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

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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 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:
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1. A crRNA to serve as translation suppressor by pairing with RBS as a part of miniToe structure.
 
1. A crRNA to serve as translation suppressor by pairing with RBS as a part of miniToe structure.
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2. A Csy4 site as a linker between crRNA and RBS  as a part of miniToe structure, which can be specifically cleaved upon Csy4 expression.
 
2. A Csy4 site as a linker between crRNA and RBS  as a part of miniToe structure, which can be specifically cleaved upon Csy4 expression.
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3. A CRISPR endoribonuclease Csy4.
 
3. A CRISPR endoribonuclease Csy4.
  
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  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 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.                                         
 
                                          
 
                                          
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  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 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.                                       
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  <br /><h4 ><font size="3">1.3 The characteristics of miniToe</font></h4>
 
  <br /><h4 ><font size="3">1.3 The characteristics of miniToe</font></h4>
 
                                          
 
                                          
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1. 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.
 
1. 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.
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2. 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.
 
2. 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.
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3. Compare to the toehold switch, miniToe don't need to redesign crRNA over and over again because the crRNA is not paired with CDS.                                         
 
3. Compare to the toehold switch, miniToe don't need to redesign crRNA over and over again because the crRNA is not paired with CDS.                                         
 
                                          
 
                                          

Revision as of 11:39, 11 October 2018

Team OUC-China: Main
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Demonstrate

1. The first system miniToe


1.1 New method —— miniToe

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:

1. A crRNA to serve as translation suppressor by pairing with RBS as a part of miniToe structure.

2. A Csy4 site as a linker between crRNA and RBS as a part of miniToe structure, which can be specifically cleaved upon Csy4 expression.

3. A CRISPR endoribonuclease Csy4.

Fig.1 The structure of miniToe.


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.

Fig.2 The two plasmids of miniToe test 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 working process of miniToe.



1.2 Proof of function

There are two problems we need to prove about miniToe’s structure and function.
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.

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.


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.

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).



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.

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.

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).


Fig.7 The result from other four teams which have proved our conclusions.

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 here!

1.3 The characteristics of miniToe

1. 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.

2. 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.

3. Compare to the toehold switch, miniToe don't need to redesign crRNA over and over again because the crRNA is not paired with CDS.

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