Difference between revisions of "Team:OUC-China/Results"

Revision as of 16:34, 16 October 2018

Team OUC-China: Main

Results

1. The results of first system: miniToe

1.1 Plasmid construction

First, we use an inducible promoter Tac to regulate the expression of Csy4 (pCsy4). Without the inducer isopropyl-β-d-thiogalactoside (IPTG), Csy4s stop producing. Otherwise, Csy4 will be produced. Also, we use the promoter J23119 from Anderson family which is a constitutive promoter to regulate the pReporter circuit which contain miniToe structure. So we can test the fluorescence intensity to know whether miniToe system works well.

Fig.1-1 The two plasmids of miniToe test system. The pCsy4 is constructed for the expression of Csy4. The pReporter contains miniToe struture.

1.2 Selective Medium Assay

After the process of circuit construction to get two plasmids pCsy4 and pReporter, we transformed both of them into E. coli DH5 Alpha and got the miniToe-test strain successfully. The recombinant strains are cultured in M9 medium. As preliminary experiment, the growth rate measurements are essential for both engineered strain and the negative control . As a result, the curve below demonstrates that the strain with miniToe system has almost the same OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of strain. The metabolic stress by two plasmids is not harmful to the recombinant strain.

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 superfold 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 miniToe system including two plasmids) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity by recombinant strain (with the miniToe system including two plasmids) without IPTG (0mM). The green line refers to another control group which only has pRepoter without the pCsy4 in strain. The result 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 refers to a test group with IPTG (0.125mM). The blue line refers to a control group without IPTG (0mM). The green line refers to a control group with only one plasmid (pReporter).

The first problem is whether miniToe structure can fold exactly. 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 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 places of miniToe structure in the whole circuit.

In reality as the result showed in Fig1-3, a control group (the green line) is relatively stable during the whole process comparing with two other strains. It means the miniToe without Csy4 folds well on the level of RNA and also keep OFF state so the changes of fluorescence intensity can not be detected.

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). It means miniToe system can work successfully.

At the same time, 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) which is stable and has low expression of sfGFP, the leakage may result from the inductive promoter Ptac. Even though the control group has leakage of sfGFP sometimes, we can prove the function of our system successfully. But in the future, there will be more time for us to find a better promoter for miniToe system.

We also test miniToe system by flow cytometric and 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 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).

1.4 Discussion

Combining the biology and math, we discuss the dynamics of sfGFP in the Fig.1-3 now. 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

In the Fig.1-3, the red line which represents the dynamics of sfGFP which increases in the beginning and then drop down to a stable level. The reason is that the capability of Csy4's cleavage is stronger. And the capability of mRNA's production() is relate weaker which results in the decline of after 10 hours. Before we add IPTG to induce the Ptac, the is accumulated because it is under controlled by a constitutive promoter. After we add IPTG, the initial concentration of plays an important role in the production of sfGFP during the first 10-hour. Even though the rate of cleavage is faster than the production of , the concentration of mRNA keeps increasing. But once the original is consumed, the stop increasing and drop down to a stable level. So the balance of the product rate and decay rate can kept. This is the reason why the level of sfGFP keep stable finally in Fig1-3.

See more details in model! Click here !

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1.5 collaborations

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! Click here to see more details!

2. The results of second system

2.1 Plasmid construction

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 circuits of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4. 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.

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 fluorescence intensities of sfGFP vary with Csy4s' capacities. It means that miniToe family members present various expression of target genes.

Prediction

Before the experiments, model have proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. The models help us to know our improvement deep this year!

Fig.2-3 The predication: the fluorescence intensities by different Csy4 mutants along with time

2.2.2 The result by Microscope

First, we have tested five different Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP have been accumulated during the cultivation period so the fluorescence can be observed by microscope. Because the five Csy4s have different capabilities of cleavage, the different intensities of fluorescent will be seen. The five strains have culture for the same time which both have same miniToe-WT but different Csy4 mutants. In Fig.2-4, there are fluorescence images by fluorescent microscope which indicate Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and 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.

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.2-4 The fluorescence images by fluorescent microscope. From top to bottom, the images 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 image. The picture on the left are

2.2.3 The result by flow cytometer

The qualitative experiment is not enough to analyze the Csy4s. So we test miniToe family system by flow cytometer after ten hours in M9 medium. The expression of five groups' sfGFP is showed in Fig.2-5, and they are Csy4-WT&miniToe, Csy4-Q104A&miniToe, Csy4-Y176F&miniToe, Csy4-F155A&miniToe and Csy4-H29A&miniToe. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A. 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.

2.2.4 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 expectations.

So we tested five Csy4s individually by microplate reader 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. In Fig.2-7-A, the Csy4-WT shows the same result with the first system. The switch turns off when the system without IPTG (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. 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 matchs 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.

By all the experiments mentioned before, we have proved that our Csy4 mutants work as expectations successfully. And the original part Csy4 has been submitted by other teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.

2.3 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.

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.

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 name 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 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 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.

3. The result of the 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 structure into circuits, more than one gene can be regulated. In this system, we focus on the ratio of gene expression. We tested polycistron system by two target genes, sfGFP and mCherry. Two kinds of groups have been set. One is the bicistron circuit without miniToe structures. The other group is for test which have miniToe system.

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.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 have been changed by miniToe family. The group A is a control group without miniToe family. 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 structure 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 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.4-1 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.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 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.4-4 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.4-5 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.

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.

reference

[1] Ravichandar J D, Bower A G, Julius A A, et al. Transcriptional control of motility enables directional movement of Escherichia coli in a signal gradient[J]. Scientific Reports, 2017, 7(1).

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   <div align="center"><p >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.
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-	<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/3c/T--OUC-China--JCHE.png" height="600"> </div>
+	<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/3c/T--OUC-China--JCHE.png" height="400"> </div>
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   <div align="center"><p >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.