Team:OUC-China/Results
Results
First of all, it is necessary to say some important concepts before we discuss the results. Four systems we design:
1) miniToe system
2) miniToe family system (The miniToe family refers to the whole second system below)
3) miniToe polycistron system (The miniToe polycistron refers to the whole third system below)
4) miniToe based motility detection system
The first system (miniToe system) is the basics of other three systems. To avoid confusion, on the level of DNA, we design a miniToe part and construct it to circuits. On the level of RNA, we focus on miniToe structure. In the miniToe family system, we focus on Csy4 hairpin which is a part of miniToe part. In fact, the Csy4 hairpin is the miniToe target region, which is a cis-repressive RNA.
Also, the miniToe system is a kind of translation activator. In miniToe family, the capabilities of different Csy4 mutants reflect their activation efficiency to turn on translation. So the fluorescence intensity reflect activation efficiency which is a comprehensive ability including the capabilities of recognition and cleavage.
1. The results of first system: miniToe
1.1 Plasmid construction
First, we use an inducible promoter Ptac to regulate the expression of Csy4 (pCsy4). Without the inducer isopropyl-β-d-thiogalactoside (IPTG), no Csy4 is produced. Otherwise, Csy4 can produce. As for another plasmid pRepoter, the superfolder green fluorescent protein (sfGFP) is the reporter gene to reflect output of our system under miniToe regulation, the expression of this gene is driven by a constitutive promoter named J23119 from Anderson family. The miniToe part is inserted between RBS and cis-repressive RNA region.
Fig.1-1 The two plasmids of miniToe test system. The pCsy4 is constructed for the expression of Csy4. The pReporter contains miniToe part.
1.2 The measurement of growth rates
After circuit construction to get two plasmids pCsy4 and pReporter, we transformed them into E. coli DH5 Alpha and got the recombinant strain with miniToe system successfully. For the sake of functional test, 5 different groups are set, the control group E. coli DH5 Alpha, the pCsy4 only group, the pReporter only group, the pCsy4&pReporter with IPTG group and the pCsy4&pReporter without IPTG group. As preliminary experiment, the growth rate measurements are essential. The curve below demonstrates all the groups have almost the same tendency of OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of recombinant strain. The metabolic stress by two plasmids is not harmful to the recombinant strains.
Fig.1-2 Growth curve of strains we used in experiments. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)
1.3 Proof of function
The microplate reader is used to test the fluorescence intensity of superfolder green fluorescent protein (sfGFP) which is changed over time. The aim is to prove that miniToe system can control the downstream gene expression during the whole cultivation period.
The following chart shows the dynamic curve measured by microplate reader every two hours. The yellow line refers to the test group which is recombinant strain (with the whole miniToe system) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity in recombinant strain (with the whole miniToe system) without IPTG (0mM). The green line refers to another control group which only has pReporter without the pCsy4 in strain. The results help us to prove two problems in miniToe system.
Fig.1-3 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups. The yellow line refers to a test group with IPTG (0.125mM). The blue line refers to a group without IPTG (0mM). The green line refers to a control group only with pReporter. Error bars represent standard deviation of three biological replicates. (Measured by microplate reader)
The first problem is whether miniToe structure can fold exactly on the level of RNA. In order to deal with the problem, the prediction of secondary structure is needed by using mfold and RNAfold. The result of prediction shows miniToe structure can fold correctly after transcription.
Fig.1-4 The structure prediction of full-length transcript of this circuit as well as miniToe target region. The miniToe target region is on the left of picture and the full-length transcript of this circuit is on the right of picture. The red frame indicates the places of miniToe target region in the full-length transcript of this circuit.
The second problem need to prove is whether miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig1-3, 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). These data strongly support that the increased expression of the target gene sfGFP is indeed due to cleavage of Csy4 site that exposed the RBS to restore translation. It means miniToe system can work successfully.
We also tested miniToe system by flow cytometric. In Fig.1-5, it's easy to distinguish the two groups (blue & white) and the test group (+IPTG) has the obvious increase compared with the control group (-IPTG). The result shows the same conclusions mentioned before.
Fig.1-5 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 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).
1.4 Discussion
Combining the biology and math, we discuss the dynamics of sfGFP in the Fig.1-3. In order to explain in detail, Fig.1-6 presents the dynamics of all species in the miniToe system.
Fig.1-6 The dynamics of all species in the miniToe system
See more details in model! Click here !
1.5 collaborations
Fig.1-7 The result from other four teams which proved our conclusions. Error bars represent standard deviation of four biological replicates.
We also collaborated with other 4 teams, and they helped us in proving our results by wet experiments in their labs. Thank you! Click here to see more details!
2. The results of second system: miniToe family
2.1 Plasmid construction
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. By analyzing the sensitivity of the GFP level in the system to cleavage rate by model, it is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. It means we may change the expression level of sfGFP if we employ different mutants of Csy4 proteins.
There are two ways in second system which can help to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe mutants. There are all the plasmids we used in Fig.2-1 and Fig.2-2.
Fig.2-1 The plasmids of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4 and we also use it in first system. The pCsy4-Q104A is a plasmid which contains Csy4-Q104A. The pCsy4-Y176F is a plasmid which contains Csy4-Y176F. The pCsy4-F155A is a plasmid which contains Csy4- F155A. The pCsy4-H29A is a plasmid which contains Csy4- H29A.
Fig.2-2 The plasmids of miniToe mutants. The pReporter is a plasmid which contains miniToe-WT and we also use it in first system. The pReporter-1 is a plasmid which contains miniToe-1. The pReporter-2 is a plasmid which contains miniToe-2. The pReporter-3 is a plasmid which contains miniToe-3. The pReporter-4 is a plasmid which contains miniToe-4. The pReporter-5 is a plasmid which contains miniToe-5.
After plasmid construction, we proved the functions of Csy4 mutants by wet experiments first.
2.2.1 Proof of functions about Csy4 mutants
In this part, three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. Our expectation is that by using new Csy4 mutants, the expression level of sfGFP vary with Csy4s' capabilities. It means that miniToe family members present various expression of target genes.
Prediction
Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. The models help us go further this year!
Fig.2-3 The predication: the fluorescence intensities by different Csy4 mutants along with time
We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.
2.2.2 The result by Microscope
First, we tested the capabilities of five Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. In Fig.2-4, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown 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. The Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.
Fig.2-4-1 The expression of sfGFP by Csy4-WT&miniToe.
Fig.2-4-2 The expression of sfGFP by Csy4-Q104A&miniToe.
Fig.2-4-3 The expression of sfGFP by Csy4-Y176F&miniToe.
Fig.2-4-4 The expression of sfGFP by Csy4-F155A&miniToe.
Fig.2-4-5 The expression of sfGFP by Csy4-H29A&miniToe.
Fig.2-4 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows E. coli without fluorescence excitation. The images on the right represent situation when fluorescence excitation.
2.2.3 The result by flow cytometer
The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.2-5, and their intensities of fluorescence are from strong to weak.
Fig.2-5 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.
Fig.2-6 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.
2.2.4 The result by microplate reader
Besides all the works before, we also need to know more information about the Csy4 mutants in entire cultivation period. Even though we known that our Csy4 mutants have differentiated expression level in ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectations.
So we tested five test stains individually (strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A) by microplate reader every two hours. The green lines in all the images represents strain-miniToe-only group keep stable. It means the miniToe structure fold well and lock the process of translation without Csy4. And the five test groups show different characteristics. In Fig.2-7-A, the group strain-Csy4 shows the same result with the first system. The switch turns off without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the Csy4-WT has strongest capabilities (Fig.2-7-F). In the Fig.2-7-B, the tendency of fluorescence intensities by Csy4-Q104A is similar with Csy4-WT. And the expression level is lower than Csy4-WT. The Csy4-Y176F’s capabilities ranks the third. What is special is Csy4-H29A. 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 as we can see in Fig.2-7-E. In summary, we put all the test groups together in Fig.2-7-F, the picture shows prediction by model match the result perfectly in Fig.2-8.
Fig.2-7 The fluorescence intensities of sfGFP by microplate reader. A. strain-Csy4. B. strain-Csy4-Q104A. C. strain-Csy4-Y176F. D. strain-Csy4-F155A. E. strain-Csy4-H29A. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A.
Fig.2-8 The comparison about model and result by microplate reader.
2.3 Proof of functions about hairpin mutants
We design a new cis-regulatory RNA element named miniToe in first system. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function. At the same time, the Csy4 site is a kind of hairpin (wild type).
In order to meet our goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. After testing Csy4 mutants, we insert the new hairpin mutants in the miniToe. So the miniToe (contains hairpin-WT) is change to five mutants named miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5. For the sake of convenience, we named miniToe part from first system a new name in second system, miniToe-WT.
By flow cytometry measurement, we rank them by their capabilities.The recombination strains for test both have same pCsy4 but different plasmids contain different miniToe parts in the following. The recombination strains to test the functions of miniToe mutants are strain-miniToe (pCsy4&pReporter), strain-miniToe-1 (pCsy4 &pReporter-1), strain-miniToe-2 (pCsy4&pReporter-2), strain-miniToe-3 (pCsy4&pReporter-3), strain-miniToe-4 (pCsy4&pReporter-4), strain-miniToe-5 (pCsy4&pReporter-5). The results are listed in the order: miniToe-WT>miniToe-5>miniToe-1>miniToe-4>miniToe-2>miniToe-3.
Fig.2-9 The fluorescence intensities of sfGFP about hairpin mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-miniToe (Black), strain-miniToe-5 (Red), strain-miniToe-1 (Green), strain-miniToe-4 (Blue), strain-miniToe-2 (Cyan), strain-miniToe-3 (Yellow). Crosscolumn number shows fold increase of sfGFP fluorescence.
Fig.2-10 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.
2.4 Proof of functions about miniToe family
In Design page , we found it is possible to use one system to meet diverse aims which means by using our miniToe system, people can create more flexible gene circuits with different expression level.
With the help of our model, 4 Csy4 mutants and 5 hairpin mutants are selected. We tested each mutant and got positive data supporting model prediction. Then we set up a function verification experiment with 5*6 combinations of Csy4 and hairpin including wild types. So the 30 combinations are the candidates for miniToe family. And we should test all of them to select the positive members finally.
And we tested our system by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.2-11. 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.2-12. The user-friendly system meets the flexible needs in study about regulating different levels of expression.
Fig.2-11 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.
Fig.2-12 The members of miniToe family.
3. The result of third system: miniToe polycistron
3.1 The purpose of experiment
The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.
1) First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
2) Then we selected some miniToe parts and inserted them between, before and behind sfGFP and mCherry. For example, for bi-cistron, then three miniToe parts will be inserted. for three genes in polycistron, then four miniToe parts will be inserted, and so on.
This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more miniToe polycistron based on miniToe family.
Fig.3-1 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system.
3.2 Proof of functions
The result by microplate reader has been shown in the Fig.3-2. After culturing for 10 hours, the rate of fluorescence intensities by sfGFP/mCherry was changed by miniToe family. The group A is a control group whose circuits have no miniToe part. The ratio of fluorescence intensities by sfGFP/mCherry is about 6.81 which means the gene near the promoter has much higher expression than the gene far from promoter in a normal polycistron. The test group-polycistron A has been changed by miniToe system because the ratio of fluorescence intensities decrease to 4.38. To our surprise, the test group-polycistron B shows the significant change whose rate is about 2.82. It means the ratio of gene expression can be regulated by miniToe family. In the future, the miniToe family create more possibilities in regulating the ratio of gene expression.
Fig.3-2 The ratio of fluorescence intensities by sfGFP/mCherry. Error bars represent standard deviation of three biological replicates.
4. The result of fourth system: miniToe based Motility detection system
4.1 The purpose of designing the experiment
As is shown in the first system miniToe, we created a new method to regulate the downstream gene expression. Furthermore, we proved that our system can be enlarged and then we created miniToe family system based on the mutation of miniToe part and Csy4 mutants. As and translation regulation tool, MiniToe can also be used in application scenario of molecular mechanism research. Sometimes scientists may puzzle with the functions of certain gene or protein without in-depth study. One general method to study them is knock-out or knock-in methods. In this way, organisms show some phenotypic change related to particular gene. However, if we want to know better about the functions of the gene, we may need more tool to change gene expression at different levels.
By using our system, the motility of E. coli can be regulated. We 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. 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 did a lot of wet lab works to test minToe system by applying it to the detection of E. coli motility. We construct plasmid by putting the motA behind miniToe part. So the target gene motA can be regulated by miniToe system.
Fig.4-1 The process of motility detection system
4.2 Proof of functions
Five groups are set, a test group and four control groups. And the results shown below proved that our system can work as expectation.
Fig.4-2 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.4-3 The test group C. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe part).Without the 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.4-4 The test group D. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe part but no Csy4. The circuit is on the control of miniToe part and its downstream gene motA can be regulated without Csy4. So MotA can be produced. We have three biological replicates in the experiment.
Fig.4-5 The test group E. The strain we culture in plates is miniToe-motA with Csy4. The strain have the miniToe part and Csy4 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.
Fig.4-6 The migration dimensions. The ratio of migration area /whole plate. This chart is made by numerical integration
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.
protocol
