Team:OUC-China/Design

Team OUC-China: Main

Design

1. Background

1.1 Background of miniToe Family

The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin. In type I and type III CRISPR systems, the specific Cas6 endoribonuclease splits the pre-crRNAs in a sequence-specific way to generate 60-nucleotide (nt) cis-repressive RNA (crRNA) products in which segments of the repeat sequence flank the spacer (the target "foreign" nucleic acid sequence)[1]. The inactivation of the Cas proteins leads to a total loss of the immune mechanism function.

The Csy4 protein consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4[2]. And once the Csy4/RNA complex formed, the structure will stay stable and hard to separate.

Fig.1-1 The structure of Csy4 Fig.1-2 The structure 22nt hairpin which can be recognized by Csy4. Fig.1-3 The Csy4/RNA complex.

2. The first system miniToe

2.1 New method —— miniToe

Based on Csy4's function, we design a new structure named miniToe which can be recognized by Csy4 at the same time[3]. The whole system is a translational activator including three modular parts:
1. A cis-repressive RNA (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.2-1 The structure of miniToe.

In the project, the superfold green fluorescent protein (sfGFP) is a target gene to test our system. First, the stability of miniToe structure is crucial. So before the experiment, we have predicted the structure of whole circuit as well as miniToe[4][5].

Fig.2-2 The structural prediction of the whole circuit and miniToe.

To explore the feasibility and function of miniToe, the circuit below is the test system to make sure function of miniToe structure. Ptac is the inducible promoter to control the existence of Csy4 or not. The miniToe is constructed before the sfGFP. And this circuit is controlled by a constitutive promoter form Anderson family named J23119.

Fig.2-3 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 downsteam gene can be regulated.

Fig.2-4 The mechanism of miniToe system.

By experiments, the system can work well! Click here for more details!

Fig.2-5 The result of first system.

2.2 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 turned 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[6], 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[7], miniToe doesn't need to redesign crRNA over and over again because the crRNA is not paired with protein coding region.

3. The second system miniToe family

3.1 miniToe family——The model help us go further

After testing first system, miniToe, the dry lab member explore more deeply!

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

Fig.3-1 The ODE model for the first system Fig.3-2 The model about sensitivity analysis of the sf sfGFP

3.2 The principles of designing mutants

For Csy4 mutants
1. Some key sites in the Csy4 are really crucial for keeping the stable of structure and making sure the functions including recognition and cleavage. The change of those sites may result 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".
For hairpin mutants
2. Do not to break the recognition site so the function of cleavage is relative stable. If we break the key site G20 may lead to the damage of cleavage function.
3. The stable of secondary structure is vital so we need to focus on each hairpin's Gibbs free energy after design.
4. The aim is to obtain different hairpins which have totally different rates be recognized and cleaved.

3.3 Models help us deeply!

Here the model help to design the mutants of Csy4 and hairpin. According to the principles of designing mutants, four key problems is important in the miniToe System:

1.Does the Csy4 dock correctly with the miniToe structure?
2.How about the binding ability between the Csy4 and miniToe structure?
3.How about the ability of cleavage between the Csy4 and miniToe structure?
4.Does crRNA release from the RBS?

And for the own feature of Csy4 and hairpin, different strategies is used to design.

For the Csy4 mutants, the molecular dynamics are used as our tools. Based on Jiří Šponer’s work[8], we choose the four significant symbols of mutants: the interaction matrix; the binding free energy, the distance of Ser151(OG)-G20(N2’) and the RMSD of the cleaved-product complex. By comparing the difference between Csy4 mutant and wild-type Csy4 of four significant symbols, we finally design four Csy4 mutants: Csy4-Q104A, Csy4-Y176F, Csy4-F155A, and Csy4-H29A.

For the hairpin mutants, the bioinformatics and machine learning are our tools. By exploring the commonality of hairpin in the following step: We firstly find the hairpin which is also Repeat Area[9] in CRISPR system and scoring them. With the help of the SVM algorithm[10], training a model to score the hairpin. By using the model, five hairpins are selected: miniToe 1, miniToe 2, miniToe 3, miniToe 4, miniToe 5.
Click here for more details about model!

With the help of the model, 4 Csy4 mutants and 5 hairpin mutants are selected. We have tested each mutant and have data support. Then there are 5*6 combinations including wild types. By testing all of them, 10 members work successfully as expectation. So the second system, miniToe family have 10 combinations which are designed and selected by us.

4. The third system——miniToe polycistron

4.1 Why we do

Many applications of synthetic biology need the balanced expression of multiple genes. Microorganisms with modified 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[11][12]. Indeed, pathway optimization requires to adjust the expression of multiple genes at appropriately balanced levels, for example, the synthetic of poly-3-hydroxybutyrate and Mevalonate[13].

As is done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient mean for regulating genes simultaneously. Thus, for the sake of tuning the expressions of genes within polycistron, we want to develop a tightly regulated by the miniToe structure. We name this system miniToe polycistron which contains several genes in one circuit between different miniToe structures. Our aim for this part is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.

4.2 How we do

Fig.4-1 The mechanisms of miniToe polycistron

By inserting miniToe hairpins between intergentic regions, it influences the amount of expressed proteins. First, sfGFP and mCherry is used to test system in bicistron simply. Then we select some miniToe structures and insert them between, before and behind sfGFP and mCherry. If there is bicistron, then three miniToes is inserted. If there are three genes which form polycistron, then four miniToes is inserted. And so on. The reasons why we design is below:

1. Without the Csy4, the existence of miniToe structure in polycistron will increase the stability when we don’t want to open the switch. The crRNA in miniToe is a compliment sequence of RNA to prevent the binding of ribosomes.
2. Also, the miniToe of 3’ end is a protection to prevent degradation of the nucleotide chain.
3. Each miniToe structure works at special recognition and cleavage rates. That 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 the Csy4, the polycistron will be cut into many chains with RNA/Csy4 complex in the 3’ end and –OH in the 5’ end. So the capability of protecting RNA is much stronger. Because more energy is needed which partly provided by ATP to degrade the RNA/Csy4 complexes in the 3’ end. So the degradation rate of RNA is much lower. 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 [14] has proved that OH-mRNAs exhibit higher gene expression than 5’ PPP-mRNAs.

Also, our work this year is an improvement based on 2016 OUC-China. The comparison is in demonstration page, the chapter 5. Click here to see more!

5. miniToe Motility detection system

5.1 The application of miniToe——Regulation of motA

MiniToe is also a good tool which can be used to study of molecular mechanism. Scientists may puzzle with the functions of certain gene or protein when first discover it. Now one common method to study is knock-out or knock-in. In this way, organisms show some flaws related to the gene which is knocked out. But if we want to know better about the gene functions, we may need the different levels of the gene expressions.

This year, we used miniToe system to control the motions of Escherichia coli. We have transformed the miniToe system into E. coli whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque[15]. 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. Expression of motA from a plasmid has been shown to restore motility in ΔmotA strains. So miniToe system can control motA protein expression with different levels. We get a strain without motA by knocking out and then transform motA protein under the control of miniToe family. The E. coli will restore motility. It seems that miniToe system has more applications such as regulation of motA.

5.2 How we do

E. coli RP6666 (knocked out motA) lacks capacity of motion. By using hairpin detection system to control the translation of the downstream gene, we have constructed a circuit and put motA downstream of miniToe, then transfer the plasmid into E. coli RP6666. By inducing the production of Csy4, motA could be controlled indirectly, thus making E. coli RP6666 strain regain the capacity of motion.

Fig.5-1 The process of motility detection system

6. In the future

In conclusion, we have demonstrated the design of modular translational activators with CRISPR endoribonuclease Csy4 named miniTo and design four systems which are improved step by step.

In the future, there are some works to perfect project. First, we would like to enlarge project by finding more and more mutants. By finding and designing more mutants a larger library may be created which can enlarge the function of toolkit. Second, we have tested the ratio of regulation in miniToe polycistron. In the future, man-made setting can be used to make calibration curve which will help us to know project deeply.

REFERENCE

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