Research Optimization

Why a new fusion promoter?

For our first device, the detection circuit, challenges were presented in the promoter region. When we talk about promoters, the detection mechanism goes through transcription. Therefore, the presence of RDX would determine the production of the mRNA that results from the transcription of the entire device. Although the AlgD promoter from last year’s cycle was inducible under the presence of RDX characterization of such mechanism showed that many regulators of the alg operon were not confirmed present in the sequence used. This doubt made us search for homologous regulators and recognition sites in the E.coli genome. Since we were unable to determine which regulators were essential the team opted to search literature for new biosensors or promoters inducible by RDX. This led us to find the promoter of the hmp (nitric oxide dioxygenase) and hcp (high-affinity nitric oxide reductase) in the E. coli genes (Lifshitz et al., 2021). According to Lifshitz et al. (2021), when these promoters are fused in tandem (hmp::hcp) a higher and more sensitive response to RDX was observed. The paper states the detection limit in liquid media was 0.126 mg/L. Additionally, the authors state that the induction of the hcp promoter is likely driven by nitric oxide (NO) produced in the pathway of RDX degradation since the detection began simultaneously when the RDX began to decline. Although it is important to evaluate this behavior the team decided this promoter would work for the initial purposes. Moreover, if other elements such as a riboswitch are added to the device it would be more selective for the biosensing of RDX.

Riboswitch Biosensor

Another improvement that we plan on pursuing is the implementation of an aptamer-based riboswitch for our device. Riboswitches are non-coding mRNA that are capable of both recognizing a specific small molecule compound and regulating the transcription and translation of downstream genes (Garst et al., 2010). With this addition, we wish to have better control over the gene expression of our device. This detection mechanism is also translational where the aptamer adds a three-dimensional structure to the RNA that can interact with the ribosomal binding site (RBS). The RDX acts as a ligand and if there is no RDX bound to the aptamer binding domain a three-dimensional conformation prevents access of the ribosome to the RBS. On the other hand, if the ligand is present the aptamer conformation relaxes such as the RBS is made accessible and allows the ribosome to translate. This would improve regulation of the detection device since the action of translation would be dependent on the presence of RDX. According to Mayo et al. (2020), the results of their modeling of the riboswitch biosensor of RDX showed that longer exposure time will produce a higher response in presence of low levels of RDX. However, they also recall that challenges for implementation, such as rapid growth rates limit the use of the biosensor in the environment. Moreover, the riboswitch developed by Eberly et al. (2019) provided a semi-qualitative method for an RDX biosensor harbored in the E. coli strain with a detection limit of approximately 0.44 µmol l^-1. Although aptamer-based riboswitches pose their own challenges for environmental implementation, more research regarding this topic could lead to the solution to this incognitive.

Justification of the change of promoter T7-LacO Promoter

While characterizing the parts of our killswitch device it was observed that the second promoter T7-LacO Promoter part BBa_K2406020 is induced by IPTG (iGEM17_Edinburgh_UG, 2017) not only T7 RNA polymerase as originally stated in our Wiki page the past cycle. It is crucial for our promoter to be induced directly by T7 RNA polymerase since the genes before our desired promoter (supD and T7ptag) would be producing the inducible agent of said promoter. Part BBa_I712074 is a T7 promoter induced by T7 RNA polymerase (iGEM07_Ljubljana, 2007), this part is ideal for our device which will start operating in the presence of formaldehyde and nitrite.