Difference between revisions of "Part:BBa K2615019"

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===Background of 2018 OUC-China' project——miniToe family===
 
===Background of 2018 OUC-China' project——miniToe family===
 
<p>
 
<p>
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This year, we design a toolkit named miniToe family focused on translational regulation, which is composed of a RNA endoribonuclease ([https://parts.igem.org/Part:BBa_K2615003 Csy4]) and a RNA module (hairpin). In our project, the cleavage function of Csy4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.  
+
This year, we designed and achieved a gene regulatory toolbox based on CRISPR RNA endonucleases Csy4 for post-transcriptional regulation. A rational designed modular RNA fragment named miniToe was utilized for precise and efficient translational regulation. The miniToe module was constructed through inserting a 22 nt Csy4 recognition site between RBS and a cis-repressive RNA element, which is able to mask the RBS region and inhibit translation initiation. By up-regulating the level of Csy4 in cell as input, the miniToe module will be cleaved and releases an exposed RBS for output translation. As our innovation, we further designed four Csy4 mutants and five mutated miniToe module in a predictable way by modeling, which aims at enriching our toolkit for diverse regulation ranges on target genes. The whole toolbox includes ten combinations of different Csy4 mutants and miniToe modules, which is called miniToe family. Based on this tool, we can achieve various expression profiles on a polycistron.
 
<br>
 
<br>
 
[[Image:T--OUC-China--JH--complex.png|center|thumb|400px|'''Fig.1  Csy4 and hairpin can form a stable structure.''']]
 
[[Image:T--OUC-China--JH--complex.png|center|thumb|400px|'''Fig.1  Csy4 and hairpin can form a stable structure.''']]
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</p>
 
</p>
  
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===miniToe polycistron===
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===The new methed to tune multple genes===
 
<p>
 
<p>
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Having designed different hairpin structures and finished experimental verification, we think it is a good idea to apply it to the polycistronic to achieve more functions by using miniToe. The polycistronic structure can meet the requirements of simultaneous and large-scale expression of different genes. We insert the hairpin structure(miniToe) into the polycistron and apply the interaction between the hairpin structure and the Csy4 enzyme to achieve the regulation of different ratio of multiple gene expression levels, meeting the requirements of practical application.
+
Many applications of synthetic biology need to regulate the expression profile of multiple genes. Microorganisms with programmable and engineered metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway [1][2]. Indeed, pathway optimization requires to adjust the expression of multiple genes at appropriately pattern, for example, the synthetic of poly-3-hydroxybutyrate and Mevalonate [3].  
 
<br>
 
<br>
 
  <br>
 
  <br>
 +
As has been done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient method for regulating genes simultaneously. Thus, for the sake of tuning the expression pattern of genes within polycistron, we hope to develop a powerful regulation tool by the miniToe system. We name this system miniToe polycistron which contains several genes in one circuit with different miniToe design. Our aim for this part is achieving different expression profile of the genes by miniToe in polycistrons compared with normal polycistrons.
 +
<br>
 +
<br>
 +
[1].Pfleger, B.F., et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol, 2006. 24(8): p. 1027-32.
 +
<br>
 +
[2].Xu, C., et al., Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization. Nat Commun, 2015. 6: p. 6900.
 +
<br>
 +
[3].Smolke, C.D. and J.D. Keasling, Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng, 2002. 80(7): p. 762-76.
 +
</p>
 +
 +
 +
===How to do===
 +
<p>
 +
By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.
 +
<br>
 +
* First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
 +
* 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>
 +
The reasons why we design like this are below:
 +
<br>
 +
1. 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>
 +
2. 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. And this function is based on the high stability and affinity between Csy4 and target RNA structure [1].
 +
<br>
 +
3. The function of each miniToe has specific recognition and cleavage rates, which will make it possible to regulate the gene flexibly.
 +
<br>
 +
<br>
 +
All in all, miniToe polycistron system has two components, Csy4 and the circuit of polycistron. With Csy4 protein, the polycistron will be cut into several mRNA chains with RNA/Csy4 complex at the 3’ UTR. The capability of RNA degradation protection will be much stronger, because of the high stability and affinity of Csy4 binding, which increase energy threshold for RNA degradation from 3’ UTR. So, the RNA degradation rate will be much lower. For the 5’ end degradation, the Csy4 cut will leave a OH- at 5’ end. the cleavage capability of RNase E will be much lower because there is no pyrophosphate in the 5’ end. Qi’s work [1] has proved that OH-mRNAs exhibit higher gene expression than 5’ PPP-mRNAs.
 
[[Image:T--OUC-China--principle2.jpg |center|thumb|500px|'''Fig.2 The working process of miniToe polycistron.'''  ]]
 
[[Image:T--OUC-China--principle2.jpg |center|thumb|500px|'''Fig.2 The working process of miniToe polycistron.'''  ]]
 +
From now, we construct two polycistron(miniToe polycistron A and miniToe polycistron B) by inserting three miniToes before, between and behind two CDS. We use miniToe-WT(BBa_K2615020) before and behind miniToe polycistron B(BBa_K2615018), meanwhile the miniToe-6(BBa_K2615017) is used between two CDS. And we use sfGFP and mCherry to test our system. The result shows we can totally change the origin ratio of two genes.
 +
<br>
 +
[1].Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature Biotechnology, 2012, 30(10):1002.
 +
</p>
 +
 +
 +
 +
=='''''Result'''''==
 +
 +
===The purpose of experiment===
 +
<p>
 +
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>
 +
1)First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
 +
<br>
 +
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.
 +
<br>
 +
<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.
 +
<br>
 +
[[Image:T--OUC-China--principle4.jpg|center|thumb|650px|'''Fig.3 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system.'''  ]]
 +
</p>
 +
 +
===Proof of functions===
 +
<p>
 +
The result by microplate reader has been shown in the Fig.4. 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>
 
<br>
−
From now, we construct two polycistron([https://parts.igem.org/Part:BBa_K2615018 miniToe polycistron A] and miniToe polycistron B) by inserting three miniToes before, between and behind two CDS. We use [https://parts.igem.org/Part:BBa_K2615020 miniToe-WT(BBa_K2615020)] before and behind miniToe polycistron B(BBa_K2615018), meanwhile the [https://parts.igem.org/Part:BBa_K2615017 miniToe-6(BBa_K2615017)] is used between two CDS. And we use sfGFP and mCherry to test our system. The result shows we can totally change the origin ratio of two genes.
+
[[Image:T--OUC-China--POLY.jpg|center|thumb|500px|'''Fig.4 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates.'''  ]]
 
</p>
 
</p>
  

Latest revision as of 02:32, 18 October 2018


MiniToe polycistron B: a new system using miniToe.

Background of 2018 OUC-China' project——miniToe family

This year, we designed and achieved a gene regulatory toolbox based on CRISPR RNA endonucleases Csy4 for post-transcriptional regulation. A rational designed modular RNA fragment named miniToe was utilized for precise and efficient translational regulation. The miniToe module was constructed through inserting a 22 nt Csy4 recognition site between RBS and a cis-repressive RNA element, which is able to mask the RBS region and inhibit translation initiation. By up-regulating the level of Csy4 in cell as input, the miniToe module will be cleaved and releases an exposed RBS for output translation. As our innovation, we further designed four Csy4 mutants and five mutated miniToe module in a predictable way by modeling, which aims at enriching our toolkit for diverse regulation ranges on target genes. The whole toolbox includes ten combinations of different Csy4 mutants and miniToe modules, which is called miniToe family. Based on this tool, we can achieve various expression profiles on a polycistron.

Fig.1 Csy4 and hairpin can form a stable structure.


We design miniToe family to meet the aim, "One system, diverse expression". This means by using one system we can even achieve flexable expression of target gene. So we further design four Csy4 mutants by point mutation( Csy4-Y176F, Csy4-H29A, Csy4-F155A, Csy4-Q104A). At the same time, we redesign 5 different hairpin mutants named miniToe(5 different types of Csy4 recognition sequence) which can be recognized and cleaved by Csy4 mutants. ( miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5, miniToe-WT).


The new methed to tune multple genes

Many applications of synthetic biology need to regulate the expression profile of multiple genes. Microorganisms with programmable and engineered metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway [1][2]. Indeed, pathway optimization requires to adjust the expression of multiple genes at appropriately pattern, for example, the synthetic of poly-3-hydroxybutyrate and Mevalonate [3].

As has been done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient method for regulating genes simultaneously. Thus, for the sake of tuning the expression pattern of genes within polycistron, we hope to develop a powerful regulation tool by the miniToe system. We name this system miniToe polycistron which contains several genes in one circuit with different miniToe design. Our aim for this part is achieving different expression profile of the genes by miniToe in polycistrons compared with normal polycistrons.

[1].Pfleger, B.F., et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol, 2006. 24(8): p. 1027-32.
[2].Xu, C., et al., Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization. Nat Commun, 2015. 6: p. 6900.
[3].Smolke, C.D. and J.D. Keasling, Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng, 2002. 80(7): p. 762-76.


How to do

By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.

  • First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
  • 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.


The reasons why we design like this are below:
1. 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. 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. And this function is based on the high stability and affinity between Csy4 and target RNA structure [1].
3. The function of each miniToe has specific recognition and cleavage rates, which will make it possible to regulate the gene flexibly.

All in all, miniToe polycistron system has two components, Csy4 and the circuit of polycistron. With Csy4 protein, the polycistron will be cut into several mRNA chains with RNA/Csy4 complex at the 3’ UTR. The capability of RNA degradation protection will be much stronger, because of the high stability and affinity of Csy4 binding, which increase energy threshold for RNA degradation from 3’ UTR. So, the RNA degradation rate will be much lower. For the 5’ end degradation, the Csy4 cut will leave a OH- at 5’ end. the cleavage capability of RNase E will be much lower because there is no pyrophosphate in the 5’ end. Qi’s work [1] has proved that OH-mRNAs exhibit higher gene expression than 5’ PPP-mRNAs.
Fig.2 The working process of miniToe polycistron.

From now, we construct two polycistron(miniToe polycistron A and miniToe polycistron B) by inserting three miniToes before, between and behind two CDS. We use miniToe-WT(BBa_K2615020) before and behind miniToe polycistron B(BBa_K2615018), meanwhile the miniToe-6(BBa_K2615017) is used between two CDS. And we use sfGFP and mCherry to test our system. The result shows we can totally change the origin ratio of two genes.
[1].Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature Biotechnology, 2012, 30(10):1002.


Result

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

Proof of functions

The result by microplate reader has been shown in the Fig.4. 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.4 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 13
    Illegal NheI site found at 36
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 197