Team:PASantiago Chile/Description

Description

This project involves the modification of the bacterium Escherichia coli, through Synthetic Biology, which is based on the creation or modification of existing organisms, making them have the characteristics that we want. This organism will sense the high levels of alpha, beta, gamma rays and X-rays emitted, factors that cause a mutation in the DNA of said bacterium that will give a purple coloration and the aroma to lemon. To validate that the research is necessary and supported by specialists in the area, a survey was conducted to radiology students, medical technologists and radiology center workers, obtaining an 83% approval, on the need for better dosimeters that are reliable results .

The sensor and its parts

For this through a scientific investigation, a sensor was designed that is based on a biphasic switch, device that can be turned off or on according to the concentration of the imput. This switch naturally regulates lambda phage, which in this case when activated the Promoter pRM gives way to the transcription of the Cl repressor, which in turn will cause inhibition of the Pr promoter and can not be transcribed Cro, LuxR and LuxI. In contrast, if DNA damage is detected with the help of the RecA protein, the Cl repressor is removed and the pRM promoter is stopped, giving rise to pR (promoter) activation and to the transcription of Cro, LuxR and LuxI .

This circuit is based on the bacteriophage (phage) lambda lysogenic, double-stranded DNA that infects E. coli and was discovered in 1951 by Esther Lederberg. Its cohesive ends of the genetic material, after the infection causes its genome to be circulated, and if it continues with the lysogenic cycle behaves like a plasmid taking advantage of the enzymes of the recombination of the bacterium integrating itself in the genome of the latter. In the lysogenic cycle, the virus is inserted at a specific point in the genome of the bacterium and replicated when it does so, passing its genes to E. coli. Duplicates. The virus synthesizes from the Cl repressor, which inhibits the expression of the rest of the genes, where in conditions of cellular stress the bacterium activates the SOS response, acting RecA to inhibit the activity of the Cl repressor that ends a cascade response that causes the Integrated virus switch to lithic route. This is where the cell is usually infected, producing viral particles that are released into the medium, once the host bacterium is lysed (breaks the cell membrane), killing it in the process. With the activation of RecA and the transcription of the Cro, LuxR and LuxI genes, the activation of the second plasmid with the LuxR / HSL promoter will be done, which will allow the color to be transcribed in the same way as the purple color, And the lemon-smelling gene. This will be the warning sign for our specialist.

The operation of the circuit is inspired by the work already done by the group of IGEM 2011, Penn States, which, similarly detect the harmful radiation rates, but unlike ours, RecA detects damage, prevents repair and connects To a reporter to know where the damaged DNA is. (States, 2011)

Why does the RecA protein serve us?

RecA is a multifunctional protein, which is essential for different biological processes. Coordinated regulation of unrelated gene expression in response to damaged DNA, also known as SOS response, is what matters for this project. The regulatory role of RecA works with an SOS operon of approximately 20 unrelated genes inducible by overexposure to DNA damaging agents. The encoded enzymes that induce these genes work to cut out the damaged DNA and facilitate the repair of possible damage occurring in DNA recombination.

Expression of SOS genes is controlled by the repressor LexA that represses its response, binds to the group of inducible SOS genes and limits its transcription. After the event with high radiation rates has occurred, the co-protease activity of RecA is activated, due to the generation of a DNA of only one strand, either by the action of the nucleases or because the replication fork is Stagnant This single-stranded DNA (ssDNA) binds to RecA in the presence of ATP, promoting the formation of a nucleoprotein that separates the LexA repressor and induces the SOS genes, including the same RecA. Genes that weakly bond to LexA are the first to be fully expressed. If the damage persists or is very high, the concentration of the RecA protein increases, and other operons are affected by being bound to LexA. Normally, RecA is repressed at a basal level of 1000 molecules per cell. Once the LexA repressor is removed, it is rapidly increased by 20 times the amount of the protein (10 molecules per second), peaking at one hour from the occurrence of the damage event. Quantities of the RecA protein return to baseline levels within 4 to 6 hours, since the event. This decrease, causes the removal of signal-induced repair of DNA damage, eliminating the agent that activates the RecA protein, resulting in increased concentration of the LexA repressor. The repression of the SOS system is reestablished and the cell returns to its initial state. (Bianco & Kowalczykowski, 1998)

Why was it chosen to work on this project?

The interest in improving the radiation detection system for people working in the area of ​​Health was due to the fact that members of the group were interested in the idea of ​​studying Medical Technology or Radiology, so that most of them knew about the risks and protocols Which should be maintained in order to minimize the possibility of serious damage to the radiation emitted by the equipment used, such as the use of dosimeters, which measure the radiation exposed in the medium. But through research we discover a need in this area, because these instruments mentioned above are very sensitive to light, moisture and heat; Factors that would alter their results. In addition, the most important point was that there was no immediate delivery of information from the device, since, according to Chilean legislation, dosimeters used in laboratories are sent to review the SEREMI, monthly, quarterly and even semiannually, depending on the institution And that, in the event of an overexposure, it will be a late warning and damage will have already been caused. Adding information to this, the dosimeters of better quality currently have a very high value in the market, so not all laboratories can access these devices. Also, the lack of safety for radiologists and medical technologists also encouraged our team to create the system that would provide accurate information that will warn the professional who is in danger. The bacteria detects ionizing radiation at harmful levels and when this occurs it gives off a lemon scent and a violet pigment, alerting the specialist that he is in an environment with levels of radiation harmful to his body and also can evacuate the area in time to reduce the amount of damages possible