Team:Shanghai High School/Results

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.

Results

Results
Cell-free expression
(1) Cell-free expression system
pSB1C3-T7-X0-mCherry and pSB1C3-T7-Y0-mCherry were conducted the first test of cell-free expression.

(2) The test expression was carried out in a water bath at 35°, and the result was no reaction. The factor was that the official plasmid vector was not suitable for the cell-free expression system, and the second bath temperature may be too high to cause the reaction enzyme to fail. T7-X0-mCherry+ T7-Y0-mCherry was not expressed in the kit, but the positive control was expressed

Results & Conclusions:
Since the failures appeared with pSB1C3 plasmids, we decided to switch the plasmids with approached speculationè Construction of the PET28a plasmid (sequence experiment according to the instructions) continued to test the cell-free expression of Y0-Y19
Y0-Y19 are 20 samples with different RNA thermometers, and each to them is designed previously. In the next experiment, our team focus on creating PET28a with these 20 samples with different features.

 

 

[•TXTL cell-free transcription–translation]
（1）Taking out the kit stored at -80 ° C and thaw it on ice.
（2）System (to be completed on ice):

Components	Trial samples x18	Negative control reaction x1	Positive control reaction x1
Reaction mix	17.2µL	17.2µL	17.2µL
Template DNA	10µLPCR result	0µL	10µL
E．coil cell extract	7.4µL	7.4µL	7.4µL
ddH2O	15.4µL	25.4µL	15.4µL

（3）Placed in a 30 ° C PCR machine for 2-3 hours.
[•Microplate reader: testing RNA thermometer fluorescence]
Temperature setting: 22-40 ° C


Results & Conclusions:
In order to test the qualifications of those RNA thermometer samples, we decided to use Microplate reader to exam the degree of fluorescence of each sample. As the data table shows, our team raised 2℃ each hour and recorded the data (degree of fluorescence) changes each time. The first data in the table is buffer, the second one is negative control, the third one is positive control, and the rest are the representative samples that choosing from Y0-Y19.
According to the data, the sample data that having ten times above the negative control is considered effective. During different temperatures, we could see the degree of fluorescence changes in each sample which means one specific temperature actually causes the transcriptions of genes and makes chromoproteins. Therefore, the data of transmittance demonstrates the concentration of those chromoproteins. Most of data that we collected demonstrated the results of experiments clearly.

 

Future approch
Through this experiment, our experimental results did not meet the expectation of perfect. The originally expected bright color was not very obvious. The feasibility of our experiment was not as good as expected, and the results seemed to be disappointing.
In order to keep the project smoothly and reach the standard we expected, we still have the project in the future we will continue to improve the space, we can on existing protein and DNA sequencing, where observation out, to find the corresponding methods to solve these problems, such as to repair and improvement of available RNA sequences, use more efficient enzyme, or increase the concentration of the amino acids to increase reaction intensity, and so on.
In the future, we will be able to make RNA thermometers more accurate, so that the accurate temperature sensing system can be used not only for vaccine transportation, but also for the transportation of medical materials that require extremely high degree of freshness, such as organs for organ transplantation, fresh spinal cord, blood, or frozen living bodies... For those tissue, which is sensitive to temperature, the temperature change will significantly lead the protein denature which makes the organ can not be transplanted, RNA thermometer can accurate perception of the organization in transit temperature change, even in a very short period of time, as long as the temperature is greater than the environment temperature limit, RNA thermometer can react immediately to temperature, make the hospital medical materials in a timely manner to realize these materials may have quality problems, timely to do quality inspection, to avoid losses and medical accident.
At the same time, we can control the time required for the reaction by editing the activity of the enzyme, and control the temperature required for the reaction by editing the length of the RNA card structure, so as to adapt to the different requirements of temperature sensitivity of different materials.

 

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