Part:BBa_K2481108
MelA expression construct
The melA gene is coding sequence of tyrosinase from Rhizobium etli.This tyrosinase converts tyrosine to dopaquinone. Dopaquinone is intermediate product of melanin biosynthesis pathway that will polymerizes in an enzyme-independent reaction to form melanin. Melanin production in E. coli requires supplemental tyrosine and CuSO4.
The part for the melA CDS was originally made by Kazuaki Amikura of the iGEM09_Tokyo_Tech group (2009-10-14, BBa_K193600). Previously, it had been used in an expression construct using the P(lac)IQ promoter (BBa_I14032) and B0040 RBS (composite part: BBa_K193602). We have developed a different expression construct specifically for use in E. coli BL21(DE3) which takes advantage of the T7 RNA Polymerase expression construct in the DE3 cassette. This will provide tighter epxression control and help to prevent leaky expression of the tyrosinase. Additionally, the double terminator B0015 was added to increase control over the system. In order to maximize production of the tyrosinase and limit unnecessary energy expenditure by the cell, a transcriptional terminator will ensure energy is not wasted on transcribing an overly-long mRNA transcript.
"Figure 1. The pathway from L-tyrosine to Melanin with the use of the melA gene.
We have shown successful incorporation of the melA expression construct into pSB1C3 that we have submitted to the Registry.
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Figure 2. 1% agarose gel of a colony PCR amplifying the melA and indB construct inserts from pSC1C3. Successfully amplified melA and indB bands are boxed.
We transformed the melA_pJET plasmid into E. coli BL21(DE3) and attempted to overexpress the MelA tyrosinase (~54kDa) and produce the pigment melanin. Four colonies from the transformation were picked and used to produce pre-cultures, which were then used to incoulate test expression cultures. During the test expression, cultures were also supplied with CuSO4 and extra tyrosine, as tyrosine is the substrate for MelA and Cu2+ is a cofactor of the enzyme. Cultures were induced with IPTG at OD600 ~1 and a 1OD sample was taken (T0). Another 1OD sample was taken after the cultures were left to grow overnight (TON). The cultures were allowed to grow another three days (supplemented with tyrosine and ampicillin) to see if pigment would form, but we were unable to detect any melanin. The 1OD samples were run on a 12% SDS-PAGE to check for melA overexpression (Figure 3). The MelA tyrosinase is ~54 kDa in size. A faint band of approximately 54 kDa appears in the TON lane of culture 3. This indicated that we were successful in expressing the MelA tyrosinase from the pJET plasmid. Before the Jamboree, we will attempt another overexpression of MelA from the pSB1C3 plasmid.
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Figure 3. 12% SDS-PAGE of the overexpression of the MelA tryrosinase from melA_pJET. MelA expression can be observed in the TON lane of colony 3.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1390
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1242
Illegal BsaI.rc site found at 547
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