Part:BBa_K4171018

Ptrc-melA

Background

This part is constructed for tyrosinase biosynthesis. pSB4KI-P_trc-melA was used to express MelA (BBa_K274001), the tyrosinase that is essential for melanin production.

Usage

This part is used to produce tyrosinase, an enzyme that catalyzes the oxidation from tyrosine to melanin. In our project, we used MelA to produce melanin. After the addition of selenocysteine (Sec), selenomelanin was expected to be produced.

Fig. 1. Melanin synthesis pathway

Characterization

The tyrosinase gene melA (BBa_K274001) from Rhizobium etli was synthesized and placed under the control of the P_trc in vector pSB4KI. The plasmid containing P_trc and melA gene was transformed into E. coli DH5α and completed the construction.

Fig. 2. Confirmation of pSB4KI-P_trc-melA (BBa_K4171018) by double digestion. M: Marker; Lane 1: pSB4KI-P_trc-melA without digestion (negative control); Lane 2: pSB4KI-P_trc-melA (6707 bp)

Fig. 3. The SDS-PAGE result of pSB4KI-P_trc-melA in MelA (67 kDa) expression with and without IPTG induction. M: Marker; Lane 1: With IPTG induction, whole cell; Lane 2: With IPTG induction, supernatant; Lane 3: Without IPTG induction, whole cell; Lane 4: Without IPTG induction, supernatant; Lane 5: Wild Type.

As Fig. 3 shows, there was slightly more MelA protein expression compared with wild-type DH5α, demonstrating that we had successfully expressed exogenous melA gene in E. coli.

After successfully synthesizing tyrosinase, the precursor tyrosine as well as the cofactor Cu²⁺ were added into culturing medium LBYT. After overnight incubation, we observed that the medium turned dark with brown and diffusible pigments gradually accumulated for days. Besides liquid culture, the bacteria were also cultured in agar plates with the addition of tyrosine and Cu²⁺, and it darkened as expected.

Fig. 4. Melanin production in E. coli DH5α. (A) Melanin production in LBYT agar plate. (B) Melanin production in M9Y2 medium (centrifuged cells)

Function test experiments were conducted to verify the identity of pigments in both solid and liquid medium. Melanized and non-melanized bacteria were put under UV-B and their survival rates were tested via CFU. The result shown below proves that melanin makes bacteria more tolerant to radiation.

Fig. 5. Survival rate of bacteria with melanin and without melanin (A) Survival rate (B) CFU comparison

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1421
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1273
  Illegal BsaI.rc site found at 578