Team:Beijing 4ELEVEN/Engineering
To achieve co-expression of adhesive/cohesive protein and tyrosinase, we need a new inducible system different from T7-LacI inducible promoter to control the expression of tyrosinase. Thus, we constructed BBa_K3089042 and BBa_K3089043, which consist EilR repressor and PJExD promoter and use cationic dyes as inducer.
Figure 1 The overall design of BBa_K3598042 and BBa_K3598043.
We first tested the inducible system through the expression of sfGFP and measured fluorescence with a microplate reader. Seven cationic dyes AO (Acridine orange), MG (Malachite green)，PB (Pyronin Y), MB (Methylene blue), VB (Victoria blue R), CV (Crystal violet), and NR (Neutral red) were used as inducer with the concentration gradient of 0, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50 μM separately. Since the effective concentration of NR is relatively high (more than 10 μM), the concentration gradient of NR was 0, 0.1, 0.5, 1, 5,10, 20, 50, 80, 100 μM.
Figure 2 Fluorescence of E. coli BL21 with BBa_K3598042 using different dyes as inducer with growing inducer concentration.
We used 1 μM CV as inducer for the expression of tyrosinase. We added 7*His at the end of tyrosinase, then extracted and purified it from the cells. The protein was verified by SDS-PAGE and quantified through BCA assay. From the results, we can see that under the induction of 1 μM CV, the gel band of tyrosinase is correct and the expression level of tyrosinase reached 1.44 mg/mL.
Figure 3. SDS-PAGE gel analysis and BCA assay of BBa_K3598043 using 1 μM CV as inducer.
