Team:Shanghai High School/Design

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.

Design

Overview
The safety problem of vaccines troubled chinese citizens since the non-working vaccine have been find out. For fully solving this problem, our team wanted to design a product which can check whether vaccines are safe enough to use or not. The RNA themometer first came up into our mind because of its characteristics: natural based, harmless to human, and able to be mass producing. the final goal of this project is to construct an indicator which will change the color in different temperature.
expectation
-The expectation of RNA Thermometer:
To achieve our goal of designing the RNA Thermometer, we expected to find out several specific sequences of RNA with high sensitivity of detecting the surrounding temperature. By testing abundant trials of RNA sequences and the most suitable temperature of several different vaccines, we were looking forward to matching each vaccine with its suitable RNA Thermometer since each vaccine had their own favorite temperature. By doing so, we were able to ensure each vaccine had their own RNA Thermometer and to know if it would deteriorate during the transportation. Making sure the qualities of any vaccines was the fundamental purpose of our RNA Thermometer Design. Medical personnel could easily and accurately identify the safety of vaccines that were equipped with RNA Thermometer by checking the color changing on the Thermometer. For our further expectation of this equipment, we would like to develop different color changings to match their corresponding surrounding temperatures. In the future research, in order to transmit more precise information of vaccines, we were looking for one way that RNA Thermometer could express different color changing after exposing to the environments with higher temperature.

Process
For our project, we need to ensure each vaccine has their own RNA Thermometer and to know if it would deteriorate during the transportation. So that we can make sure the qulity of the vaccines which is the foundamental purpose of our design of RNA themometer. Therefore, Medical personnel could easily and accurately identify the safety of vaccines that were equipped with RNA Thermometer by checking the color changing on the Thermometer.

Principle explaination
An RNA thermometer is a temperature-sensitive non-coding RNA molecule which       regulates gene expression.

How this works: Increased temperature → Unbinding of RNA structure → RBS exposed → Protein synthesis initiated → Change of color

We use reporter protein as a mechanism to display information.To quantify the strength or activity of upstream gene expression parts such as promoters and ribosome binding sites (RBS) .

Modeling could be used to select several different RNA thermometers that respond to  different temperatures.One base changes to the thermometer sequence could generate the desired diverse sets of temperature responses. In addition, NUPACK is utilized to compute their free energies and to melt profiles.

To construct the plasmid, we went through the following steps.
Primer design

• Select appropriate vectors, cleavage sites and RNA sequence.
• Confirm the base sequence of the target fragment again on NCBI.
• Submit to the company to make primer.

PCR→ Endonuclease digestion, ligation→Transformation→Plasmid extraction→Collect→Resuspend→Lysis→Wash→dissolution

 

TXTL cell-free expression method was employed to the final product of our RNA thermometer, that is to produce protein using biological machinery without the use of living cells.

Furture plan
For our further expectation and future investigation, we would like to develop different color changings to match their corresponding surrounding temperatures. In order to transmit more precise information of vaccines, we will be looking for a way that RNA Thermometer could express different color changing when it exposing in a more volatile environment circustances.

 

Reference：
https://baike.baidu.com/item/长春长生疫苗事件
Storz,G. (1999) An RNA thermometer. Genes Dev., 13, 633–636.
Johansson,J., Mandin,P., Renzoni,A., Chiaruttini,C., Springer,M.and Cossart,P. (2002) An RNA thermosensor controls expression ofvirulence genes in Listeria monocytogenes. Cell, 110, 551–561.
Shaunak Sen, Divyansh Apurva, Rohit Satija, Dan Siegal, and Richard M. Murray
ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017
Downloaded from http://pubs.acs.orgon April 27, 2017

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