Team:Beijing 4ELEVEN/Improvement
Old Part：BBa_K3089006
Improved Part: BBa_K3598022
When required of an expression system of tyrosinase, we searched in 2019 Greatbay_SCIE's project StiKit and found their part BBa_K3089006, a coding part that is designed for "mTyr-CNK protein purification and in vitro/in vivo DOPA modification of all Mfp containing recombinant proteins". Although the part proved to be capable of expression and purification of mTyr-CNK and modification of Mfp-related recombinant proteins, its yield of mTyr-CNK is only 0.7mg/mL, which is not enough for us. Therefore, we aimed to boost its production of tyrosinase with the construction of an RBS library.
Figure 1. Overall design of improvement part
We began by randomly arranging six pairs in the system's RBS sequence TAAGTATAAGNNNNNNATAT, and verifying the resulting RBSs' strengths with RBS calculator (https://salislab.net/software/login). 15 of the strongest RBSs from verification is taken into our RBS library. These RBSs are then integrated into the expression system of mTyr-CNK through Goldengate, transformed into E. coli BL21 along with the original part BBa_K3089006 as control group. Then the strains were cultivated in LB medium with 0.5mM IPTG, 25℃, for 20 hours. Proteins purified and verified with SDS-PAGE and BCA assay.
Figure 2 SDS-Gel results of improvement part.
From the SDS-PAGE, we can clearly identify correct bands of mTyr-CNK (Figure 2). The yield of tyrosinase can be inferred from BCA assay results (Figure 3). In our library, 6 of 15 RBS library improved tyrosinase yield, and the highest reached 2.94 mg/mL, which was more than 4 times higher than the control group of 0.70 mg/mL.
Figure 3. Figure 3 BCA assayresults of improvement part.
