Part:BBa_K3598022

LacI_LacI promoter_T7 promoter_lac operator_RBS lib_mTyr-CNK_T7 terminator

This part was improved from part BBa_K3089006--mTyr-CNK tyrosinase

This is the RBS lib construction system for mTyr-CNK expression. The T7 promoter is regulated by lacI repressor through lacO operator to control the expression of mTyr-CNK. The system is used to produce mTyr-CNK, so we construct a RBS library to improve the yield of mTyr-CNK.

Experiments and Results

We have improved the previous part BBa_K3089006 by using a series of new RBSs that differ in their expression efficiencies in expressing tyrosinase mTyr-CNK.

We began with randomly arranging six pairs in the system's RBS sequence TAAGTATAAGNNNNNNATAT, and verifying the resulting RBSs' strengths with RBS calculator. 15 of the strongest RBSs from verification are taken into our RBS library.

Figure 1. Overall design of 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.

All the strains were cultivated in LB medium with 0.5mM IPTG, 25°C, for 20 hours. 100 mL culture were collected for proteins purification. Then the products are verified by SDS-PAGE and quantified through BCA assay.

Figure 2. SDS-PAGE gel of our RBS library

In the results, we have succeeded in improving the production of mTyr-CNK. From the SDS-PAGE, we can clearly identify correct bands of mTyr-CNK (Figure 2). In BCA assay results , 6 of the 15 RBSs have improved the tyrosinase yield, the highest one is about 4 times to the control (Figure 3).

Figure 3. BCA assay of our RBS library

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2233
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1280
  Illegal BsaI.rc site found at 1270