Team:OUC-China/Results
Results
The result of first system
Plasmid construction
First, we use an inducible promoter Ptac to regulate the expression of Csy4. Without the IPTG, the circuit of Csy4 closes at the same time. On the contrary, Csy4 enzyme produce and help regulate the expression of downstream genes of miniToe structure in another plasmid as a part of our first system. Also, we use the promoter J23199 from Anderson family which is a constitutive promoter to regulate the reporter circuit. So if our system works well, we will get some curves for fluorescence intensity as our expectation.
Fig.1-1 The two plasmids of miniToe test system.
Selective Medium Assay
After circuit construction to get the two plasmids we mentioned before, we transformed both of them into E.coli DH5 Alpha and got the miniToe testing strain successfully. Because the promoter Tac has high leakage in LB medium so we culture our recombinant strain in M9 medium. We measured the growth rate of both our engineered strain and the negative control as preliminary experiment. As a result, the curve well demonstrates that the strain with our system has almost some OD600 with the negative control strain during the entire cultivation period. It means that our system has no big negative influence on the growth of strain. The metabolic stress by two plasmids is not harm to our recombinant strain.
Fig.1-2 Growth curve of strains of our recombinant strain (with the whole miniToe system including two plasmids) and negative control groups. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)
Proof of function
We use microplate reader to test the fluorescence intensity of sfGFP which is changed over time. getting data to support our idea directly that our system can control the downstream gene expression during the whole cultivation period.The following chart shows the dynamic curve measured by microplate reader. We test our system every two hours. The yellow line is the symbol of test group which is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity by a recombinant strain(with the whole miniToe system including two plasmids) without IPTG (0mM). The green line is also a control group in our system, it shows the fluorescence intensity of sfGFP by a strain with only one kind of plasmids (pReporter) which only has miniToe structure without the Csy4. The result by this curve help us to prove two functions in miniToe system.
Fig.1-3 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 first problem is whether our miniToe structure fold exactly. So first we predict our secondary structure by using mfold(http://unafold.rna.albany.edu/?q=mfold) and RNAfold(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). We predict the whole structure of our circuit and structure of miniToe to see if our structure can fold directly on the level of RNA. By the result of prediction, we just find our structure can fold directly after transcription.
Fig.1-4 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.
Fig.1-3 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).
At the same time, we find the control group without IPTG (the blue line) has leakage compared with other two group. Because the control group with only one plasmid (the green line) has the stable and low expression of sfGFP, the leakage may result from the Inductive promoter Ptac. Even though the control group has leakage of sfGFP, we can prove the function of our system successfully. But in the future, we may have more time to find a better promoter which is also suitable for our system.
We also test our system by Flow cytometric, the blue group showed in the Fig.1-5 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.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 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).
Discussion
Combining the biology and math, we discuss the dynamics of GFP in the Fig.1-3 now. In order to explain in detail, we present the dynamics of all species in the miniToe system in Fig.1-6.
Fig.1-6 The dynamics of all species in the miniToe system
See more details in model! Click here!
(https://2018.igem.org/Team:OUC-China/miniToe)
collaboration：结果对比正确/SDU/SK
Fig.1-7 The result from other four teams which have proved our conclusions.
We also have collaborations with other 4 teams, and they help us in proving our results by experiments in their labs. Thank you! See more details here! (https://2018.igem.org/Team:OUC-China/Collaborations)
The result of second system
Plasmid construction
In our second system, we find it possible to achieve our aim by using the mutants of Csy4 and mutants of miniToe structure. There are two ways in our second system which can help us to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe muntants.By point mutantion and synthesis by company, we have obtained all the circuits below.
Fig.2-1 The circuits 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 circuits 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 prove the functions of Csy4 mutants first.
Proof of functions about Csy4 mutants
In this part, we have three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. At the same time, we focus on the capacities of Csy4’s cleavage (including all the mutants we design). Our expectation is that by using our new Csy4 mutants, the fluorescence intensities of sfGFP can vary upon the rates of Csy4s’ cleavage which means that the result presents various expression of target genes.(图：此处可以有一张多酶预测图)
Prediction
Before the experiments, we have proved our ideas by model. The predication below shows the possibilities of different expression levels by different Csy4 mutants. So the model help us to know our improvement deeply this year!
Fig.2-3 The predication: The fluorescence intensities by different Csy4 mutants along with time
The result by Microscope
First, we have tested five different Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. We have cultured them in the solid medium in plates until the bacterial colonies can be observed by naked eyes. At that time, the sfGFP have been accumulated so we can see the fluorescence by microscope. Because the five Csy4s have different capabilities of cleavage, we want to see different intensities of fluorescent by eyes. As we can see in Fig.2-4, we have cultured the five different strains for same time which both have the same miniToe circuit but have totally diffenent Csy4 mutants. From top to bottom in Fig.2-4, there are fluorescence imagines by fluorescent microscope which indicate Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A in sequence. We can observe visible distinctions in these imagines. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s’ capabilities of cleavage decrease one by one. So the imagines indicate 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 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.
The result by flow cytometer
The qualitative experiment is not enough to analyze the Csy4s. So we test our system by flow cytometer when we have cultured them for ten hours in M9 medium. The expression of five groups’ sfGFP are showed in Fig.2-5, and they are Csy4-WT&miniToe, Csy4-Q104A&miniToe, Csy4-Y176F&miniToe, Csy4-F155A&miniToe and Csy4-H29A&miniToe. We find that 5 groups’ fluorescence intensities have a obvious order from Csy4-WT to Csy4-H29A which means the capabilities of cleavage decrease one by one. The strongest one is Csy4-WT. The second one is Csy4-Q104A. The third one is Csy4-Y176F. The fourth one is Csy4-F155A and the Csy4-H29A is the weakest. (此处无对照) As the Fig.2-5 shown, the relative expression level can be measured by flow cytometer at the same time.
Fig.2-5 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.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.
The result by microplate reader
Besides all the works we have done before, we also need to know more information about the Csy4s we design. Even though we have known that our Csy4 mutants have differentiated expression level after ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectation.
So we tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the imagines 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.2-7-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without IPTG works (As the blue line shows). And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT (Fig.2-7-F). In the Fig.2-7-B, 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 as we can see in Fig.2-7-E. （此处缺一个解释）In summary, we put all the test groups together in Fig.2-7-F, the picture shows our prediction by model match the result perfectly in Fig.2-8.
Fig.2-7 The fluorescence intensities of sfGFP by microplate reader. A. Csy4-WT&miniToe. B. Csy4-Q104A&miniToe. C. Csy4-Y176F&miniToe. D. Csy4-F155A&miniToe. E. Csy4-H29A&miniToe. 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.
Fig.2-8 The comparison about model and result by microplate reader.
Proof of functions about hairpin mutants
In order to meet this goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. After testing Csy4 mutants, we have tested another way that may help us to create more possibilities. We also proved that we can get some different hairpin mutants by changing the sequences of hairpin-WT.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.2-9 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.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.
Proof of functions about miniToe family
As we talk about before 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 circuit with different expression level.In order to meet this goal, there are two ways. One is designing some Csy4 mutants and two is designing some hairpin mutants. And we have proved that all of them are the good materials to form a bigger group. So we just combine all the mutants together. The combinations of different Csy4 mutants and hairpin mutants which compose a small library give us more possibilities in use. We named those combinations miniToe family which is the second system of our project.
And we have 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 this heat map, some groups have the similar expression levels. 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 meet the flexible needs in study which can help user’s need about different levels of expression.
Fig.2-11 The heat map generated from flow cytometry data reflecting 30 groups’ intensities of fluorescence by sfGFP
Fig.2-12 The members of miniToe family.
The result of the third system—— miniToe polycistron
The setting of test groups and control groups
The miniToe polycistron is a new method designed by OUC-China this year. 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 expectation, we have created the recombinant strain (control group) which only has the circuit constructed by miniToe without Csy4. The test group both have 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 tested more polycistron based on miniToe family.
Proof of functions
The result by microplate reader has been shown in the Fig.3-1.Fig.3-1
The result of miniToe Motility detection system
Proof of functions
As we shown before in the first system, we have created a new method to regulate to downstream gene expression named miniToe. And we also have proved that our system can be enlarged. So we have created miniToe family in the second system.We believe that miniToe is also a good tool which can be apply to study of molecular mechanism. Scientists may puzzled with the functions of certain gene or protein when first discover it. Now one common method to study single gene is knock out or knock in. In this way, organisms without one gene show the lack of forms or functions. But if we want to know better about the gene functions, we may need different level of the gene expressions.
By using our system, the motility of E.coli can be regulated. As we know, MotA provides a channel for the proton gradient required for generation of torque. ΔmotA strains can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
So we have done some works to test our system in dealing with the real-world problems by E.coli motility detection experiments. We construct our circuit by putting the motA behind our miniToe structure. So the motA as a target gene can be regulated by miniToe.
Five groups have been set, three test group and two control group. And the result shown below have proved that our system can work as expectation.
Fig.4-1 The control groups including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) which have motility and they move everywhere in the plates. The plates on right are ΔmotA (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 the experiment.
Fig.4-2 The test group-1. 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 strain, ΔmotA. We have three biological replicates in the experiment.
Fig.4-3 The test group-2. 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 it down stream motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.
Fig.4-4 The test group.