Difference between revisions of "Part:BBa K3594004"

Revision as of 17:24, 23 October 2020

Guaiacol Degrading Enzyme System

This composite part is used as the device to degrade guaiacol, one of the aggregation pheromones. It consists basic parts BBa_K3594006 and BBa_K3594014, which is respectively Cytochrome P450 (CYP) and Ferredoxin Reductase (FR), and Plux, which functions as a promotor.

The process we do in order to determine the expressing of protein

The analysis of SDS-PAGE of CYP

Lane 3 and 4 are plamsmids after overnight culture, and then diluted to M9 and cultured to an OD approximately equal to 0.6,which means that there is no induction. Lane 5 and 6 are plasmids adding IPTG for 3h under 37 degrees centigrade.

Pre-inoculum of PSB4K5_pTac_CYP_FR strains and control PSB4K5 strains were cultured overnight in M9 medium with 10 g/L glucose at 30 °C with orbital shaking at 220 rpm. Guaiacol assimilation experiments were carried out in 250mL shake flasks with 150mL of M9 medium and 1.5 ml inoculum in the same conditions described for the pre-inoculum with guaiacol 5mM. Aliquots were withdrawn regularly for measurement of optical density at 600nm (OD600). Then we used TPR_China’ NAHR sensor analysis of the concentration of guaiacol.

The experimental process and the comparison of fluorescence intensity between 0h and 28h

The degradation effect of CYP and FR on guaiacol in the absence (left) and supplementary carbon source (right)

OD values were measured after sampling at 0, 2, 4, 20, 24, and 28h after adding the final concentration of 5mM, indicating that the bacteria were growing normally after adding guaiacol.

After the reaction with NAHR Senor of TRP team, which is a sensor that can sense small aromatic molecules, the higher the fluorescence value was, the higher the guaiacol was, and the overall trend of our curve was downward, but there was a rise in the middle, which may be due to the induction effect of the products produced by the degradation of guaiacol. The specific metabolic mechanism needs to be further studied. Time is limited, and we will continue to experiment in the future.

The component diagram of NAHR

In the future, we determine the activity of enzymes with the synchronized lysis circuit (SLC) because we use this system to improve the degradation effect.

Sequence and Features