Part:BBa_K3598043

EilR_EilR promoter_PJExD promoter_mTyr-CNK_7*His

This part is for the expression of mTyr-CNK in our project. PJExD promoter is regulated by EilR, and several cationic dyes act as efficient low-cost inducers.

Background and Description

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_K3598043, which consists EilR repressor and PJExD promoter and uses cationic dye as inducer.

Figure 1. The overall design of BBa_K3598043

EilR promoter starts the expression of EilR, an repressor to PJExD promoter, so the expression of mTyr_CNK will not start spontaneously. Cationic dyes act as an inducer that blocks the repression of PJExD promoter, starting the expression of mTyr_CNK.
For purification of mTyr_CNK, we add 7*His tag.

Experiment and Results

We used LB medium to cultivate the E.coli BL21 chasis that contains our inducible system, and use 1μM CV as inducer, inducing the production at 25℃ for 24 hours. We get 100mL of the liquid produced. We dissociated and purified mTyr_CNK and used SDS-PAGE for authentication. W0 and W1 in Figure 2 are the unpurified liquid, which have notable strand of mTyr_CNK. E1, E2, and E3 are the liquid gained after first purification, second purification, and third purification respectively, in which the strands of E2 are comparatively intensive. We used BCA assay to measure the concentration of mTyr_CNK, which is 1.44mg/mL.

Figure 2. SDS-PAGE gel analysis of mTyr-CNK produced by our inducible system

Assembly Compatibility:

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

  Illegal BglII site found at 916
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 346
  Illegal AgeI site found at 496
  
• 1000
  COMPATIBLE WITH RFC[1000]