designing the experiment</font></h4><br />
-As we shown before in the first system, we have created a new method to regulate to downstream gene expression named miniToe. And we also have proved that our system can be enlarged. So we have created miniToe family in the second system.
+As is shown in the first system miniToe, we created a new method to regulate the downstream gene expression. Furthermore, we proved that our system can be enlarged and then we created miniToe family system based on the mutation of miniToe part and Csy4 mutants. As and translation regulation tool, MiniToe can also be used in application scenario of molecular mechanism research. Sometimes scientists may puzzle with the functions of certain gene or protein without in-depth study. One general method to study them is knock-out or knock-in methods. In this way, organisms show some phenotypic change related to particular gene. However, if we want to know better about the functions of the gene, we may need more tool to change gene expression at different levels.
 <br />
-We believe that miniToe is also a good tool which can be apply to study of molecular mechanism.
+<br>By using our system, the motility of <i>E. coli</i> can be regulated. We transformed the miniToe system into <i>E. coli</i> whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque. Δ<i>motA </i>strains (the <i>motA</i>-deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
-Scientists may puzzled with the functions of certain gene or protein when first discover it. Now one common method to study single gene is knock out or knock in. In this way, organisms without one gene show the lack of forms or functions. But if we want to know better about the gene functions, we may need different level of the gene expressions.
 <br />
-By using our system, the motility of E.coli can be regulated. As we know, MotA provides a channel for the proton gradient required for generation of torque. ΔmotA strains can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
+<br>So we did a lot of wet lab works to test minToe system by applying it to the detection of <i>E. coli</i> motility. We construct plasmid by putting the <i>motA</i> behind miniToe part. So the target gene <i>motA</i> can be regulated by miniToe system.
 <br />
-So we have done some works to test our system in dealing with the real-world problems by E.coli motility detection experiments. We construct our circuit by putting the motA behind our miniToe structure. So the motA as a target gene can be regulated by miniToe.
-<br />
-Five groups have been set, three test group and two control group. And the result shown below have proved that our system can work as expectation.
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/b1/T--OUC-China--res41.png" width="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/80/T--OUC-China--desmotA.jpg" width="800"> </div>
 <br />
-  <div align="center"><p >Fig.4-1 The control groups including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) which have motility and they move everywhere in the plates. The plates on right are ΔmotA (the motA-deletion strain) (B1, B2, B3), E.coli RP6666 which have no motility so the strains stay on the center. We have three biological replicates in the experiment.</p></div>
+  <div align="center"><p >Fig.4-1 The process of motility detection system</p></div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/5e/T--OUC-China--res42.png" height="400"> </div>
-<br />
 <br />
- <div align="center"><p >Fig.4-2 The test group-1. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E.coli cannot move. And the Csy4 have no big influence on strain compared with the strain, ΔmotA. We have three biological replicates in the experiment.</p></div>
+<br /><h4 ><font size="5">4.2 Proof of functions</font></h4>
-<div align="center"><img src="1" height="400"> </div>
+<br />
+Five groups are set, a test group and four control groups. And the results shown below proved that our system can work as expectation.<br />
+<br>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/1/17/T--OUC-China--JCFig.4-2.jpg" width="600"> </div>
+ <div align="center"><p>Fig.4-2 The control groups A and B including positive group and negative group. Plates were inoculated with <i>E. coli</i> RP437 (A1, A2, A3) that have motility and they can move arbitrarily in the plates. The plates on right are ΔmotA strains(the motA-deletion strain) (B1, B2, B3), <i>E. coli</i> RP6666, which have no motility so the strains stay on the center. We have three biological replicates in this experiment.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/59/T--OUC-China--JCFig.4-3.jpg" height="300"> </div>
   <div align="center"><p >
-Fig.4-3 The test group-2. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4). The circuit is on the control of miniToe and it down stream motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.</p></div>
+Fig.4-3 The test group C. The plates were inoculated with Csy4-Δ<i>motA</i> (the <i>motA</i>-deletion strain with Csy4 but no miniToe part).Without the <i>motA</i>, the <i>E. coli</i> cannot move. And the Csy4 have no big influence on strain compared with the Δ<i>motA</i> strain. The little round of papers indicates the places of inducer IPTG (Isopropyl β D thiogalactopy ranoside). We have three biological replicates in the experiment.</p></div>
-<div align="center"><img src="1" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/5/55/T--OUC-China--JCFig.4-4.jpg" height="300"> </div>
 <br />
   <div align="center"><p >
-Fig.4-4 The test group.</p></div>
+Fig.4-4 The test group D. The plates were inoculated with miniToe-<i>motA</i> (the <i>motA</i>-deletion strain with miniToe part but no Csy4. The circuit is on the control of miniToe part and its downstream gene <i>motA</i> can be regulated without Csy4. So MotA can be produced. We have three biological replicates in the experiment.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/bf/T--OUC-China--JCFig.4-5.jpg" height="300"> </div>
+ <div align="center"><p >
+Fig.4-5 The test group E. The strain we culture in plates is miniToe-<i>motA</i> with Csy4. The strain have the miniToe part and Csy4 which means <i>motA</i> can be regulated by miniToe. In the picture, the <i>E. coli</i> move everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene <i>motA</i> come into play. The <i>E. coli</i> can move everywhere in the plate. We have three biological replicates in the experiment.</p></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--OUC-China--24000.jpg" height="400"> </div>
+<div align="center"><p >
+ Fig.4-6 The migration dimensions. The ratio of migration area /whole plate. This chart is made by numerical integration</p></div>
+<br>As we can see, test group strains can move everywhere in the plate and the control groups strains can not move.The test group work as expectation compared to the control groups. But there is no time for us to test more miniToe mutants and Csy4 mutants in miniToe family. We want to realize the function of regulation by using different miniToe family members in the future. So we still have a lot of work to do.
+<br />	<br />
+<a href=' https://2018.igem.org/Team:OUC-China/Experiments'> protocol </a>
 		</p>
-<br /><h4 ><font size="3">reference</font></h4>
-[1] Ravichandar J D, Bower A G, Julius A A, et al. Transcriptional control of motility enables directional movement of Escherichia coli in a signal gradient[J]. Scientific Reports, 2017, 7(1).
+<br /><br /><br /><br /><br />
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