Team:OUC-China/Safety

Team OUC-China: Main

Design

Background of miniToe Family

The endoribonuclease Csy4 from CRISPR family is the main role of our toolkit. 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) crRNA products in which segments of the repeat sequence flank the spacer (the target "foreign" nucleic acid sequence). The inactivation of the Cas proteins leads to a total loss of the immune mechanism function.

The Csy4 protein consists of a 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. 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.

Also, （crispr家族蛋白的背景：相较与其他crispr家族其他蛋白质的优点）

The first system miniToe

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. The whole 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.2-1 The structure of miniToe.

In our project, we use sfGFP as a target gene to test our system. 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.2-2 The structure prediction of the whole circuit and miniToe.

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.2-3 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 working process of miniToe.

By experiments we have proved that our system can work well!

Fig.2-5 The result of our first system.

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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.

The second system miniToe family

miniToe family——The model help us go further

After testing our first system, miniToe, the dry lab member explore more deeply about our system.
After building the ODE model, we use it to simulate the dynamics of GFP. Comparing with the experimental data, we find it fits perfectly, which indicates that our model is reliable in our first system. Then we analyze sensitivity of the GFP, it is not too difficult to find that the cleavage rate has an influence in the expression of GFP, which shows that if we change the Csy4 protein we can change the expression of GFP which is a symbol of target gene.

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

The principles of designing mutants

For Csy4 mutants
1. 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".
For hairpin mutants
2. 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.
3. The stable of secondary structure is vital so we need to focus on each hairpin's Gibbs free energy after design.
4. Our aim is to obtain different hairpins which have totally different rates be recognized and cleaved.

Models help us deeply!

（此处假装有模型的简述版）Click here for more details!
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.

The third system——miniToe polycistron

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. 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.
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.

How we do

Fig.4-1 The mechanisms of miniToe polycistron

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 especially for the genes far from 5' terminal.
2. Also, the miniToe of 3' end is a protection to prevent degradation of nucleotide chain.
3. Each miniToe structure works at special recognition and cleavage rates. That will make it possible to regulate the gene behind this miniToe.

Result

Finally, we ( 此处要补一下多顺反子的结果后补充 )

miniToe Motility detection system

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 our system to control the motions of E.coli. We have transformed miniToe system into E. coli whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque. ΔMotA strains (the motA-deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation. Expression of motA from a plasmid has been shown to restore motility in ΔmotA strains. So we use our miniToe system to 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 our system has more applications such as regulation of motA.

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 construct 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

In the future

In conclusion, we have demonstrated the design of modular translational activators with CRISPR endoribonuclease Csy4 named miniToe. And we have design four systems which is improved step by step.
In the future, we still have some ways to perfect our project. First, we would like to enlarge our project by finding more and more mutants. By finding and designing more mutants we may get a larger library which can enlarge the function of our toolkit. Second, we have tested the ratio of regulation in miniToe polycistron. We can use man-made setting to make calibration curve in the future applications which will help us to know our project deeply.