Team:USTC/Results
Results
Now We Harvest
Leaders
After the preliminary investigation and the massive work on building our two systems, there comes the most exciting part of our project----the verification part. We conducted several verification experiments to test our hypothesis.
Experiment Design
For the Pro Priontein system we conducted experiments to measure the fluorescence intensity of the split sfGFP in both Escherichia coli(E. coli) and Saccharomyces cerevisiaes(S. cerevisiaes). In E. Coli, the gene for sfGFP 1-10 was expressed in BL21 on a pSB1A3 vector and the gene for sfGFP 1-10 was on a pSB1C3 vector. In S.cerevisiae (S. cerevisiae), sfGFP 1-10 was placed on YeWGAP and sfGFP11 was placed on YeUGAP.(YeWGAP and YeUGAP are two kinds of yeast plasmids which enable the transformant of yeast cells to grow on a medium lacking tryptophan and uracil respectively. )
Firstly, we compared the fluorescence intensity of the E. coli respectively containing sfGFP1-10 and sfGFP11 with that of the E. coli in which sfGFP1-10 and sfGFP11 are coexpressed to see whether or not our spilt sfGFP system can work as an effective indicator in real condition.
What we expected is that the E. coli can produce much brighter fluorescence when the two separate part of sfGFP protein (sfGFP1-10 and sfGFP11) are expressed in the same bacteria.
And then we changed our chassis to S. cerevisiae to verify the function of the kill switch we designed, which works like this: heat shock can cause the aggregation of Sup35 while GdnHCl can disaggregate them. We tried to detect the aggregation level of Sup35 under temperature and GdnHCl concentration gradient by measuring the fluorescence the S. cerevisiae produced. As what we’ve already mentioned before, the aggregation condition of Sup35 is indicated by the fluorescence intensity. The low level of fluorescence intensity can indicate high level of aggregation of Sup35, this is because when sup35 aggregates, sfGFP11, which is fused with sup35, will fail to bind with sfGFP1-10. According to the mechanism of split sfGFP system, it can lead to weaker fluorescent intensity. Briefly speaking, the brighter the fluorescence is, the weaker the proteins' aggregation is.
We conducted experiments to compare the different fluorescence intensity induced by heat shock under temperature gradient. And in further experiment, we added GdnHCl solution to the system and then we measured the fluorescence to see if GdnHCl can disaggregate the protein. So that we could know if our kill switch can works effectively.
The expected results are fluorescence intensity will decrease under heat shock. But if we add GdnHCl solution to the system the fluorescence will turn back due to the curing function of GdnHCl.
Experiment Results
1. The spilt sfGFP experiment in E. coli
To test if the split sfGFP can function as our expectation, we transform the plasmids containing sfGFP1-10 and the plasmids containing sfGFP11 respectively in BL21, cultivating the bacteria at 37°C and shacking at 250 rpm/min overnight. We use fluorescence microscope to observe the bacteria under 100X objective lens. From these fluorescent images, we find that fluorescence of sfGFP1-10 is stronger than the fluorescence of sfGFP11 and both of them are weak. It corresponds with our expectation that either of separate part of sfGFP have part function of complete sfGFP and sfGFP1-10, which is longer, may be brighter after excitation.
However, when the pSB1C3 carrying the part of sfGFP11 and the pSB1A3 carrying the part of sfGFP1-10 ware expressed together in E. coli , it doesn't present stronger fluorescence than either plasmid is expressed in E. Coli respectively. the possible reasons are as follows. Firstly, different metabolic stress of two plamids causes indistinct results. The pSB1C3 carrying sfGFP11, whose gene length is shorter, may have higher expression level than pSB1A3 carry sfGFP1-10. Secondly, there is obvious fluorescence quenching after excitation. Therefore, results of fluorescent images and fluorescent images appear that co-expression of sfGFP1-10 and sfGFP11 has weaker fluorescence intensity. Thirdly, because sfGFP1-10 and sfGFP11 may as well not be expressed in E. Coli at 1:1 ratio, the collision probability is lower than our expectation.
2.The spilt sfGFP experiment in Yeast(S. cerevisiae,W303)
Firstly, we transformed the two kinds of plasmids into the S. cerevisiae W303 to get three types of S. cerevisiae respectively containing sfGFP1-10, PR-sfGFP11 and both. Then we cultivated the cells at 30 ℃ for 24 hours.
(1) Testing the effect of heat shock
We measured fluorescence intensity of two groups of S. cerevisiae cultivated separately at 30,34,38,42 ℃ without GdnHCl. The first group was cultivated for 1 hour and the other one for 2 hours.Here are original images of the two groups’ fluorescence experiment.
By using Image Processing method described in the appendix, we got these two diagrams.
The data of the points above is showed below.
| Par1 | Par2 | Par3 | Par4 | Par5 | Average | |
|---|---|---|---|---|---|---|
| 30°C | 169.0165 | 160.7907 | 173.7442 | 177.6913 | 177.0472 | 171.658 |
| 34°C | 167.8331 | 164.8636 | 133.7497 | 153.8358 | 143.118 | 152.68 |
| 38°C | 121.4902 | 120.289 | 105.2324 | 124.6197 | 132.0618 | 120.7386 |
| 42°C | 116.3674 | 107.4996 | 112.4971 | 114.5681 | 106.1929 | 111.425 |
Heat Shock 1h
| Par1 | Par2 | Par3 | Par4 | Par5 | Average | |
|---|---|---|---|---|---|---|
| 30°C | 153.2803 | 116.601 | 131.8142 | 122.8715 | 165.9979 | 138.113 |
| 34°C | 110.8943 | 123.2279 | 106.1407 | 101.0241 | 83.48351 | 83.48351 |
| 38°C | 116.4582 | 107.2333 | 122.945 | 95.81423 | 95.33545 | 107.5572 |
| 42°C | 118.5441 | 104.3932 | 94.09721 | 94.72964 | 107.0703 | 103.7669 |
Heat Shock 2h
As shown in the Figure 1 (2 hours of heat shock) above, the brightness of the bright dots in the photo, which represents the level of sfGFP 1h after heat shock, decreases almost linearly as the temperature increases. According to the linear fitting, the brightness drops about 5 units for the increase of temperature of 1°C, which is about 3% of the level at 30 degrees Celsius. In the modeling part, we predicted a linear decreasing relationship between temperature and the sfGFP output level. So it coincides with our experimental results.
It is noteworthy that high temperature is likely to affect the binding of sfGFP1-10 and sfGFP11, and destabilize the sfGFP complex. So we must exclude the possibility that the result in the figure above can be only attributed to this factor, instead of the aggregation of Sup35. According to Zhang et al, from 30 degrees to 42 degrees, the fluorescence intensity decreases for about 20%, owing to the effect of high temperature. However, in our experiment, it decreases with 35%, which is obvious more than the effect of temperature only. So there must be another mechanism, which should be that aggregation of Sup35 blocks sfGFP11 and make it impossible or at least harder to bind with sfGFP1-10. What’s more, the difference of the decreasing ratio, 15%, is precisely identical to the predicted ratio in our modeling result. Thus we can safely draw the conclusion that the sfGFP level decreases with increasing temperature, due to or at least partly due to the aggregation of Sup35.
As for the Figure 2 (2 hours of heat shock), it shows that there is a huge decrease of fluorescence intensity when temperature increases from 30 ℃ to 34 ℃ while no obvious changes occur when temperature varies from 34 ℃ to 42 ℃. That is not fully in line with expectation. The possible explanation is that heat shock indeed causes the aggregation of Sup35 and relatively higher temperature can enhance the aggregation effect. But there is another important factor you have to notice, which is that when Sup35 is in prion state (non-prion state of Sup35 nearly don’t form aggregation), it can propagate and transform the normal Sup35 protein into its prion state. And 2 hours is so long for the cells to finish the form changing process and aggregation of all the Sup35, regardless of the little differences in the temperature of heat shock. In conclusion, the latter three points in diagram 2 have similar values because the aggregation of Sup35 had come to saturation. Now you might want to ask why group 1 doesn’t show this contradiction. The reason might be that 1 hour is not sufficient for process of injection and aggregation to finish and during that time temperature has a dominant effect on the aggregation of Sup35.
In a word, from the temperature experiments above, we provide valid evidence which showed that our system could work effectively!
(2) Testing the effect of GdnHCl
We measured fluorescence intensity of S. cerevisiae cultivated at 42 ℃ in 0.25 mM, 0.50 mM, 3 mM, 5 mM GdnHCl for 4 hours.
By using Image Processing method described in the appendix, we got this diagram.
According to statistics analysis, with the increase of concentration of GdnHCl, fluorescence intensity increase in the first stage, and then decrease. In the first stage, fluorescence became brighter because Sup35 disaggregated and our kill switch turned on again. In the latter stage, the fluorescence intensity decreases which was opposite to our modeling results. We assumed that GdnHCl, which possesses high electric charge, may lead to the misfolding of split sfGFP. It is likely to disturb the two fragments assembling and reconstituting. Thus, GdnHCl is a suitable curing reagent for Sup35 in S. cerevisiae within limited concentration range.
Experiment Design
For the Propri-ontein system we measured the fluorescence intensity of the S. cerevisiae containing the plasmid carrying pGAL+GFP without the activation of AD and BD.
For further experiment of the Propri-ontein system, we can compare the fluorescence intensity of the S. cerevisiae containing all of the three plasmids (AD, BD and pGAL+GFP) with that of the S. cerevisiae containing only pGAL+GFP. The ideal results are that the fluorescence produced by the S. cerevisiae containing AD, BD and pGAL+GFP will be much brighter than the S. cerevisiae containing only pGAL+GFP after heat shock. And if we add GdnHCl to the system, the fluorescence will decrease because it can disaggregate the protein so that AD and BD can no longer bind together, which can eliminate the expression of the downstream gene under the control of pGAL.
Experiment Results
The pGAL-GFP experiment in S. cerevisiae
For the pGAL-GFP device, we conducted several tests in S. cerevisiae.
Firstly, we transformed the recombinant plasmid containing pGAL1-GFP into the S. cerevisiae W303. Then we cultivated the cells at 30 ℃ for 24 hours. Next we measured the fluorescence of the cells under the same condition. (We used fluorescence microscope to observe cells under 100X objective lens.)
Here're the results of the fluorescence experiment.
Compared with the results from spilt sfGFP experiment, the fluorescence intensity was extremely low. And that is exactly what we expected. So we proved that the pGAL-GFP device really functions !
As for the other devices in propri-ontein system. We tried to transformed the three plasmid-AD, BD and pGAL-GFP to S. cerevisiae. Unfortunately, we didn’t get the colony on our plates. The possible reason is that transformation of three kinds of plasmid at the same time brings too much pressure to the cell, which can cause the death of cells. It was really a pity that we had to stop the experiment due to the time limitation. But we will finish the verification of the whole system in the near future.
Overview
Due to the temperature sensetive property of the SUP35NM, our system can be applied for a Biological Temperature Indicator. In the Proof of Concept section of our project, we have showed the relationship between the temperature and the intensity of the fluorescence, so we construted a standard cure using our prototype, and tested the standard curve with a specific temperature. Furthermore, if we introduce the GdnHCl, we can alter the standard curve and locate the peak of the intensity of the fluorescence in any chose temperature. In the process of industry production,engineers find that incrustation has harmful effects on the device especially the boiler. When there is a certain number of incrustation aggregate on the pipe it will cause a serious accident. Therefore,when and where we should clean the incrustation is a headache to all the engineers. Our BTI presented here provides a novel method to solve this problem.
The Prototype
As the pipes in industry are cylindrical in most cases, we also design our hardware in cylindrical to fit it.
We design a clearance in our hardware to contain the yeast medium.
In order to prevent other microbes from coming into the device, we also design a cover over the device.
We design our device by SOLIDWORKS and make it into reality by 3D printing(3DP). Considering the light transmittance and economics, the material of our hardware is transparent photosensitive resin and the material of the cover is ordinary resin.
Before we put the prototype into use, we had done lots of mathematical modeling to show the working properties of it. See more details in our modeling section.
The standard curve
We combined a thermostatic water bath and a pump to simulate the working conditions.
The room temperature was around 24 ℃. The pump was set at 63 rpm. We set the temperature of the water bath at 34 ℃, 38 ℃ and 42 ℃. At each temperature, we cultivated the yeast in our prototype for 1 hour, and then extracted the yeast for fluorescence testing. The prototype was washed thoroughly before adding new yeast to it and used for the test of another temperature.
We chose 36 ℃ as the test point. Other conditions were set as same as the points on the standard curve.
We took five photoes of each temperature through the fluorescence microscope. the photos are showed below.
Using the quantitating method we introduce in our Proof of Concept section, we get the figure below, showing the standard curve and the test point.
The data of the points above is showed below.
| 34 ℃ | 38 ℃ | 42 ℃ | 36 ℃ |
|---|---|---|---|
| 164.46 | 140.61 | 116.94 | 96.09 |
Apparently, there is a negative correlation between the intensity and the temperature just like we have showed in our modeling section. So we can conclude that the prototype of the BTI can work well in the simulated working conditions.
Project Achievements
√ We constructed the Pro Priontein system in Saccharomyces cerevisiae, verified it could work, showing a potential way to construt protein-level switch.
√ We constructed all the circuits of Propri-ontein system, a specified temperature controlled system in Saccharomyces cerevisiae.
√ We 3D printed a model to demonstrate how our engineered yeasts under simulated conditions of industrial production.
√ Using our prototype, we succeessfully got a standard curve for temperature measuring.
√ We proved that sfGFP can be splited into sfGFP1-10 and sfGFP11, making it a standardized BiFC system. See details in our Design section.
√ We constructed a set of mathamatical models to demonstrate the mechanisms of our system.
√ We wrote a software as a spin-off to assist our enzyme digestion experiments.
× We failed to observe the sfGFP split system working in Escherichia coli as expected. See detailed analysis in our Results section and Modeling section.
× We failed to transform all three circuits of the Propri-ontein system, so pitifully we could not verified it.
Medals
This year Team USTC has met all the requirements to win the gold medal. Bronze: √ Register for iGEM, have a great summer, and attend the Giant Jamboree. √ Meet all deliverables on the Requirements page. √ Create a page on our team wiki with clear attribution of each aspect of our project. See our Attributions section. √ Document 9 new standard BioBrick Parts or Devices central to our project and submit this part to the iGEM Registry. See our contribution at our part section. √ We also documented a new application of a BioBrick part from a previous iGEM year.See part BBa_J63006.
Silver:
√ Experimentally validate that our new BioBrick Parts of our own design and construction works as expected through the fluorescence microscope. Document the characterization in the Main Page section of the Parts' Registry entry. See our Results section and Parts section.
√ Submit 9 new parts to the iGEM Parts Registry.See our Parts section.
√ We helped Team BIT with their modeling. See our Collaborations section
√ We held a lot of activities ranging from education to Public engagement, from communicating to surveying. The topics included sustainability, safety and People's opnions on science.See our Human Practices Silver section.
Gold:
√ We did a survey on people's knowing and opnions of the yeast prion, meanwhile we introduced the yeast prion to those who didn't know about that. We also collected people's idea around the yeast prion, which help us to determine our applications. We communicated with Dr. Dong Men, an expert on yeast prion, to determine what apllication that our systems may be used for.See our Human Practices Gold section
√ We improved two parts this year, one is part BBa_J63006 and the other is part BBa_K1739000.
√ We verified our ideas on our Pro Priontein system would work in Saccharomyces cerevisiae. We proved the devices of this system are successful.
√ We demonstrated our work at simulated conditions by designing a model,which is 3D printed, to contain our yeasts of both system. See our Design section and Results section.
Finish the work of the Propri-ontein system
Before we started our experiments, we set a goal to finish two system we designed, one of which is a gene-level switch, and the other is a protein-level switch. We constructed our device in the plasmids of Escherichia coli and then integrated to the plasmids of Saccharomyces cerevisiae. After that, we transformed all the plasmids to the competent yeasts. However, due to the limit time, we only finished the Pro priontein system, leaving the yeast plasmids of our Propri-ontein system untransformed. With sufficient time, we believe that our Propri-ontein system will work succeessfully.Expand our system to Escherichia coli
This year, we fused the gene of SUP35NM with the activating domain of GAL4, the binding domain of GAL4, and sfGFP11. We verified the fusion of SUP35NM and sfGFP11 worked fine. It was believed that the cure of the prion aggregate might be associated with the generation propagation, however, a generation of the yeast can be very long, which may reduce the usefulness of the application.There are evidences showing that the yeast prion can work in E. coli[1]. So one of our future work falls on expanding our systems to E. coli make our system faster.Find more interacting protein pairs
This year we use the sfGFP split to construct our Pro Priontein system, but theoretically, any interacting protein pair can be exploited in this system, which could be recognized as a universal protein-level switch. In scienticfic research, especially the protein field, the interaction of proteins is frequently taken into consideration. Thus our system appears to be a very useful tool in protein study. To verify its universality, more protein pairs are required, which makes a part of our future work.Find a more convenient fluorescence reading method
Before performing our experiments, we thought the difference of the intensity of fluorescence would be distinguished by eye, however the results overthroughed our former assumption. Therefore we can only see the difference by the fluorescence microscope and process the images using Matlab. Thus a more convenient fluorescence reading method is required to make our system portable and ready-to-use.Automatically eliminate the noise
The noise of the background of the images took through fluorescence microscope would seriously influence the quantativating. However, we could not figure out how to eliminate it automatically, because we didn't know the specfic relationship between the noise and the subject. We have tryed to use subtraction and division to eliminate, but the error still remained. Thus we concluded that the relationship between the noise and the subject was very complicated. Now we can clearly see the difference of the intensity by eye but have't figure a way to precisely quantivate it.references: [1] Men, D., Guo, Y. C., Zhang, Z. P., Wei, H., Zhou, Y. F., & Cui, Z. Q., et al. (2009). Seeding-induced self-assembling protein nanowires dramatically increase the sensitivity of immunoassays. Nano Letters, 9(6), 2246-50.
Image Processing
For the original photos we got, we handled all of them in the same way. The image processing method is showed here.
Take one photo for example. This is an original photo of fluorescence experiment(Image 1).
We use the mathematical software Matlab to calculate the brightness of fluorescence according to how bright the pixel is. Then we convert the photo into a 3D plot where XY surface denotes the location of pixel and Z axis represents the brightness, which looks just like this (Image 2)
Then we converted it into a contour line where same value would show in the same curve, which is shown below (Image 3)
In this picture, different color indicates different brightness value. Then we reverse the contour figure to make it coincide with the original figure. (Image 4)
Then we only select the bright part of the figure, and omit other parts
Finally, we calculate the average brightness value of the illuminating area so that the value can represent the quantitative and precise fluorescence intensity of the original photo
All of analysis above is processed with Matlab. You can find the original code by clicking this hyperlink.
