Team:OUC-China/Demonstrate

Team OUC-China: Main

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.

2. The second system miniToe family

2.1 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.

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.

As the Fig.9 shown, the relative expression level can be measured by flow cytometer.

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.

We tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the Images represent the control group, “miniToe only” group and the green lines keep stable which means the miniToe structure can close the expression of downstream genes. And the test groups show different characteristics. As we can see in Fig.11, the Csy4-WT shows the same result with the first system. And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT. The tendency of increase of fluorescence intensities by Csy4-Q104A is almost same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What is special is Csy4-H29A. We have mentioned Csy4-H29A before. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage. In summary, the picture shows our prediction by model matchs the result perfectly in Fig.11.

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.

3. The third system——miniToe polycistron

3.1 The design of miniToe ploycistron

Many applications of synthetic biology need the balanced expression of multiple genes. For the sake of tuning the expression of genes within polycistron, we want to develop a tightly regulated by the miniToe structure. Our aim for this part is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.

Fig.16 The mechanisms of miniToe polycistron

By inserting our hairpins between intergentic regions, it influences the amount of expressed proteins. First, we decide to use GFP and mCherry to test our system in bicistron simply. Then we select some miniToe structures designed by us and insert them between, before and behind GFP and mCherry. If there is bicistron, then we need three miniToe structure. If there are three genes which form polycistron, then we need four miniToe structure. And so on.

By inserting miniToe structure into circuits, more than one gene can be regulated. So in this system, we focus on the ratio of gene expression. We tested polycistron system by two target genes, sfGFP and mCherry.

Three kinds of groups have been set. One is the bicistron circuit without miniToe structures. In order to make sure our miniToe structure folded as expectations, we have created the recombinant strain (control group) which only has the circuit constructed by miniToe without Csy4. The test group have both miniToe polycistron and Csy4. This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.

Fig.17 The three test groups. Group A is the control group without miniToe system. Group B is the control group with miniToe but no Csy4. Group C is the test group with miniToe and Csy4.

3.2 Proof of functions

We have tested our miniToe polycistron by microplate reader after culturing 10 hours. The sfGFP were measured at excitation/emission wavelengths of 485nm/520nm. The mCherry were measured at excitation/emission wavelengths of 587nm/610nm.

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