Part:BBa_K4773006
RBS-ycnE
This part is a gene encoding the ycnE enzyme from E. hirae GDIAS-5, which can oxidize indole to indigo in the presence of oxygen and NADH. This part can be used to construct biosensors for indole detection or bioreactors for indigo production. The part also contains a strong bacterial ribosome binding site (RBS) (BBa_B0034) to ensure efficient translation of the ycnE gene.
Figure 1. SDS-PAGE verification of target proteins FMO (53.1kDa, A) and ycnE (14.5kDa, B). MW： Marker, S: Supernatant, P: precipitate, Control: empty vector.
We incubated the engineered and control bacteria (i.e., E.coli transformed with an empty vector) overnight, centrifuging them and discarding the supernatant, and then re-suspending them in LB medium with or without 1 mM indole for the assay to validate the enzyme's function. The results showed that our engineered bacteria could effectively degrade endogenous indole produced by E.coli and indole from the external environment and produce isatin.
Figure 2. Isatin production (A) and measured yields (B) by engineered bacteria in LB medium with or without 1 mM indole addition. Control is the E.coli introduced with the empty vector, ycnE is the engineeredE.coli expressing FMO and ycnE, (+) indicates exogenous addition of 1 mM indole.
We assayed the enzyme activity of the ycnE variant in the same manner as before. After culturing the corresponding bacteria overnight, centrifuge and discard the supernatant, then resuspend in M9 medium containing 1mM indole and culture with shaking. After 24 hours, samples were taken to detect the isatin concentration, thus determining the enzyme activity.
Figure 3. Isatin production of wild type and mutant ycnE. The concentration of isatin was determined after 1mM of exogenous indole was added and cultured at 37°C for 24h.
Figure 4. Color of supernatant indigo red of ycnE wild type and variants after 24h incubation with 1mM exogenous indole.
The results showed that ycnE-E54S exhibited the highest enzyme activity and could produce up to 0.258mM isatin when exposed to 1mM exogenous indole. Consequently, we utilized the ycnE-E54S variant in subsequent experiments.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| protein |