This year, our team employed the yqjF promoter to detect the aromatic compounds and increased its detection threshold by 250-fold in three ways, which will potentially help other iGEM teams in their projects. Our team has built a very practical hardware detection system that can be utilized in other applications if the promoter activation system is modified. We believed that our project can inspire many other teams.
The yqjF promoter, originally involved in the metabolism of aromatic compounds in bacteria, has also been reported to be activated by aromatic compounds from explosives, particularly 2,4-Dinitrotoluene (DNT) . Through mutagenesis-based optimization, our team has reduced the DNT detection threshold of the yqjF promoter by 250-fold in three ways.
Firstly, we generated point mutations in the -35 region of the yqjF promoter, and also carried out random mutagenesis in the entire promoter sequence. The obtained new version of the promoter reduced the DNT detection threshold from 25 mg/L to 5 mg/L, leading us to generate the third-generation promoter, yqjF3rd.
Secondly, based on previous reports, DNT can be metabolized to 2,4,5-Trihydroxytoluene (THT) catalyzed by three enzymes, including NemA, NfsA and NfsB. Importantly, THT is the important inducer that binds to the transcriptional factor YhaJ and activates its transcriptional activity of the yqjF promoter. To increase the DNT-to-THT conversion, we overexpressed the three enzymes NemA, NfsA and NfsB in the engineered bacteria. As a result, with the overexpression of the three enzymes, the engineered bacteria showed reduced DNT detection threshold from 5 mg/L to 1 mg/L.
Finally, by overexpressing the transcriptional activator yhaJ in the engineered bacteria, we reduced the DNT detection threshold of the yqjF promoter from 1 mg/L to 0.25 mg/L. Additionally, we optimized yhaJ through random mutagenesis and screened the generated mutants. Consequently, we obtained an improved transcription activator yhaJ1st, which could reduce the DNT detection threshold of the yqjF promoter from 0.25 mg/L to 0.1 mg/L.
Fig. 1. The thresholds of yqjF, yqjF3rd, yqjF3rd + yhaJ and yqjF3rd + yhaJ1st.
As shown in the Fig. 1, the threshold for detection of DNT was reduced from 25 mg/L to 0.1 mg/L.
With practical landmine detection in mind, our team has created an exciting device to achieve our goal. Our device consists of four parts: shell, circuit, bottom fan and the hydrogel on the interior wall. External DNT in the air is blown into the device by the bottom fan. Our engineered bacteria encapsulated by the hydrogel can absorb the DNT in the airflow on the surface of the hydrogel, leading to activated production of green fluorescent protein in the bacteria. We designed the 3D model diagram of the device and assembled it.
In addition, our device will not only be capable of detecting landmines, but also be potentially used in other applications, such as the detection of environmental pollutants, harmful metal ions in the soil, and drug deterrence of crime scene. For example, we can put a set of sensors responsive to drug metabolites in sewer pipes, and use the modified device to detect these drug metabolic chemicals, which can be used to provide evidence for illegal drug uses. We believe that biosensors will also have a bright future in the field of detecting different chemicals. Importantly, our design can be a great inspiration to other iGEM teams.