Part:BBa_K4171019

Ptrc-melA-B0015

Background

This part is for tyrosinase biosynthesis. pSB4KI-P_trc-melA-B0015 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 which catalyzes the oxidation from tyrosine to melanin. In our project, we used MelA to produce L-dopaquinone. After the addition of selenocysteine, 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 pSUI. By adding a double terminator (BBa_B0015), it was expected to reach a higher production of tyrosinase. The plasmid containing P_trc, terminator B0015 and melA gene was transformed into E. coli DH5α and completed the construction.

Fig. 2. Confirmation of pSUI-P_trc-melA-B0015 (BBa_K4171019) by double digestion. M: Marker; Lane 1: pSUI-P_trc-melA-B0015 without digestion (negative control); Lane 2: pSUI-P_trc-melA-B0015 (5505 bp).

Besides the addition of a terminator, there were a few experiments conducted to reach a better production of melanin. To maximize the production of melanin, we cultured these two strains in two kinds of medium, LBYT and M9Y2, respectively. Melanin production was measured by OD₄₀₀ and the result is shown below.

Table 1. Melanin production in each condition.

Fig. 3. Culture condition and result.

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