We used the T7-lacO as a promoter because it is the easiest to control. The RBS is our binding site. TBG and TTR are the genes that is going to codify our transporter proteins. We are planning to replace T7-lacO with an estrogen promoter in a later version of the project, but we chose to use T7-lacO because it is a commonly used promoter and we already know the way it works.
Figure 7: operon including promoter T7-lacO BBa_K1402010; RBS BBA J61110; the codifying gene for the protein TGB/Serpin A7; and terminator BBA_B0015.
Figure 8: operon including promoter T7-lacO BBa_K1402010; RBS BBA J61110; the codifying gene for the protein TTR; and terminator BBA_B0015.
What Are Alginate Beads?
Alginate beads are made of sodium alginate and calcium chloride. The alginate is extracted from the seaweed and broken into pieces before being blended with an alkali solution. The mixture has to be filtered afterwards in order to separate the alginate from the cellulose and remaining residues. The product is a sodium alginate powder that in contact with calcium chloride acquires a gel like texture. This happens because the Calcium ions crosslink the polymers in alginate and gradually give the mixture a more solid physical state.
Figure 9. Jelly Beads! Procedure. Extracted from http://www.omsi.edu/sites/all/FTP/files/chemistry/U3JellyBeads.pdf
How Are They Made?
In this case the protein would be put into a mixture with the alginate in a micropipette that will drop the solution into a beaker with calcium chloride in constant motion. The size of the bead will be regulated either by modifying the pipette or adjusting the distance from which it is dropped. There are various technologies that can be used in the production of the beads. Jet cutter, vibration, electrostatic technology and coaxial air flow are the principal ones. There can also exist other arrangements like the different landings in the case of Jet cutter.
Normal landing is a 90 degree fall and soft landing means a diagonal throw with low risk of deformation.
The parameters to compare the different technologies shall be the size and the shape of the beads as well as the amount of beads produce and the efficiency of each method at different mass percentages of alginate depending on the amount of beads required.
The most efficient in terms of production of all the methods is the Jet Cutter in the normal landing mode, although it has the disadvantage that it won’t produce beads until there is a 1% concentration of alginate unlike the other methods that only require .5%. The jet cutter technology widely surpasses the others in bead production, size of the beads and in the case of the soft landing, even though a bit efficiency is sacrificed the product has a higher quality and it is still more efficient than the other methods. In lack of a cutting tool vibration has a good production rate and does not require as much material.
Alginate bead production. Example of electrostatic stainless steel bead production. Ltd, Raffles Bioscience Pte. Raffles Bioscience Pte Ltd. 2016, http://www.rafflesbioscience.com/products-applications/product-1/296-2.
What Can They Be Used For?
This can also be done with other gel like technologies, the most common is polyacrylamide because as it is not ionic, the change in the enzymatic properties is minimal although the materials required are really toxic and that is why calcium alginate beads are the better choice. The retention of original enzymatic activities is between 25 and 50% in relation to the original. The enzymes are not consumed by the reactions so they can be reused as long as the enzyme remains active. The gel also adds resistance to changes like pH and temperature to the enzyme. The use of entrapment technologies such as calcium alginate beads block the exit of reaction products.
The immobilization is used widely nowadays because the enzymes have a more stable operational capacity and it can be used as a filter, the enzyme can be easily removed from a product, an example of this is found in the production of lactose free milk.
Figure 10. Enzyme immobilization Research Lines. http://www.franciscoploulab.eu/p/research-lines.html. Accessed 7 Oct. 2016
TBG and TTR intact structures Manager, Thyroid Disease. Thyroid Hormone Serum Transport Proteins - Thyroid Disease Manager. Thyroid Disease Manager, 7 June 2015, http://www.thyroidmanager.org/chapter/thyroid-hormone-serum-transport-proteins-2/
6.How are We Planning to Use the Alginate Beads With the Protein?
In the process of creating the alginate beads the human proteins TBG and TTR would be put into mixture and immobilized within the calcium alginate gel beads. This protein would bind to toxins released by plastics (BPA, phthalates, PCB, PBDE). Taking advantage of the entrapment process properties, the toxins would react with the enzyme and then they would be stored inside the bead. It would work as a filter and the beads are to be replaced periodically.
In order to create the bioremediator, the bacteria will enter an alginate pearl, where the protein and the bacteria will remain inside. The alginate pearl is a semi permeable barrier that will allow the toxins inside it. The proteins will bind to the toxins inside the pearl and won’t let them go. Therefore, when the bacteria are killed, the water will be decontaminated.
Left imgage first trail. Right image final trails.
We tried different concentrations of calcium chloride (CaCl2) to see which one gave the best results. Using natural dyes, we compared the amount of dye contained inside the pearls after a day. In our first trial, the pearls lost all colour after the first day. Afterwards, we tried with a higher concentration of CaCl2 and different percentages of alginate: 2.5 M CaCl 2%2 , 1 M CaCl2 2%, and 2.5 M CaCl2 4%. The ones that retained the most dye were the 2.5 M CaCl2 4%, but the alginate pearls still let the dye go through, proving to be a semi-permeable barrier. We will use this concentration in our project because it has shown to get the best results. The decision to use these pearls is based on the biosensor developed by.