Team:Nanjing 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

Our goal is to construct cells which can record the length of time that they are exposed to light. In order to insert a light detection system in the cells, we genetically modified some plasmids and transformed them into BL21AI cells for the expression of certain proteins. The light detection system consists of two parts, which are blue sensor and fixk2 promoter. Blue sensor is a composite ofYF1, a blue-light-sensitive histidine kinase, and its cognate response regulator FixJ. In the absence of blue light, YF1 phosphorylates FixJ, which then drives robust gene expression from the fixk2 promoter. In the presence of blue light, net kinase activity of YF1 is greatly reduced. Therefore, gene expression driven by fixk2 promoter is reduced significantly. Knowing the mechanism of the light detection system, we plan to construct our target gene downstream of fixk2 promotor, so that expression of the gene can be regulated by blue light. The carrier cell of such light detection system was BL21-AI E.coli, as it is an arabinose-inducible strain designed to give the maximum protein expression with the tightest regulation available from a T7 expression system. 
To create the cells which meet our goals and test their effectiveness, we build the following cells with or without the Light detection System:
BL21-AI cells with pCDF-T7-Cas1Cas2:
These cells are constructed by transforming pCDF-T7-Cas1-Cas2 into BL21-AI cells. T7 is an inducible promoter that controls the gene expression downstream. Only when Isopropyl β-d-1-thiogalactopyranoside (IPTG) is added will T7 promoter starts to allow the proteins downstream, in this case Cas1 and Cas2, to express. After the expression is initiated, Cas1-Cas2 complex, the combination of E.coli Cas1 and E.coli Cas2 proteins, cuts alien DNA sequence and inserts the DNA fragments into the CRISPR array of the cells, expanding the array.  Theoretically, there is a positive correlation between the acquisition of new spacers of the CRISPR array and the amount of time the expression is induced. Since the spacer acquiring ability of these cells is not influenced by blue light, it serves as the control group for the experiment. We plan to collect samples of this type of bacterial cells after the gene expression is induced for different amounts of time. PCR is then done on these samples to make more copies of the cells so that the PCR products can be run in agarose gel. We would then be able to see whether new DNA fragments are inserted into the CRISPR array under different inducing time and compare the results with those of other light detection systems.
BL21-AI cells with pSB1C3-fixk2-Cas1-Cas2 and pCDF-T7-Blue Sensor:
These cells are constructed by transforming pSB1C3-fixk2-Cas1Cas2 and pCDF-T7-Blue Sensor into BL21-AI cells. T7 promoter is used here to control the Blue Sensor. Under blue light, the ability of Cas1-Cas2 complex to cut and insert DNA fragments is restricted as a result of the promoter fixk2 not being initiated. Therefore, the expasion of CRISPR array in the cells is also limited. However, in the absence of blue light, Cas1 and Cas2 are expressed, thereby allowing the complex to cut and insert DNA fragments onto the CRISPR array. We plan to have samples from the bacterial cells when cells are exposed to blue light for different amounts of time. PCR is then used to make more copies of the cells so that the PCR products can be run in agarose gel. Whether CRISPR array has expanded can be told by the length of the CRISPR array.


>BL21AI cells with pET28a-fixk2-TfusCas3, pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor:
These cells are constructed by transforming pET28a-Fixk2-TfusCas3, pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor into BL21-AI cells. J23100 promoter is a constitutive promoter. Blue sensor under J23100 will express continuously. Thermobifida fusca Cas3 (Tfus Cas3) is a protein found to have a function of restraining E.coli Cas1-Cas2 expression. Tfus Cas3 is built downstream of fixk2, the light regulated promoter, so the expression of Tfus Cas3 is regulated by blue light.  In the presence of blue light, Tfus Cas3 expression will be greatly reduced, which leads to the expression of Cas1-Cas2 if IPTG is added to induce T7 promoter. The probability of catching DNA sequences increases as Cas1 and Cas2 proteins are expressed, leading to observable CRISPR array expansion. In the absence of blue light, Tfus Cas3 is expressed. Therefore, Cas1-Cas2 expression will be restricted. As a result, CRISPR array will not expand in this condition. We plan to have samples from the bacterial cells when cells are exposed to blue light for different amounts of time. PCR is then used to make more copies of the cells so that the PCR product can be run in agarose gel. Whether CRISPR array has expanded can be told by the length of the CRISPR array.

 

In contemporary world, designs like anti-blue light glasses which prevent customers from over expose to blue light are prevalent, but designs that are capable of visualizing the amount of time they spent with blue laser are rare. We want to design a cheap but effective product to monitor the amount of the blue light using our micro-organism. Through data analysis, our customers may clearly be aware of the amount of time they spend in front of electronic devices and take actions to protect themselves from the harm of the blue light. For example, we think of a kind of necklace to contain our micro-organism. It is a hollow small ball made of glasses, and we can put our bacteria liquid into the ball. When wearing this necklace, blue light can be caught by the funstion of the micro-organism in the necklace. After a day or a week of work, our customers can send the necklace back to our laboratory, let us to examine the total time, and change new bacteria liquid. If customers want a more beautiful style, we can provide things like flowers to decorate the necklace. Moreover,if pur customers think that the necklace is inconvenient, we also offer small glass bottles that can be place on the desk to record the time.

Future product
In contemporary world, designs like anti-blue light glasses which prevent customers from over expose to blue light are prevalent, but designs that are capable of visualizing the amount of time they spent with blue laser are rare. We want to design a cheap but effective product to monitor the amount of the blue light using our micro-organism. Through data analysis, our customers may clearly be aware of the amount of time they spend in front of electronic devices and take actions to protect themselves from the harm of the blue light. For example, we think of a kind of necklace to contain our micro-organism. It is a hollow small ball made of glasses, and we can put our bacteria liquid into the ball. When wearing this necklace, blue light can be caught by the funstion of the micro-organism in the necklace. After a day or a week of work, our customers can send the necklace back to our laboratory, let us to examine the total time, and change new bacteria liquid. If customers want a more beautiful style, we can provide things like flowers to decorate the necklace. Moreover,if pur customers think that the necklace is inconvenient, we also offer small glass bottles that can be place on the desk to record the time.

References:
1.      Martinez-Garcia, J.F., Huq, E. and Quail, P.H. (2000) Direct targeting of light signals to a promoter element-bound transcription factor. Science, 288, 859-863.
2.      Levskaya, A., Chevalier, A.A., Tabor, J.J., Simpson, Z.B., Lavery, L.A., Levy, M., Davidson, E.A., Scouras, A., Ellington, A.D., Marcotte, E.M. et al. (2005) Synthetic biology: engineering Escherichia coli to see light. Nature, 438, 441-442.
3.      Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. and Moglich, A. (2012) From dusk till dawn: one-plasmid systems for light-regulated gene expression. Journal of molecular biology, 416, 534-542.
4.      Wang, X., Chen, X. and Yang, Y. (2012) Spatiotemporal control of gene expression by a light-switchable transgene system. Nature methods, 9, 266-269.
5.      Fernandez-Rodriguez, J., Moser, F., Song, M. and Voigt, C.A. (2017) Engineering RGB color vision into Escherichia coli. Nature chemical biology, 13, 706-708.
6.      Xiao, Y., Ng, S., Nam, K.H. and Ke, A.J.N. (2017) How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration. 550, 137.
7.     Moglich, A., Ayers, R. A., and Moffat, K. (2009). Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol, 385(5), 1433−1444.

©2019 Microsoft Corporation. All rights reserved.