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 the project, the superfold green fluorescent protein (sfGFP) is used as a target gene to test our system. To explore the feasibility and function of miniToe, we designed the circuit ((pCsy4) below. The Ptac is the inducible promoter of Csy4 to control the existence of Csy4 or not. At the same time, the miniToe is constructd before the sfGFP. And this circuit (pReporter) 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 mechanism of miniToe.

1.2 Proof of function

There are two problems we need to prove about miniToe’s structure and function.

First, the stability of structure and the formation of hairpin (The Csy4 site) is crucial. So before the experiment, we predict the 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.

In fact, 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 is that whether miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig.5 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 isopropyl-β-d-thiogalactoside (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 miniToe system can work successfully.

We also test miniToe 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.

There are three teams we collaborate with and they help us in proving the previous 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. The 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 and have tested each mutant. Then there are 5*6 combinations including wild types. By testing all of them, we finally select 10 of them work successfully as expectation. So the second system, 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.

1. The expression of sfGFP by Csy4-WT&miniToe.

2. The expression of sfGFP by Csy4-Q104A&miniToe.

3. The expression of sfGFP by Csy4-Y176F&miniToe.

4. The expression of sfGFP by Csy4-F155A&miniToe.

5. The expression of sfGFP by Csy4-H29A&miniToe.

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 (Black), 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 (Black), 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.

Fig.18 The rate of fluorescence intensities by sfGFP/mCherry.

4. The result of miniToe Motility detection system

4.1 The purpose of designing the experiment

As is shown in the first system miniToe, we have created a new method to regulate the downstream gene expression. Furthermore, we have proved that our system can be enlarged and then we created miniToe family system based on the mutation of miniToe structure. It is believed that miniToe is also a good tool which can be applied to the study of molecular mechanism. Now the normal method to study the function of single gene is to "knock-out" or "knock-in". In this way, defective strain will lose some functions. But if we want to know better about the effect of a gene on the strain, we may need to explore the different level of gene expression.

By using our system, the motility of E.coli can be regulated. As we all know, 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.

So we have done a lot of works to test our minToe system by applying it to the detection of E.coli motility. We construct our circuit by putting the motA behind our miniToe structure. So the target gene motA can be regulated by our miniToe system.

Fig.19 The process of motility detection system

4.2 Proof of functions

Five groups have been set, a test group and four control groups. And the results shown below have proved that our system can work as expectation.

Fig.20 The control groups A and B including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) that have motility and they can move arbitrarily in the plates. The plates on right are ΔmotA strains(the motA-deletion strain) (B1, B2, B3), E.coli RP6666, which have no motility so the strains stay on the center. We have three biological replicates in this experiment.

Fig.21 The test group C. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E.coli cannot move. And the Csy4 have no big influence on strain compared with the ΔmotA strain. The little round of papers indicates the places of inducer IPTG (Isopropyl β D thiogalactopy ranoside). We have three biological replicates in the experiment.

Fig.22 The test group D. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4. The circuit is on the control of miniToe and its downstream gene motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.

Fig.23 The test group E. The strain we culture in plates is miniToe-motA with Csy4. The strain have the whole miniToe system which means motA can be regulated by miniToe. In the picture, the E. coli move everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene motA come into play. The E. coli can move everywhere in the plate. We have three biological replicates in the experiment.

As we can see, test group strains can move everywhere in the plate and the control groups strains can not move.The test group work as expectation compared to the control groups. But there is no time for us to test more miniToe mutants and Csy4 mutants in miniToe family. We want to realize the function of regulation by using different miniToe family members in the future. So we still have a lot of work to do.

5. Improvement based on 2016 OUC-China

The 2016 OUC-China have a method to regulate the expression of polycistron.
They concentrate on stem-loops inserted into the intergenic regions. When transcribed as one polycistron, digested and separated into several independent fragments, cistrons with 3' end stem-loops will get different stability. They have designed a lot of different stem-loop. And they have measured and standardized a series of native and designed stem-loops, transforming into a toolkit for a broad use.
Click here for more details about 2016 OUC-China’s project. https://2016.igem.org/Team:OUC-China/Design

Fig.24 The three stem-loops designed by 2016 OUC-China

The new system named miniToe polycistron is an important improvement based on 2016 OUC-China. The idea using stem-loop to regulate the gene expression is creative and inspired us to do more. Enlighted by 2016 OUC-China, we find miniToe is also a good stem-loop that can be inserted to polycistron. And we test our system by bicistron first. We insert three miniToe hairpins to circuit when we need to tune the expression of two genes. It means that for each target gene we have two miniToe hairpins. The one is downstream of the gene, and the other is upstream of the gene. Our system is flexible and stable. We make an enhanced version based on previous project.

1.The 2016 OUC-China only put the hairpins between two genes, and we put the hairpins downstream and upstream for each gene. It is a stable structure. Without the existence of Csy4, the system by us is stable and the switch is closed. We add a new switch function besides the previous system.

2. The Gibbs free energy of miniToe mutants is much lower than the stem-loops designed by 2016 OUC-China. Our 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 we need more energy 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 has proved that OH-mRNAs exhibit higher gene expression than 5’ PPP-mRNAs.