Project Safety
Potential experiment to escape frequency
Testing the escape frequency of our organism can be done in vitro. Because our kill switch is temperature dependent, we can setup an experiment in which we quantify colony growth at varying temperatures and time, compared to a baseline. From this a ratio can be determined at varying conditions. Our kill switch should differentiate between across 33°C. This is because we want our organism to kill itself outside of the body, which typically maintains a temperature of 37°C. From this we can setup growth conditions for body temperature, at, above, and below 33°C, and possibly other temperatures. We can monitor their growth to determine whether growth is inhibited at lower temperatures, which will allow us to determine an escape frequency compared to baseline, non-engineered strain. More specifically, the aim of our RNA thermometer is that it inactivates the antitoxin of a toxin/antitoxin system upon leaving the body (at a lower temperature than the average human body temperature). To make sure the lab can still detect the results, the antitoxin and the toxin will first be cloned in different plasmids. Furthermore, the toxin will be placed under the control of a tetracycline inhibitable promoter. Additionally, multiple stop codons are added flanked by a restriction enzyme site so that it can be removed after successful cloning. Before analyzing the sample, a buffer with anhydrate tetracycline is added to make sure the cell is only intact for diagnostic purposes. How do we determine effectiveness of kill switch? Grow the two constructs in different temperatures and see whether they took up the plasmid using flow cytometry. GFP should be translated if they took up the plasmid
Dose-dependent biosensor response
We will develop a whole-cell biosensor, using Lactobacillus as a model organism, to detect colon cancer in vivo and include a novel kill switch when the cell leaves the body. To achieve this, we will first try to make the constructs work in E. coli (strains DH5-α Z1, DH10-β, BL21) before transferring them to Lactobacillus spp. Most biosensors can detect cancer biomarkers. However, these are also expressed in normal cells in lower concentration risking non-specificity. Thus, our team develop a dose-dependent biosensor response based on split T7 RNA polymerase and the activation of a transcription factor with a membrane kinase receptor, that only reacts when the biomarker concentration reached a certain threshold. As our proof of concept, we are using NarX-NarL two component system and naringinen promoter system to control the dose dependency. In other words, our biosensor will ignore the naringenin concentration expressed by the normal cells and will activate its Shoot part at high concentration indicative of tumor or cancerious environment. Naringenin is the molecule we use as a proof of concept, once applied it would be replaced by the adequate biomarker. During this part the biosensor will produce a therapeutic or reporter molecule in order to attack the tumor. We use mKATE (red fluorescence protein) as a substitute of the therapeutic molecule that would be released in the final model, we do so in order to be able to measure the expression (fluorescence). However, since it will create a fluorescent signal, it can also be used for detection.The expression of this molecule is controlled by the NarX which depends on the concentration of naringenin, but also by the NarX mutant, inactive version of NarX. Hence, NarX must have a high enough concentration to express the downstream gene. In this way, we try to go over the hurdle of utilizing gut bacteria in patients which mainly is related to their lack in robust sensing mechanism.
