Troubleshooting-Harnessing the power of transcription factors
To amplify the expression of a protein, the most common way is to directly over-express its correlated target gene. However, in dealing with nitrite reductase, we have learned that the nrfA gene is jointly regulated by the nrfABCDEFG operon. Simply over-express NrfA will only drain out the whole system. However, it’s also way too sophisticated to transform the entire operon.
Our team turns to a relatively novel perspective that utilizes transcriptional factors to boost the expression of the whole system. Specifically, by transforming a plasmid that could improve the expression level of NarP, the expression level of its downstream nitrite reductases could be promoted. According to our experimental results, engineered BL21 behaves with an enhanced nitrite-dealing ability though enzyme activity assay, marking the correctness and feasibility of our design.
In the future, other iGEM teams could use our part to construct E. coli that can metabolize nitrite at high levels in an oxygen-free environment and then study areas related to nitrogen metabolism. Our engineering success further proves the flexibility of controlling a protein expression by regulating its upstream transcription factors, offering a new perspective to applying synthetic biology.
Modeling - Playing with oxygen: Constructing a more comprehensive regulatory network to nitrite reductases
Expressing nitrite reductases of high efficiency and sufficient amount are of great significance in our project. The regulating role of oxygen-responsive protein makes anaerobic a nonnegligible prerequisite. To better understand how do FNR and CRP function to convert the E. coli from its anaerobic to aerobic metabolism to guide our experimental design, we analyzed the promoter sequences of two nitrite reductases and successfully unraveled all probable binding sites. Other researchers could refer to these steps to predict the motif binding sites of other regulatory pathways. Click here to see more details
At this stage, the regulatory pattern for two oxygen-responsive transcription factors on nitrite reductases is solved, allowing us and future iGEM teams to use oxygen more flexibly to achieve regulation. To begin with, our experiment explores several conditions to create a qualified anaerobic condition, which could be a referenced by future research. On the one hand, RNAi or directed-evolution could be introduced to change the function of these transcription factors, making it possible for further experiments to be conducted in aerobic conditions and therefore simplify the experiment requirements; on the other hand, we could utilize this property in the opposite way. Transforming motif binding sites of either CRP or FNR to the bacteria could make the oxygen a threshold to control the expression of the target proteins, which increases the safety of the engineered organism as well as making the experiments more controllable.
Here we show an attempt to alter the function of FNR:
Hardware - A combination of Arduino-based electrochemical detection system and screen-printed electrode sensing system
Our sensor design could be divided into an Arduino-based electrochemical detection system and a screen-printed electrode sensing system. For point-of-care testing, electrons produced from nitrite reduction could directly transfer to the screen-printed electrode surface. We further utilize the simple Arduino-based electrochemical station to perform cyclic voltammetry to generate the electrical signal readout. The “nitrisensor” has shown fundamental selectivity for nitrite detection based on our experiments. The successful construction of our sensor sets a model for small-quantity detection.
Biosensor – Cell coating
Our project innovatively immobilizes E. coli on the SPE surface, bringing several nonnegligible advantages.
a) It simplifies the procedures to extract the nitrite reduction enzyme and reduces cost.
b) It could improve detection efficiency and allow for continuous enzyme expression during detection.
c) Immobilized cells help maintain enzyme activity, prolonging the product lifetime and facilitating storage.
Safety Concern
Our design also prevents the accidental spread of the engineered bacteria by using the hydrogel since the entrapment material could not provide any nutrients to support the survival of cells and can be regarded as a time-dependent suicide system.
Software – Leading a more intelligent lifestyle
Considering the probable but invisible existence of nitrite in water and food that everyone may encounter, this project points out the importance of portable nitrite detection. It provides a feasible method that makes people could take control of their food security. Put the test sample on the sensor, and the detection result and the following suggestions appear on the application. Using the power of synthetic biology and biomedical engineering, our “nitrisensor” leads a step toward a more intelligent, proactive, and environmentally friendly lifestyle.
