Team:Lethbridge HS/Results

Results

 Part Development

We decided to prioritize the assembly of one construct, the melanin construct, because black is the most commonly used ink colour. We designed a g-block to contain assembled promoter, RBS, melA and terminator parts, including the BioBrick prefix and suffix sequences. We first cloned that into the pJET plasmid using the ligation kit from Thermo-Fisher Scientific. We chose this method because this kit is very efficient and it would improve our chances of successful assembly. We did succeed in inserting the melA construct into pJET (Figure 1).

Figure 1. 1% agarose gel of melA_pJET digested with EcoRI and PstI. Plasmids 1 and 3 had the melA construct successfully insert into pJET without disruption of the cut sites.

We then proceeded through cloning methods to finally insert the melA expression construct into pSB1C3 for submission to the iGEM registry. Were able to generate two successful clones (Figure 2, melA_pSC1C3 5 and 6, boxed). One of these clones was sequence confirmed and submitted to the Parts Registry (BBa_INSERT LINK).

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.

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.

 Biological Pigment Ink - Proof of Concept

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