Description of the project

This project consists in modifying a bacterium called Escherichia coli, through synthetic biology, which is based on the creation and modification of organisms that already exist, managing that they have the characteristics that we want. These organisms will sense the high levels of alpha and beta particles, gamma and x-ray emitted, factors that cause a mutation in the DNA of the already said bacterium, which will give a purple coloring and a lemon scent.

To validate that the investigation is necessary and it is supported by different specialists in the area, we made a survey to radiologist’s students, medical technologists, and workers of radiological centers, obtaining an 83% of approval about the need of better dosimeters with trustworthy results.

The sensor and its parts

For this, through a scientific investigation, a sensor was designed, which is based on a biphasic switch, device that can be turned off or on according to the concentration of the input. This switch naturally regulates lambda phage, which in this case, when the pRM promoter is activated, gives a rise to the transcription of the Cl repressor, which in turn will provoke inhibition of the Pr promoter and will not be able to transcribe 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 ceases of being activated, giving a rise to pR (promoter) activation and to the transcription of Cro, LuxR and LuxI. (Plasmid 1)

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, it behaves like a plasmid taking advantage of the enzymes of the recombination of the bacterium integrating itself into 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 duplicate E. coli. The virus synthesizes (object) 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 chain reaction that causes the virus is integrated into lithic route. It is here where the cell is usually infected, producing viral particles that are released into the medium, once the host bacterium is lysed the cell membrane breaks, 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 given, 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 signal for our specialist. (Plasmid 2)

The operation of the circuit was taken from the work already done by a 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 the SOS genes is controlled by the LexA repressor that represses its response, binds to the group of inducible SOS genes and limits its transcription. After the event with high levels of radiation has occurred, the co-protease activity of RecA is activated, due to the generation of a single-stranded DNA (ssDNA), 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 repressor LexA and induces the SOS genes, including RecA. Genes that weakly bind 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 linked to LexA.

Normally, Reca is repressed at a basal level of 1000 molecules per cell. Once the LexA repressor is separated, is rapidly increased by 20 times the amount of the protein (10 molecules per second), reaching its maximum in an hour since the damage event occurred. Quantities of the RecA protein return to basal levels within 4 to 6 hours since the event. This decrease causes the elimination of signal-induced repair of DNA damage by removing the agent that activates the RecA protein, resulting in an increase of the concentration of lexA repressor. The repression of the SOS system is restored and the cell returns to its initial state. (Bianco & Kowalczykowski, 1998)

Why did we choose to work on this project?

The interest to improve the system of detection of radiation for people who work in the health area, it was mainly because there were some students of the team who were interested in the idea of studying medical technology or radiology, whereby, the majority know the risks and protocols of security which have to be maintained to minimize the possibilities of suffering serious damages due to the radiation emitted from the usage of medical devices, such as the utilization of dosimeters, which measure the radiation of the medium. Thanks to this investigation, we have discovered that this area is the one that needs more help, because the devices already mentioned are very sensitive to the light, humidity and heath, factors that will modify the results.
Also, the most important point was that there was not an immediate delivery of information from the device, because, according to the Chilean Legislation, the dosimeters utilized in the laboratories have to be checked for the SEREMI, monthly, quarterly or even biannually, depending on the institution. And, in the case of the overexposure, will be a late advice and there will be a damage. In addition to information, the dosimeters of the best quality actually have a very high value, so not the most of the laboratories can be access to the devices.
In addition, the empty in the security for the radiologists and medical technologists, incentivized the team to create the system that will provide accurate information that will warn if the professional is at risk.
The bacteria detect ionizing radiation in harmful levels, and when this happens, a lemon scent and a purple coloring is emitted, warning the specialist is in a medium with harmful radiation for the body, and to evacuate the zone in time to minimize the quantity of possible damages.

Parts utilized in the project