Our final product from the cells will be the pigments we require to make ink, but before we can use them we must extract them from the cells. We will be producing the pigments in the cells using a method called large batch overexpression. We will have large amounts of liquid LB media in which we will have our cells with the constructs in them. We will let the cells replicate and mature before we induce the pigment production with IPTG. The way we will be extracting the pigment is via organic extraction. We will first centrifuge the cells and the liquid LB broth media in order to get the cells and pigment in the pellet. This will allow us to pour off the supernatant which will be the media to get the cells to work with. We will then add the solvent for the organic extraction and depending on the pigment it will be either acetone or methanol. This will lyse the cells as well as have our pigment dissolved in it. We will then homogenize the mixture in order to destroy all of the rest of the cell membranes and any living cells. This homogenized solution will then be put through a series of filters in order to get out the larger proteins and all of the parts of the cells, this will leave us with pigment and the solvent and all that is left is the evaporation of the solvent.
Figure 1: This image shows the process of our pigment extraction from the cells starting with live cells with pigment inside of them and ending with our pigment.
Now that we have our pigment isolated from the cells that produced it, we need to add it to the other components of ink to allow us to use it. The Three main components of ink are the Resin the Solvent and our portion, the colorant and in our case pigment. For our resin and solvent, we have selected gum arabic and acetone as our research revealed that these are some common materials that will perform as necessary for a proof of concept. The resin must evenly spread the colorant throughout the ink and across the substrate. The solvent contains the other parts and allows for the ink to be stored in liquid form as well and it will evaporate rapidly as they are volatile and will leave the resin and solvent on the substrate. If we were to ever apply our pigments to printer ink we would need to very carefully control the proportions of pigments resin and solvent as printer require a specific and controlled viscosity in order to correctly use the 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 we do a "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 2). Both solvents were able to extract pigment, but they resulted in different shades and intensities of colour. (Figure 2 and 3). 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 2. 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 3. 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, we used a syringe to draw a line with each ink across Whatman paper (Figure 4). 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 4. 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.
Quality Control of Ink
After our interview with Dave Warwick of Warwick Printing in our hometown Lethbridge AB, he raised a few questions about the quality of products he uses. He stated that a few of the qualities a consumer is looking for in ink are: consistency in color, a variety of colors, lack of bleeding and bleaching, and the cost. For more information concerning the Warwick tour and interview, click here. We intend to solve or address all of these qualities and hopefully surpass the existing inks in any and/or all of them. For the consistency of color, we intend to make sure that we have added the correct proportions of pigment to resin to solvent and to mix thoroughly to ensure an even spread of all substances in any amount of ink. For color variety, we addressed this issue in our research, we have made pigments that correspond to the CMYK in any color printer. CMYK stands for cyan, magenta, yellow and black and these four colors can make any color in existence. To address bleeding we will be testing our produced ink on many substrates to ensure that the volatile solvent evaporates before the ink can bleed through the substrate, and if there are any problems we will need to change the proportions of our ink. For bleaching we will test our pigments against current inks in a UV Photobleaching test, in which we place our pigments on a substrate and place it under extreme UV light, if it is good then it will not fade over time. to address cost we will need to investigate the cost of large-scale production of our pigments in order to see how much it would cost.