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

Latest revision as of 02:26, 18 October 2018

Team OUC-China: Main

1. THE FIRST SYSTEM： MINITOE
2. THE SECOND SYSTEM： MINITOE FAMILY
3. THE THIRD SYSTEM: MINITOE POLYCISTRON
4. THE FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM
5. IMPROVEMENT BASED ON 2016 OUC-CHINA

Demonstrate

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 first system: miniToe

1.1 New method: miniToe

Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4 [1]. The whole system works as a translational activator including three modular parts:

1. A cis-repressive RNA (crRNA) served as a translational suppressor by pairing with RBS as the critical part of miniToe structure.
2. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
3. A CRISPR endoribonuclease Csy4.

Fig.1 The composition of miniToe system.

To verify the feasibility and function of miniToe, the following circuits were designed for testing the function of miniToe. An inducible promoter P tac controls the expression level of Csy4. The cis-repressive RNA coding sequence is inserted at the upstream of reporter (sfGFP) gene, which is controlled by a constitutive promoter from Anderson family named J23119.

Fig.2 The two plasmids of miniToe system.

Without Csy4, the crRNA pairs with RBS very well, so the switch just turns off, which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downstream gene can be regulated.

Fig.3 The mechanism of miniToe system.

1.2 Proof of function

There are two problems that need to be proved about miniToe system.

Firstly, the stability of miniToe structure is crucial. Hence, before wet experiment, we predicted the structure of full-length transcript of this circuit as well as miniToe structure [2][3].

Fig.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 Fig.5, 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.

Fig.5 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains 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).

The second problem that needs to be proved is whether miniToe system can work successfully and regulate the downstream genes expression as a switch. Obviously, in the Fig.5, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with 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 the fact that the expression of target gene sfGFP increases indeed due to cleavage of Csy4 site, thus exposing the RBS and restarting translation. It means miniToe system can work successfully.

We also test miniToe system by flow cytometric. In Fig.6, it's easy to distinguish the two groups (blue & white).The intensity of sfGFP of the test group (+IPTG) increases obviously compared with the control group (IPTG=0). The result shows the same conclusions mentioned before.

Fig.6 Flow cytometric measurement the intensity 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 intensity. 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).

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

There are four teams we collaborated with and they helped us prove the previous results by experiments in their labs. Thank you! See more details here!

1.3 The characteristics of miniToe system

1. The Csy4 protein and miniToe structure have high binding affinity [4]. It shows that the miniToe system may control the state of expression in a digital-like way (ON/OFF). When the switch is at OFF state, the downstream gene expression is tightly blocked, the reaction grows very slowly in the beginning but accelerating rapidly once the Csy4 protein truncates the cis-repressive RNA element.

2. Compared to the small RNA based on riboswitch [5], the insertion of hairpin provides Csy4 with a recognition and cleavage site so that the Csy4 may enhance the steric hindrance effect between cis-repressive RNA and RBS when we need to release the cis-repressive RNA for opening the downstream gene expression, which could promote translation activation.

3. Compared to the toehold switch [6], miniToe does not need to redesign cis-repressive RNA case by case because the cis-repressive RNA is not paired with protein coding region.

2. The second system: miniToe family

2.1 The principles of designing mutants

For Csy4 mutants
1. Some key sites in the Csy4 are really crucial for keeping the stability of the structure and maintaining the functions of recognition and cleavage. Mutations on those sites may have a serious influence on our system. By point mutation, we hoped to get a library of mutants, which could provide several Csy4 mutant candidates with recognition and cleavage rates showing as a "ladder".

For hairpin mutants
2. We need to avoid breaking the recognition site and keep the function of cleavage relatively stable. If we break the key site G20, it may lead to the damage of cleavage function.

3. The stability of secondary structure is vital, so we need to focus on each hairpin's Gibbs free energy during designing.

4. The aim is to obtain different hairpins which have various activity for Csy4 recognition and RNA cleavage.

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. By testing all of them, 10 combinations work successfully as expectation. So, our second system, miniToe family, has 10 combinations which were well designed and selected.

2.2 Proof of functions

First, we tested the capabilities of five Csy4 mutants:

1. The result by Microscope
2. The result by flow cytometer
3. The result by microplate reader

Second, six different hairpin mutants were tested by microplate reader.

Finally, all the 30 groups' intensities of fluorescence is tested. We ranked them by the heat map and then selected 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 user-friendly system meets the flexible needs in study about regulating different levels of expression. The final 10 members of miniToe family are shown below.

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.

2.2.1 Proof of functions: Csy4 mutants

The qualitative experiment by Microscope. We can observe visible distinctions 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. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A.

1. The expression of sfGFP by strain-Csy4-WT.

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

3. The expression of sfGFP by strain-Csy4-Y176F.

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

5. The expression of sfGFP by strain-Csy4-H29A.

Fig.8 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.

As the Fig.9 shown, the relative expression level can be measured by flow cytometer.

Fig.9 The intensities of sfGFP of 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.10 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 proves that they have different capabilities.

We tested five Csy4s individually by microplate reader every two hours. The test groups show different characteristics. As we can see in Fig.11, the Csy4-WT shows the same result as the first system. And the expression level is the highest among all the test groups which indicates the highest enzyme activity. The tendency of fluorescence intensities increasion by Csy4-Q104A is almost the same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What's special is Csy4-H29A. We 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. Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage. In summary, the picture shows our prediction by model matchs the result perfectly in Fig.11.

Fig.11 The comparision about model and result by microplate reader. The intensities of sfGFP by microplate reader on the left when the model is on the right.

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.

2.2.2 Proof of functions: Hairpin mutants

We also redesigned 5 hairpin mutants and tested them by flow cytometry and ranked them by their capabilities. The results are listed in the order: miniToe-WT>miniToe-5>miniToe-1>miniToe-4>miniToe-2>miniToe-3.

Fig.12 The 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.13 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 proves that they have different capabilities.

2.2.3 Proof of functions: MiniToe family

And the whole system is tested by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.14. We ranked them by the heat map and then selected 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 gave up some combinations and then selected the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.15. The user-friendly system meets the flexible needs in study which can meet user's need about different levels of expression.

Fig.14 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.

Fig.15 The members of miniToe family.

3. The third system: miniToe polycistron

3.1 The design of miniToe ploycistron

Many applications of synthetic biology need the balanced expressions of multiple genes. For the sake of tuning the expression of genes in polycistron, we planed to realize a tightly regulation by the miniToe structure. Our aim is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.

Fig.16 The mechanisms of miniToe polycistron.

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.

Two kinds of groups are set. One is the bicistron circuit without miniToe parts. The other is the test group which have miniToe parts. This year, we designed two kinds of miniToe polycistrons, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.

Fig.17 The two groups in experiment. Group A is the control group without miniToe system. Group B is the test group with miniToe system.

3.2 Proof of functions

We tested our miniToe polycistron by microplate reader. The sfGFP were measured at excitation/emission wavelengths of 485nm/520nm. The mCherry were measured at excitation/emission wavelengths of 587nm/610nm.

Fig.18 The rate of fluorescence intensities by sfGFP/mCherry.

4. THE FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM

4.1 The purpose of designing the experiment

As a translation regulation tool, miniToe can also be used in application scenario of molecular mechanism study. Sometimes scientists may puzzle about the functions of certain gene or protein without in-depth study. One general method to study them is to "knock-out" or "knock-in". In this way, organisms show some phenotypic changes related to particular gene. However, if we want to know better about the functions of the gene, we may need more tools to realize different levels of gene expression.

By using our system, the motility of E. coli can be regulated. As we all know, MotA provides a channel for the proton gradient required for generation of torque [8]. 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.

We did a lot of works to test our minToe system by applying it to the detection of E. coli motility. We constructed circuit by putting the motA behind miniToe part. So the target gene motA can be regulated by our miniToe system.

Fig.19 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.20 The control groups A and B including positive group and negative group. Plates were inoculated with E. coliRP437 (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.21 The test group C. 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 ΔmotA strain. The little round of papers indicates the places of inducer IPTG (Isopropyl-β-d-thiogalactoside). We have three biological replicates in the experiment.

Fig.22 The test group D. 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 its downstream gene motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.

Fig.23 The test group E. The strain we cultured in plates is miniToe-motA with Csy4. The strain have the whole miniToe system which means motA can be regulated by miniToe part. In the picture, the E. coli moved everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene motA comes into play. The E. coli can move everywhere in the plate. We have three biological replicates in the experiment.

Fig.24 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 cannot move. The test group works 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.

5. Improvement based on 2016 OUC-China

The 2016 OUC-China had a method to regulate the expression of polycistron.
They concentrated on stem-loops inserted into the intergenic regions. When transcribed as one polycistron, digested and separated into several independent fragments, cistrons with 3' end stem-loops will get different stability. They designed a lot of different stem-loop. And they measured and standardized a series of native and designed stem-loops, transforming into a toolkit for better uses.
Click here to see 2016 OUC-China.

Fig.25 The three stem-loops designed by 2016 OUC-China

The new system named miniToe polycistron is an important improvement inspired by 2016 OUC-China. The idea using stem-loop to regulate the gene expression is creative and inspired us to do more. We found miniToe part is also a good stem-loop that can be inserted to polycistron. We inserted three miniToe parts to circuit when we need to tune the expression of two genes. It means that for each target gene we have two miniToe hairpins. The one is downstream of the gene, and the other is upstream of the gene. We made an enhanced version based on previous project.

1.The 2016 OUC-China only put the hairpins between two genes, and we put the hairpins downstream and upstream for each gene. Without Csy4, the existence of miniToe structure in polycistron will inhibit the gene expression when we don’t want to open the switch. The cis-repressive RNA in miniToe has complementary sequence of adjacent RNA region (RBS) to prevent the binding of ribosomes.

2. After cleavage, in the 5' end, the capabilities of cleavage by RNase E is much lower because there is no pyrophosphate in the 5' end. Qi's work has proved that OH-mRNAs exhibit higher gene expression than 5' PPP-mRNAs [7].

3. Also, the RNA secondary structure of miniToe with Csy4 binding keeping at 3’ UTR after Csy4 cleavage is a protection mechanism to prevent RNA degradation. Because more energy is needed which partly provided by ATP to degrade the RNA/Csy4 complexes in the 3' end, the degradation rate of RNA is much lower. And this function is based on the high stability and affinity [7] between Csy4 and miniToe structure.

4. The function of each miniToe structure has specific recognition and cleavage rates, which will make it possible to regulate the gene flexibly.

ACHIEVEMENTS

★Our engineered system worked under realistic conditions, which is proved by our different forms of experimental datas, including the qualitative experiment by microscope, flow cytometric measurements and microplate reader. Furthermore, we applied our system into motility detection system and it worked well. Other four iGEM teams also helped us test our system successfully in their lab.
★We put safety in the first place all the time, ensuring the safety of our project, the safety of our lab work and the safety of biobrick shipment. Go to see Safety page!
★Our miniToe polycistron system derives from 2016 OUC-China. Based on their project, we put the hairpins downstream and upstream for each gene rather than only put the hairpins between two genes. In our system, the degradation rate of RNA hairpin is much lower than theirs, and the gene regulation is more flexible, which improved a lot compared to their project.
★We have designed four systems this year. We measured functions of these four systems in detail and describe it clearly.

Go to see design page!

Go to see result page!

REFERENCE

[1]. Du P, Miao C, Lou Q, et al. Engineering Translational Activators with CRISPR-Cas System[J]. Acs Synthetic Biology, 2016, 5(1):74.
[2].Hofacker I L. Vienna RNA secondary structure server[J]. Nucleic Acids Research, 2003, 31(13):3429-3431.
[3].M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-3415, 2003.
[4].Przybilski R, Richter C, Gristwood T, et al. Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.[J]. Rna Biology, 2011, 8(3):517-528.
[5].Mandal M, Breaker R R. Gene regulation by riboswitches.[J]. Nature Reviews Molecular Cell Biology, 2004, 5(6):451-63.
[6].Green A, Silver P, Collins J, et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
[7].Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature Biotechnology, 2012, 30(10):1002.
[8].Ravichandar J D, Bower A G, Julius A A, et al. Transcriptional control of motility enables directional movement ofEscherichia coliin a signal gradient[J]. Scientific Reports, 2017, 7(1).

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 			<!-- Main -->
-					<div id="main" class="container">
+						<div class="sticky">
+<div class="topleft2">
+<br /><br /><br />
+<ul class="side-nav" style="width:150px">
+  <li><a href="#tips1"id="thu">1. THE FIRST SYSTEM： MINITOE</a></li>
+  <li><a href="#tips2"id="thu">2. THE SECOND SYSTEM： MINITOE FAMILY
+</a></li>
+  <li><a href="#tips3"id="thu">3. THE THIRD SYSTEM: MINITOE POLYCISTRON
+</a></li>
+  <li><a href="#tips4"id="thu">4. THE FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM
+</a></li>
+	<li><a href="#tips5"id="thu">5. IMPROVEMENT BASED ON 2016 OUC-CHINA
+</a></li>
+</ul>
+</div>
+</div>
+								<div class="nb">
-							<div class="12u">
+							<div class="sk">
 								<!-- Features -->
 									<section class="box features">
-										<h2 class="major"><span>Demonstrate</span></h2>
+										<h2 class="major"><span><font size="7"><span>Demonstrate</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><font size="4">
+<br />To help you enjoy our result in a more convenient and smooth way, here are some key concepts in our discussion.
+<br />
+) 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 />
 <p>
-<h3>1. The first system miniToe</h3>
+<a id="tips1"></a><h3><font size="6">1. The first system: miniToe</font></h3>
-<br /><h4 ><font size="3">1.1 New method —— miniToe</font></h4>
+<br /><h4 ><font size="5">1.1 New method: miniToe</font></h4>	<br />
-Based on Csy4’s function, this year we design a new structure named miniToe which can be recognized by Csy4. Our system is a translational activator including three modular parts:
+Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4 [1]. The whole system works as a translational activator including three modular parts:
 <br /> <br />
-. A crRNA to serve as translation suppressor by pairing with RBS as a part of miniToe structure.
+. A cis-repressive RNA (crRNA) served as a translational suppressor by pairing with RBS as the critical part of miniToe structure.
-<br /> <br />
+<br />
-. A Csy4 site as a linker between crRNA and RBS  as a part of miniToe structure, which can be specifically cleaved upon Csy4 expression.
+. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
-<br /><br />
+<br />
 . A CRISPR endoribonuclease Csy4.
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f5/T--OUC-China--design2-1.png" height="400">
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/20/T--OUC-China--demonstrate-1.png" height="400">
 										</div>
 <br />
-  <div align="center"><p >Fig.1 The structure of miniToe.</p></div>
+  <div align="center"><p >Fig.1 The composition of miniToe system.</p></div>
 <br />
- In our project, we use sfGFP as a target gene to test our system. To explore the feasibility and function of miniToe, we designed the circuit below as our test system in order to test the function of miniToe structure. We use Ptac as the inducible promoter of Csy4 to control the existence of Csy4 or not. At the same time, we construct the miniToe before the sfGFP which is a symbol of target gene in our circuit. And this circuit is controlled by a constitutive promoter form Anderson family named J23119.
+To verify the feasibility and function of miniToe, the following circuits were designed for testing the function of miniToe. An inducible promoter P <i>tac </i> controls the expression level of Csy4. The cis-repressive RNA coding sequence is inserted at the upstream of reporter (sfGFP) gene, which is controlled by a constitutive promoter from Anderson family named J23119.
   <div align="center"><img src="https://static.igem.org/mediawiki/2018/1/16/T--OUC-China--design2-3.png" width="700" >
 </div>
 <br />
-  <div align="center"><p >Fig.2 The two plasmids of miniToe test system.</p></div>
+  <div align="center"><p >Fig.2 The two plasmids of miniToe system.</p></div>
   <br />
- Without Csy4, the crRNA pairs with RBS very well, so the switch just turns off, which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downstream gene can be regulated.
+Without Csy4, the crRNA pairs with RBS very well, so the switch just turns off, which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downstream gene can be regulated.
   <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/ce/T--OUC-China--design2-4.png" width="700" >
 </div>
 <br />
-  <div align="center"><p >Fig.3 The working process of miniToe.</p></div>
+  <div align="center"><p >Fig.3 The mechanism of miniToe system.</p></div>
   <br />
-<br /><h4 ><font size="3">1.2 Proof of function</font></h4>
+<br /><h4 ><font size="5">1.2 Proof of function</font></h4>	<br />
-There are two problems we need to prove about miniToe’s structure and function.
+There are two problems that need to be proved about miniToe system.
 <br /><br />
-First, we need to make sure the stability of our structure and the formation of hairpin (The Csy4 site) is also crucial. So before the experiment, we focus on the structure of miniToe. We have a prediction of structure of whole circuit as well as the structure of miniToe.
+Firstly, the stability of miniToe structure is crucial. Hence, before wet experiment, we predicted the structure of full-length transcript of this circuit as well as miniToe structure [2][3].
+<br /><br />
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/5/5c/T--OUC-China--design2-2.png" width="700" >
 </div>
 <br />
-  <div align="center"><p >Fig.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.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>
   <br />
-But in fact, we also need to prove that our miniToe can fold directly in reality by experiments. As the result showed in Fig.5, a 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.
+As the result showed in Fig.5, 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.
 <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.5 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.5 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains 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>
@@ Line 194: / Line 277: @@
- 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 Fig.5 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.
+The second problem that needs to be proved is whether miniToe system can work successfully and regulate the downstream genes expression as a switch. Obviously, in the Fig.5, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with 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 the fact that the expression of target gene sfGFP increases indeed due to cleavage of Csy4 site, thus exposing the RBS and restarting translation. It means miniToe system can work successfully.
   <br /> <br />
-  We also test our system by flow cytometric and the blue group showed in the Fig.6 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.
+  We also test miniToe system by flow cytometric. In Fig.6, it's easy to distinguish the two groups (blue & white).The intensity of sfGFP of the test group (+IPTG) increases obviously compared with the control group (IPTG=0). 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.6 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.6 Flow cytometric measurement the intensity 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 intensity. 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>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e4/T--OUC-China--res7.png" height="250"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/4c/T--OUC-China--res72.jpg" height="750"> </div>
 <br />
-  <div align="center"><p >Fig.7 The result from other four teams which have proved our conclusions. </p></div>
+  <div align="center"><p >Fig.7 The results from other four teams which proved our conclusions.  Error bars represent standard deviation of four biological replicates. </p></div>
-  We also have collaborations with other 3 teams, and they help us in proving our results by experiments in their labs. Thank you! See more details <a href="https://2018.igem.org/Team:OUC-China/Collaborations">here!</a>
+  There are four teams we collaborated with and they helped us prove the previous results by experiments in their labs. Thank you! See more details <a href="https://2018.igem.org/Team:OUC-China/Collaborations">here!</a>
   <br />
-  <br /><h4 ><font size="3">1.3 The characteristics of miniToe</font></h4>
+  <br /><h4 ><font size="5">1.3 The characteristics of miniToe system</font></h4> <br />
-. The Csy4/RNA complexes become really stable once they have formed. It shows that the miniToe can control the state of expression like a switch (ON/OFF). When the switch is turn OFF, the downstream gene expression is completely closed, the reaction grows very slow in the beginning but accelerating rapidly once the complexes have formed.
+. The Csy4 protein and miniToe structure have high binding affinity [4]. It shows that the miniToe system may control the state of expression in a digital-like way (ON/OFF). When the switch is at OFF state, the downstream gene expression is tightly blocked, the reaction grows very slowly in the beginning but accelerating rapidly once the Csy4 protein truncates the cis-repressive RNA element.
                                          <br /><br />
-. Compare to the small RNA, the insertion of hairpin provides Csy4 with a recognition and cleavage site so that the Csy4 may enlarge the steric hindrance between crRNA and RBS when we need to release the crRNA for opening the downstream gene expression.
+. Compared to the small RNA based on riboswitch [5], the insertion of hairpin provides Csy4 with a recognition and cleavage site so that the Csy4 may enhance the steric hindrance effect between cis-repressive RNA and RBS when we need to release the cis-repressive RNA for opening the downstream gene expression, which could promote translation activation.
                                          <br /><br />
-. Compare to the toehold switch, miniToe don't need to redesign crRNA over and over again because the crRNA is not paired with CDS.
+. Compared to the toehold switch [6], miniToe does not need to redesign cis-repressive RNA case by case because the cis-repressive RNA is not paired with protein coding region.
@@ Line 228: / Line 313: @@
 <p>
-<h3>2. The second system miniToe family</h3>
+<a id="tips2"></a><h3><font size="6">2. The second system: miniToe family</font></h3>
-<br /><h4 ><font size="3">2.1 The principles of designing mutants</font></h4>
+<br /><h4 ><font size="5">2.1 The principles of designing mutants</font></h4><br />
 For Csy4 mutants
 <br />
-. Some key sites in the Csy4 is really crucial for keeping the stable of structure and making sure the functions including recognition and cleavage. The change of those sites may results in big influence on design. By point mutation, we want to get an amount of mutants whose recognition and cleavage rates shows as a "ladder".
+. Some key sites in the Csy4 are really crucial for keeping the stability of the structure and maintaining the functions of recognition and cleavage. Mutations on those sites may have a serious influence on our system. By point mutation, we hoped to get a library of mutants, which could provide several Csy4 mutant candidates with recognition and cleavage rates showing as a "ladder".
 <br /> <br />
@@ Line 240: / Line 325: @@
 <br />
-. Do not to break the recognition site so that we can ensure the function of cleavage. If we break the key site G20 may lead to the damage of cleavage function.
+. We need to avoid breaking the recognition site and  keep the function of cleavage relatively stable. If we break the key site G20, it may lead to the damage of cleavage function.
 <br /> <br />
-. The stable of secondary structure is vital so we need to focus on each hairpin's Gibbs free energy after design.
+. The stability of secondary structure is vital, so we need to focus on each hairpin's Gibbs free energy during designing.
 <br /> <br />
-. Our aim is to obtain different hairpins which have totally different rates be recognized and cleaved.
+. The aim is to obtain different hairpins which have various activity for Csy4 recognition and RNA cleavage.
 <br /> <br />
-With the help of model, we finally select 4 Csy4 mutants and 5 hairpin mutants. We have tested each mutant and have data support. Then there are 5*6 combinations including wild types. We have tested all of them and have proved that 10 of them work successfully as we expect. So the second system of our project, miniToe family consist of 10 combinations which is designed and selected by us.
+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. By testing all of them, 10 combinations work successfully as expectation. So, our second system, miniToe family, has 10 combinations which were well designed and selected.
 <br /> <br />
-<br /><h4 ><font size="3">2.2 Proof of functions</font></h4>
+<br /><h4 ><font size="5">2.2 Proof of functions</font></h4><br />
-First, we have tested five different Csy4 mutants by three ways:
+First, we tested the capabilities of five Csy4 mutants:
 <br /> <br />
 . The result by Microscope
-<br /> <br />
+<br />
 . The result by flow cytometer
-<br /> <br />
+<br />
 . The result by microplate reader
 <br /> <br />
-Second, we have tested six different hairpin mutants by microplate reader.
+Second, six different hairpin mutants were tested by microplate reader.
 <br /> <br />
-Finally, we have tested all the 30 groups’ intensities of fluorescence. We rank them by the heat map and then select the groups from different expression levels. 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 below.
+Finally, all the 30 groups' intensities of fluorescence is tested. We ranked them by the heat map and then selected 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 user-friendly system meets the flexible needs in study about regulating different levels of expression. The final 10 members of miniToe family are shown below.
-<br />
+<br /> <br />
+<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 />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/7/7f/T--OUC-China--plasmid.jpg
+" height="200"> </div>
-<br /><h5 ><font size="3">2.2.1 Proof of functions: Csy4 mutants</font></h5>
+<br /><h5 ><font size="5">2.2.1 Proof of functions: Csy4 mutants</font></h5>  <br />
-The qualitative experiment by Microscope. We can observe visible distinctions 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. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A.
+The qualitative experiment by Microscope. We can observe visible distinctions 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. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A.
 <br /> <br />
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/a/a2/T--OUC-China--res24.png" width="400"> </div>
+<div>
-<br />
-  <div align="center"><p >Fig.8 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.
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/28/T--OUC-China--JCYWT.png" width="800"> </div>
-</p></div>
+<div align="center"><p >1. The expression of sfGFP by strain-Csy4-WT.</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 >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 >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 >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 >5. The expression of sfGFP by strain-Csy4-H29A.</p></div>
+<div align="center"><p >Fig.8 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>
 As the Fig.9 shown, the relative expression level can be measured by flow cytometer.
 <br /> <br />
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e8/T--OUC-China--fig2-5z.png" height="400"> </div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e8/T--OUC-China--fig2-5z.png" height="450"> </div>
 <br />
-  <div align="center"><p >Fig.9 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.9 The intensities of sfGFP of 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.10 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>
+  <div align="center"><p >Fig.10 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 proves that they have different capabilities.  </p></div>
-We tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the Images 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.11, the Csy4-WT shows the same result with the first system. And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT. 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. In summary, the picture shows our prediction by model matchs the result perfectly in Fig.11.
+We tested five Csy4s individually by microplate reader every two hours. The test groups show different characteristics. As we can see in Fig.11, the Csy4-WT shows the same result as the first system. And the expression level is the highest among all the test groups which indicates the highest enzyme activity. The tendency of fluorescence intensities increasion by Csy4-Q104A is almost the same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What's special is Csy4-H29A. We 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. Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage. In summary, the picture shows our prediction by model matchs the result perfectly in Fig.11.    <br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/ba/T--OUC-China--res28.png" height="300"> </div>
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/b/ba/T--OUC-China--res28.png" height="400"> </div>
 <br />
-  <div align="center"><p >Fig.11 The comparison about model and result by microplate reader. The fluorescence intensities of sfGFP by microplate reader on the left when the model is on the right.
+  <div align="center"><p >Fig.11 The comparision about model and result by microplate reader. The intensities of sfGFP by microplate reader on the left when the model is on the right.
-</p></div>
+</div>
+<br />
+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 />
+    </p>
-<br /><h5 ><font size="3">2.2.2 Proof of functions: Hairpin mutants</font></h5>
+<br /><h5 ><font size="5">2.2.2 Proof of functions: Hairpin mutants</font></h5><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.
+We also redesigned 5 hairpin mutants and tested them by flow cytometry and ranked them by their capabilities. 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>
+<br /><br />
 <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.12 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.12 The 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.13 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.
+  <div align="center"><p >Fig.13 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 proves that they have different capabilities.
 </p></div>
-<br /><h5 ><font size="3">2.2.3 Proof of functions: MiniToe family</font></h5>
+<br /><h5 ><font size="5">2.2.3 Proof of functions: MiniToe family</font></h5> <br />
-And we have tested our whole system by flow cytometry. All the 30 groups’ intensities of fluorescence are shown in Fig.14. 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.15. The user-friendly system meets the flexible needs in study which can help user’s need about different levels of expression.
+And the whole system is tested by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.14. We ranked them by the heat map and then selected 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 gave up some combinations and then selected the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.15. The user-friendly system meets the flexible needs in study which can meet user's need about different levels of expression.
 <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.14 The heat map generated from flow cytometry data reflecting 30 groups’ intensities of fluorescence by sfGFP.
+  <div align="center"><p >Fig.14 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.
 </p></div>
@@ Line 339: / Line 465: @@
 <p>
-<h3>3. The third system——miniToe polycistron</h3>
+<a id="tips3"></a><h3><font size="6">3. The third system: miniToe polycistron</font></h3>
-<br /><h4 ><font size="3">3.1 The design of miniToe ploycistron</font></h4>
+<br /><h4 ><font size="5">3.1 The design of miniToe ploycistron</font></h4><br />
-Many applications of synthetic biology need the balanced expression of multiple genes.
+Many applications of synthetic biology need the balanced expressions of multiple genes.
-For the sake of tuning the expression of genes within polycistron, we want to develop a tightly regulated by the miniToe structure. Our aim for this part is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.
+For the sake of tuning the expression of genes in polycistron, we planed to realize a tightly regulation by the miniToe structure. Our aim is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.
 <div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f2/T--OUC-China--design4-1.png" height="500">
 										</div> 	<br />
-  			<div align="center"><p >Fig.16 The mechanisms of miniToe polycistron
+  			<div align="center"><p >Fig.16 The mechanisms of miniToe polycistron.
 	 </p></div>
-By inserting our hairpins between intergentic regions, it influences the amount of expressed proteins. First, we decide to use GFP and mCherry to test our system in bicistron simply. Then we select some miniToe structures designed by us and insert them between, before and behind GFP and mCherry. If there is bicistron, then we need three miniToe structure. If there are three genes which form polycistron, then we need four miniToe structure. And so on.
+<br />
-<br /><br />
+By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins. <br />
-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.
+)	First, sfGFP and mCherry is used as a test system in bi-cistron circuit. <br />
+)	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 />
 <br /><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 expectations, we have created the recombinant strain (control group) which only has the circuit constructed by miniToe without Csy4. The test group have both miniToe polycistron and Csy4. This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.
+Two kinds of groups are set. One is the bicistron circuit without miniToe parts. The other is the test group which have miniToe parts. This year, we designed two kinds of miniToe polycistrons, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.
 <br /><br />
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/9/91/T--OUC-China--fig17.jpg" height="500">
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/a/a6/T--OUC-China--res31.jpg" width="700">
 										</div> 	<br />
-  			<div align="center"><p >Fig.17 The three test groups. Group A is the control group without miniToe system. Group B is the control group with miniToe but no Csy4. Group C is the test group with miniToe and Csy4.
+  			<div align="center"><p >Fig.17 The two groups in experiment. Group A is the control group without miniToe system. Group B is the test group with miniToe system.
 	 </p></div>
-<br /><h4 ><font size="3">3.2 Proof of functions</font></h4>
+<br /><h4 ><font size="5">3.2 Proof of functions</font></h4><br />
-We have tested our miniToe polycistron by microplate reader after culturing 10 hours. The sfGFP were measured at excitation/emission wavelengths of 485nm/520nm. The mCherry were measured at excitation/emission wavelengths of 587nm/610nm.
+We tested our miniToe polycistron by microplate reader. The sfGFP were measured at excitation/emission wavelengths of 485nm/520nm. The mCherry were measured at excitation/emission wavelengths of 587nm/610nm.
-<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/45/T--OUC-China--fig18.jpg" height="500">
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/4/42/T--OUC-China--res322.png" height="500">
 										</div> 	<br />
-  			<div align="center"><p >操你妈
+  			<div align="center"><p >Fig.18 The rate of fluorescence intensities by sfGFP/mCherry.
 	 </p></div>
@@ Line 379: / Line 506: @@
+<p>
+<a id="tips4"></a><h3><font size="6">4. THE FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM
+</font></h3>
+<br /><h4 ><font size="5">4.1 The purpose of designing the experiment</font></h4><br />
+As a translation regulation tool, miniToe can also be used in application scenario of molecular mechanism study. Sometimes scientists may puzzle about the functions of certain gene or protein without in-depth study. One general method to study them is to "knock-out" or "knock-in". In this way, organisms show some phenotypic changes related to particular gene. However, if we want to know better about the functions of the gene, we may need more tools to realize different levels of gene expression.
+<br />
+<br>By using our system, the motility of <i>E. coli</i> can be regulated. As we all know, MotA provides a channel for the proton gradient required for generation of torque [8]. 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.
+<br />
+<br>We did a lot of works to test our minToe system by applying it to the detection of <i> E. coli</i> motility. We constructed circuit by putting the <i>motA</i> behind miniToe part. So the target gene <i>motA</i> can be regulated by our miniToe system.
+<br />
+<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.19 The process of motility detection system</p></div>
+<br />
+<br /><h4 ><font size="5">4.2 Proof of functions</font></h4>
+<br />
+<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.20 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 Δ<i>motA</i> strains(the <i>motA</i>-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.21 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 structure).Without the gene <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-thiogalactoside). We have three biological replicates in the experiment.</p></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.22 The test group D. The plates were inoculated with miniToe-<i>motA</i> (the <i>motA</i>-deletion strain with miniToe structure but no Csy4. The circuit is on the control of miniToe and its downstream gene <i>motA</i> can be regulated without Csy4. So the expression of downstream gene <i>motA</i> keep closing. 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.23 The test group E. The strain we cultured in plates is miniToe-<i>motA</i> with Csy4. The strain have the whole miniToe system which means <i>motA</i> can be regulated by miniToe part. In the picture, the <i>E. coli</i> moved everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene <i>motA</i> comes 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="300"> </div>
+<div align="center"><p >
+ Fig.24 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 cannot move. The test group works 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 />
+		</p>
+<p>
+<a id="tips5"></a><h3><font size="6">5. Improvement based on 2016 OUC-China</font></h3>
+<br />
+<br />
+The 2016 OUC-China had a method to regulate the expression of polycistron.
+<br />They concentrated on stem-loops inserted into the intergenic regions. When transcribed as one polycistron, digested and separated into several independent fragments, cistrons with 3' end stem-loops will get different stability. They designed a lot of different stem-loop. And they measured and standardized a series of native and designed stem-loops, transforming into a toolkit for better uses.
+<br /><a href='https://2016.igem.org/Team:OUC-China/Design'> Click here to see 2016 OUC-China. </a>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d5/T--OUC-China--pr-3-6.jpg" height="400"> </div>
+<br />
+<br />
+ <div align="center"><p >Fig.25 The three stem-loops designed by 2016 OUC-China</p></div>
+<br />
+The new system named miniToe polycistron is an important improvement inspired by 2016 OUC-China. The idea using stem-loop to regulate the gene expression is creative and inspired us to do more. We found miniToe part is also a good stem-loop that can be inserted to polycistron. We inserted three miniToe parts to circuit when we need to tune the expression of two genes. It means that for each target gene we have two miniToe hairpins. The one is downstream of the gene, and the other is upstream of the gene. We made an enhanced version based on previous project.
+<br /><br />
+.The 2016 OUC-China only put the hairpins between two genes, and we put the hairpins downstream and upstream for each gene. Without Csy4, the existence of miniToe structure in polycistron will inhibit the gene expression when we don’t want to open the switch. The cis-repressive RNA in miniToe has complementary sequence of adjacent RNA region (RBS) to prevent the binding of ribosomes.
+<br /><br />
+. After cleavage, in the 5' end, the capabilities of cleavage by RNase E is much lower because there is no pyrophosphate in the 5' end. Qi's work has proved that OH-mRNAs exhibit higher gene expression than 5' PPP-mRNAs [7]. <br /><br />
+. Also, the RNA secondary structure of miniToe with Csy4 binding keeping at 3’ UTR after Csy4 cleavage is a protection mechanism to prevent RNA degradation. Because more energy is needed which partly provided by ATP to degrade the RNA/Csy4 complexes in the 3' end, the degradation rate of RNA is much lower. And this function is based on the high stability and affinity [7] between Csy4 and miniToe structure. <br /><br />
+. The function of each miniToe structure has specific recognition and cleavage rates, which will make it possible to regulate the gene flexibly. <br /><br />
+<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/ff/T--OUC-China--project-_overview.png" width="800"></div>
+<h3><font color="#008B45">ACHIEVEMENTS</font></h3>
+★Our engineered system worked under realistic conditions, which is proved by our different forms of experimental datas, including the qualitative experiment by microscope, flow cytometric measurements and microplate reader. Furthermore, we applied our system into motility detection system and it worked well. Other four iGEM teams also helped us test our system successfully in their lab.
+<br />★We put safety in the first place all the time, ensuring the safety of our project, the safety of our lab work and the safety of biobrick shipment.<a href=' https://2018.igem.org/Team:OUC-China/Safety'>  Go to see Safety page! </a>
+<br />★Our miniToe polycistron system derives from 2016 OUC-China. Based on their project, we put the hairpins downstream and upstream for each gene rather than only put the hairpins between two genes. In our system, the degradation rate of RNA hairpin is much lower than theirs, and the gene regulation is more flexible, which improved a lot compared to their project.
+<br />★We have designed four systems this year. We measured functions of these four systems in detail and describe it clearly.
+<br /><br />
+<a href=' https://2018.igem.org/Team:OUC-China/Results'> Go to see design page! </a>
+<br /><br />
+<a href=' https://2018.igem.org/Team:OUC-China/Results'> Go to see result page! </a>
+<br /><br /><br />
+<h3>REFERENCE</h3>
+[1]. Du P, Miao C, Lou Q, et al. <i>Engineering Translational Activators with CRISPR-Cas System</i>[J]. Acs Synthetic Biology, 2016, 5(1):74.
+<br />
+[2].Hofacker I L. <i>Vienna RNA secondary structure server</i>[J]. Nucleic Acids Research, 2003, 31(13):3429-3431.
+<br />
+[3].M. Zuker. <i>Mfold web server for nucleic acid folding and hybridization prediction. </i>Nucleic Acids Res. 31 (13), 3406-3415, 2003.
+<br />
+[4].Przybilski R, Richter C, Gristwood T, et al. <i>Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.</i>[J]. Rna Biology, 2011, 8(3):517-528.
+<br />[5].Mandal M, Breaker R R. <i>Gene regulation by riboswitches.</i>[J]. Nature Reviews Molecular Cell Biology, 2004, 5(6):451-63.
+<br />[6].Green A, Silver P, Collins J, et al. <i>Toehold switches: de-novo-designed regulators of gene expression.</i>[J]. Cell, 2014, 159(4):925-939.
+<br />[7].Qi L, Haurwitz R E, Shao W, et al. <i>RNA processing enables predictable programming of gene expression</i>[J]. Nature Biotechnology, 2012, 30(10):1002.
+<br />[8].Ravichandar J D, Bower A G, Julius A A, et al. <i>Transcriptional control of motility enables directional movement ofEscherichia coliin a signal gradient</i>[J]. Scientific Reports, 2017, 7(1).
+<br /><br />
+<br /><br />
+<br /><br /><br /><br />
+        </p>
                                  </section>
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