The Part
Modified NarK promoter with extra half-binding site: BBa_K4735006
Our designs tested 3 promoters, narGp, narKp and dmsAp each modified to possess an extra FNR half binding site [TTGAT] (1.5 total binding sites each) with the intention of increasing FNR sensitivity to maximize yield in low O2 conditions (with FNR being deactivated in >2% oxygen).
In our e.coli, the fnr gene is has a constant base rate of transcription, allowing a consistent amount of FNR to roam within the cytoplasm and hence either upregulate gfp transcription in low oxygen levels (by binding to a promoter), or be deactivated in high oxygen and cease upregulation. When FNR is exposed to high levels of oxygen naturally, it will break down and not bind to the FNR binding sites, whereas when desired levels of oxygen are present, FNR will bind to its binding site and act as a medium for RNA polymerase to locate and start translating the following genetic code (gfp in this case). This will hence increase the unit of superfolder gfp synthesized which can be measured by monitoring the frequency and intensity of fluorescence in colonies under certain uv conditions.
Thus the prediction was that all three promoters would behave this way in regulating transcription, however experimentally NarG exhibited no sensitivity to oxygen whereas NarK and DmsA were roughly as expected (A, B, C is NarG, NarK, DmsA respectively)
Our experiment thus showed that FNR was shown to be successful as an oxygen sensor with promoters B (NarK) and C (DmsA) whereas it showed no O2 sensitivity in promoter A (NarG). Also, for the purposes of our project we’ve determined that promoter B (NarK) performed best as, between it and promoter C (DmsA) it exhibited more stable, consistent fluorescence across different oxygen intervals with a more compact spread of results than promoter C (DmsA) between the biological and technical replicates.
Therefore testing indicates our basic part, a modified NarK promoter, responds very well to oxygen as it shows no GFP fluorescence anaerobically or aerobically but gives GFP fluorescence at 0.5% oxygen and 2% oxygen.
Importance to the Community
This new basic part has significant usefulness to both the iGEM community and wider society.
The success of the NarK and DmsA promoters as effective biosensors to oxygen opens up a wide range of new possibilities for future iGEM teams. An oxygen biosensor could be applied to a vast variety of designs, such as bioreactor monitoring or metabolic engineering of bacteria, some frequently explored in iGEM. The basic part could also be combined with other parts, to develop and continue on a similar vision of an effective fertiliser agent, which future teams could work on combining the oxygen sensor with a carbon source sensor. Ultimately, the oxygen sensor is something that can be utillised and applied in many different situations, to benefit future generations of iGEM.
Furthermore, we see this new part as being able to be applied in wider scientific research and medical advancements. Oxygen biosensors can be integrated into these synthetic organisms to monitor environmental parameters, such as oxygen concentration, and provide insights into environmental health and pollution. In the medical field, these NarK and DmsA promoters could be employed in synthetic systems to detect and respond to hypoxic regions within tumours, which are often resistant to traditional cancer treatments. This opens up a potential for a new way of identifying cancerous tumours for early intervention
