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 parts 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.