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.
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.
2. The second system miniToe family
2.1 The principles of designing mutants
For Csy4 mutants1. 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.
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.
2.2 Proof of functions
First, we have tested five different Csy4 mutants by three ways:1. The result by Microscope
2. The result by flow cytometer
3. The result by microplate reader
Second, we have tested six different hairpin mutants by microplate reader.
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.
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.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.
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.
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.
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.
2.2.2 Proof of functions: Hairpin mutants
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.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.
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.
2.2.3 Proof of functions: MiniToe family
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.Fig.14 The heat map generated from flow cytometry data reflecting 30 groups’ intensities of fluorescence by sfGFP.
Fig.15 The members of miniToe family.