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_K2481108).
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
When we were investigating what our proof of concept should be, we talked to the business liaison at the University of Lethbridge Greg Vilk, and he proposed the "simple showcase". He explained that in order to show that our project can work as intended, we need only to prove the concept in the easiest way (for more information concerning our interview with Greg Vilk, click here). For our proof of concept, we intend make an ink with pigments extracted from an alternate source and put them into fountain pens. We bought common supplement tablets (Lutein tablets, which contain lutein and zeaxanthin and cranberry extract tablets, which contain anthocyanin). We attempted an extraction of the pigments using either acetone or water (Figure 4). Both solvents were able to extract pigment, but they resulted in different shades and intensities of colour. (Figure 4 and 5). Guar gum was added to a final concentration of 0.5% to increase the viscosity of the ink and help it bind to surfaces. The guar gum thickened the water based inks, but precipitated in the acetone based inks (Figure 2).
Figure 4. Extraction of pigment from lutein and cranberry tablets using water or acetone. (A) Acetone extraction of red pigment from cranberry tablets. (B) Water extraction of red pigment from cranberry tablets. (C) Acetone extraction of pigment from lutein tablets (contain lutein and zeaxanthin). (D) Water extraction of pigment from lutein tablets. Pigment did not mix well with pigment and caused the contents to coat the tube. The water based solution colour is shown in the petri dish.
Figure 5. Ink solutions prepared from acetone and water extractions of cranberry and lutein tablets. Guar gum was added to the solutions to a final concentration of 0.5% and allowed to set. Water based inks became thick and more opaque than the acetone based inks. Guar gum precipitated in the acetone based inks.
Finally, used a syringe to draw a line with each ink across Whatman paper (Figure 6). The acetone based inks were more prone to bleeding than the water based inks. The water extraction of the cranberry tablets yielded a more vibrant colour on paper than the acetone extraction, and the water extraction of lutein/zeaxanthin was more uniform in colour an darker than the acetone extraction.
Figure 6. Demonstration of the inks application to Whatman paper.
Using the ink in old fountain pens was attempted, but we found contamination with previously used ink in the pens were interfering with our results. The creation of our ink for use in fountain pens does not need to be as precise in terms of viscosity as it would need to be for printers. This is because there are no mechanical or moving parts or significant movement of ink within the pen, all we need is for the ink to be able to come out while keeping the amount of solvent as low as possible to prevent bleeding of the ink. This allows us to just attempt a few proportions of pigment, resin, and solvent for the pens.