Team:Shanghai High School/Description

The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

Description

Inspiration
Getting vaccinated is a method of preventing diseases that are being highly regarded by parents. In the November of 2017, an issue claimed by Chinese National Medical Products Administration, which eventually became our inspiration of this project, raised high awareness in parents: a batch of freeze-dried rabies vaccines for human use produced by Changchun Changsheng Biology Technology Inc. were reported of fraud. The approximate number of vaccines that were involved was 113,000. Even though the incorporation claimed that all those problematic vaccines were called back and not yet introduced to the market, the issue went beyond the limits of morality. Due to the jeopardy of unqualified supervision for making and transporting vaccines, we are spurred to provide a convenient method to ensure that citizens are getting vaccinated in a safe environment. To ensure the vaccine quality, WHO recommended the temperature ranges (2˚C to 8˚C) that best preserve the vaccines. The system, “the cold chain”, is used for storing and transferring vaccines at health facility level with proper monitoring. Vaccines that are sensitive to extreme temperatures may loose vaccine potency quite easily. In other words, if vaccines lost its potency, they could no longer adequately protect the patients from being at risk of getting diseases. Strictly following the traditional regulations of Controlled Temperature Chain, health workers ensure the temperature of the cold chain stays between 2C˚and 8C˚. Furthermore, the frequency of vaccine wastage should be reduced because unused vaccines worth a significant amount of the public sector of a nation.

As being described above, most kind of vaccines will denature as the temperature increases. So it is essential for applying the controlled temperature chain to the whole transportation and storage chain. Some categories of vaccines are kinds of pathogens that are still “alive”. When increasing the environment temperature, these pathogens such us bacterias will become active and return to the state of continuous reproduction. This will bring back the toxicity of the target pathogen, and, at the same time, probably loose the antigen needed during the repeated reproduction. Others include inactivated vaccine and toxoid. These are often made up of protein and polysaccharides which will denature when exposed in high temperature.

To sum it up, temperature is a key factor in examining the activity of vaccines. Health facilities often expect for a mean to examine the quality when the vaccine reaches the medical center. There has been a chemical method for detecting the temperature change during transportation.

Description
Our project aims to design a color indicator based on the mechanism of RNA thermometer for vaccines using TXTL Cell-Free Expression method. TXTL Cell-Free Expression method is the production of protein using biological machinery without the use of living cells. Cell-Free transcription and translation systems are used for testing and facilitating the implementation of circuits in cells. TXTL reactions in microcompartments enable prototyping of molecular communication and integration into genetic circuits by copying critical cell features. We want to make this project into a product— a small reaction kit that can be easily used outside and has a long preservation time at room temperature. The product will be made into a business, and applied to human practices. Eventually, we intend to turn the business into social contribution to make the world a better place to live, such as to minimize unnecessary vaccine wastage during transportation and reduce the health risk of using damaged vaccines.

One key ingredients in our experiment is the reporter protein, which is used as a mechanism to display information. The chromoprotein is a conjugated protein that contains a pigmented prosthetic group, and quantifies the activity of upstream gene expression parts such as promoters and ribosome binding sites.

To model for our RNA thermometers, we select different RNA thermometers that respond to different temperatures. We find out that one base changes of the RNA in the thermometers can generate the desired outcome of temperature changes. The increased of temperature can cause the melting of RNA structure in thermometers. Thus, RBS will be exposed, initiating protein synthesis. As a result, we will see a color change that indicates a rise of temperature. The signal of temperature change can warn people of certain vaccine degeneration.

This is a picture of a kind of temperature-sensitive chemical stickers. As shown in the picture above, the color of the square in the center darkens as the temperature increases. With reference to the color around, it is easy to determine whether the vaccines has  Although seem to be perfect over time, it still has a number problems. First, the color darkens as the heat accumulates, meaning that it would change color even if the temperature haven’t reach the denature point. Second, its maximum sensitivity is 2 days, which is relatively too long for the well developed transportation system world.

As a result, we were inspired to create a kind of temperature indicator which is sensitive enough for the present use. This turns our eye sight to the subtlety of the RNA thermometer. RNA thermometer uses a kind of basic character that RNA molecules are unstable. During low temperatures it will form hydrogen bonds between the complementary bases. As the temperature increases to a certain temperature, hydrogen bonds breaks and exposes the RBS(Ribosome binding site) on the single strand of RNA. This activates the translation process and produce the amino sequence which codes for a protein, such as Mcherry. This protein shows color both in UV light and natural light, which enables it to show color and thus tell the temperature vaccine had been in.

Reference

1. Sun Z Z, Hayes C A, Shin J, et al. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology[J]. JoVE (Journal of Visualized Experiments), 2013 (79): e50762.
2. Sen S, Apurva D, Satija R, et al. Design of a Toolbox of RNA Thermometers[J]. ACS synthetic biology, 2017, 6(8): 1461-1470.
3. Garenne D, Noireaux V. Cell-free transcription–translation: engineering biology from the nanometer to the millimeter scale[J]. Current opinion in biotechnology, 2019, 58: 19-27.

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