Team:RHS-Calgary/Experiments
Experiments
Experiment 1:
Testing Hydrogen Peroxide with the Colorimetric Assay Kit
In the very first steps of our experimentation, we began by fabricating an outline to test each of the components of our project at their most fundamental levels. Prior to attempting to conduct any genetic modifications, or making our design more convoluted, to create our fully fleshed out desired outcome, we did extensive research to pin-point a reaction that best suits our required reactants and products. With this, we also had to ensure that we target an enzyme that is substrate specific for Beta-D-Glucose. Once our research had been completed, we discovered that our forthcoming experiments will be solely based on the following reaction: B-D-Glucose + O2 → H₂O₂ + D-Glucono-1.5-lactone, catalyzed in the presence of an enzyme, Glucose Oxidase. The H₂O₂ produced will then form a secondary biochemical reaction with a working reagent kit containing the dye, Xylenol orange. As mentioned, our first step was to ensure our purchased kit produces a sufficient reaction when exposed to hydrogen peroxide. With this, we created 3 different solutions of H₂O₂ combined with Working Reagent, in different concentrations. These solutions were composed of 0.03% H₂O₂, 0.3% of H₂O₂, and 0% H₂O₂ (which served as a control), respectively. This test was conducted in two, 6-well plates, with 1 trial of each of the variables in both plates.
Experiment 2:
Testing the production of hydrogen peroxide through GOx
After ensuring that the individual components of our kit yielded the results that we were seeking, we set out to incorporate our enzyme into our reaction to begin testing the efficiency and success of the enzymatic production of H₂O₂. As the enzyme we are currently using is manufactured as a commercial product, its packaging states that at a minimum, a concentration of 26 units/mL is required to produce a reaction under highly specific conditions. Due to this, we were required to conduct various complex stoichiometric and mathematical calculations to determine the precise volume of GOx required to produce a reaction. In the calculations we ensured that we considered all factors that were at play. In such scenarios, mathematical and stoichiometric computations are extremely crucial in applications to synthetic biology, and they are methods frequently used in a majority of labs. In the end, we layed out a design that allowed us to have varying concentrations of GOx to test its efficiency and rate of activation energy required to produce a reaction. The varied amounts of GOx, however, mainly aided in creating dilutions that would produce 1 unit of GOx per 1 micromole (µmol) of solution. Our final volumes of GOx in a 1unit/1µmol sample were 85µL, 128µL, and 267µL. We began this experiment by making two solutions of glucose, one with a higher concentration than the other, so we can begin to form a colour gradient that is indicative of the different amounts of glucose that could possibly be present in a cat's urine. We created a solution of 5000mg/L of glucose, and a second solution containing 8000mg/L glucose, both of which were required to be in a final volume, with the added GOx and Working Reagent, of 50mL. Then, we proceeded by calculating the volume of working reagent required to produce the desired final volume of solution. The kit our team ordered for the reagent, from Thermo Scientific, contains a Working Reagent A and a Working Reagent B. In order for the product to function correctly, a ratio must be upheld between reagent A and reagent B, stated as 1µL:100µL, respectively. With this, we were also required, by the instructions of the kit, to ensure a secondary ratio of 10 volumes of working reagent to 1 volume of sample, which we classified as 1mL W.R.:100µL sample, after further stoichiometric calculations. Next, we prepared the working reagent solution according to Thermo Scientific's Kit Protocol, with 110µL of Reagent A and 11000µL of Reagent B. The last step in the process was to recalculate the volume of each of the three main components required to obtain a final volume of 0.05L in each well in 2, 6-well plates, which can be seen in Figure 1 below.
Figure 1: Chart demonstrating an experimental design set-up to test the reaction between GOx, W.R., and Glucose.
Experiment 3:
Testing the reaction between WR, GOx and Glucose, with multiple control trials
One of the main experimental designs our team has fabricated thus far allowed us to ask the principle question: can we detect high levels of glucose through a visible colour change with the use of GOx and a colorimetric peroxide assay? Having been able to say yes to this fundamental question allowed us to then research precise concentrations of glucose in the urine of diabetic cats, as provided to us by vet technician, Ms. Emily Chorwood. We conducted this experiment multiple times, each time in two 24-well plates with two trials of each variable in both plates. The purpose of this experiment was to produce a colour gradient that illustrates the concentration of glucose in a minimally diabetic cat's urine, to a severely diabetic cat's urine. This range of concentrations initially included, in order from minimally diabetic to severely diabetic, [2.8M], [5.5M], [14M], [28M], and [55M], where ‘M’ refers to Moles/Litre. Once we completed performing the above experiment, we narrowed down our variables by simply using the lowest concentration of glucose ([2.8M]) and the highest concentration ([55M]), as we needed to solidify our proof of concept, ensuring that our negative controls do not change color. With a new goal in mind we asked ourselves: Can we prove that we can successfully trigger a colour change in our positive controls and not our negative controls when we combine our biological components of Glucose Stock Solution (5000mg/L Glucose combined with Synthetic Urine to create a [55mM] concentration of Glucose), Synthetic Urine, GOx and Working Reagent under certain conditions and in particular dilutions? To answer this, we set out to create an experimental design which we referred to in our processes several times, which can be seen below in Figure 2.
Figure 2: Chart demonstrating an experimental design that tests the maximum and minimum concentrations of glucose and all possible controls.
Experiment 4:
Creating a Plasmid Construct and Protein Purification
overview
Our team's ambition is to assemble our own plasmid and create a construct that can be contributed to iGEM, using the genome sequence of a GOx enzyme. We acquired a His-tagged sequence of GOx from independent research studies that an Isreali iGEM team previously conducted. The plasmid we construct will then be transformed into BL21-E.Coli bacterial cells. This particular strain of E.Coli is specifically engineered to efficiently express high levels of protein. After the transformation of BL21 with the constructed plasmid, the cells will be induced, and through the processes of transcription and translation, a GOx protein will be synthesized. Finally, we will conduct a protein purification protocol to isolate the GOx protein. Once we have genetically engineered the protein, we will also develop our own colorimetric peroxide assay kit.
parts and resources
Initially, we intended to order Wild-Type (WT) GOx, a part fabricated by iGEM Manhattan College. Due to the fact that it no longer exists on the iGEM database, our team decided to attempt to re-create the part and hopefully re-enter it into the database for future use by other iGEM teams. With this, we intend to construct our plasmid, a process most commonly performed through in-vitro digestion of DNA fragments using endonucleases (restriction enzymes), which are specific to certain restriction sites. The resulting fragments are then re-connected by a ligase enzyme. At that point, we will still have to manufacture the insert itself, as we were unable to order it, and we will then add it to our plasmid. As mentioned, our construct will have to be created in two separate components; the fundamental plasmid will be received from iGEM and the insert is from another plasmid, tailored to our desired endonucleases, retrieved from Integrated DNA Technologies (IDT). The process of removing one insert from a construct in order to place it into another is known as subcloning, otherwise known as the transfer of DNA from one vector to another to gain the ability and functionality to study or express a gene of interest. Throughout our processes, we navigated through many bioinformatics sources including Expasy, an online bioinformatics resource operated by the Swiss Institute of Bioinformatics (SIB), providing us access to over 160 databases and software tools. Expasy enabled us to translate our nucleotide sequence to a polypeptide, otherwise known as a protein or sequence of amino acids.
construct and purification
Our design for creating a highly specific plasmid construct begins with sending the sequence of GOx and our required restriction sites, Xba1 and Spe1, to IDT to clone the protein sequence and manufacture it into a custom plasmid. The insert that will be a component of the IDT brand plasmid will contain the WT-GOx sequence with a 6x His-Tag site, a TEV site, and two restriction sites. Once we receive the plasmid from IDT, the Xba1-GOx-Spe1 insert needs to be cleaved through the use of endonucleases using enzymes corresponding to specific restriction sites (Xba1 and Spe1). Restriction sites are only recognized by endonucleases that correspond to the palindromic sequence of DNA in the specific site. Succeeding this phase, we will take the pSBC3-BBa_K2238000 plasmid, from iGEM, and the restriction sites will be located. These sites are contained in a larger area called the Multiple Cloning Site, which contains a wide variety of sites for endonucleases to recognize.
Furthermore, we will then take the insert with the fabricated sticky ends and use a Ligase enzyme to connect it to the pSBC3 plasmid. By doing this step, the final plasmid construct is manufactured. Following the construction of our full plasmid, we will preliminarily have to transform it into DH5-alpha competent E.Coli bacterial cells. DH5a cells are genetically optimized for the increased and efficient replication of DNA, and this replication is to be done through a QIAprep Miniprep procedure. Then, another transformation will be completed in BL21 E.Coli cells, which are genetically modified and optimized to express high levels of protein. The expression of our target GOx protein will then be induced and we will grow high amounts of the final bacteria in our lab. These bacteria will then be Lysed and purified. The substance will undergo the process of elution in a Nickel column, which will separate our desired protein from all other cellular components. To ensure we have done this correctly, we will analyze the column using methods of spectrophotometry, which measures the amount of chemicals in a solution using a light beam that passes through samples.
References
1.1.3.4: Glucose oxidase. BRENDA. (n.d.). Retrieved October 21, 2021, from https://www.brenda-enzymes.org/all_enzymes.php?ecno=1.1.3.4&table=Temperature_Stability.
Berg, J. M. (1970, January 1). The purification of proteins is an essential first step in understanding their function. Biochemistry. 5th edition. Retrieved October 21, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK22410/.
The BioBricks Foundation:standards/technical/formats. OpenWetWare. (n.d.). Retrieved October 21, 2021, from https://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats.
BL21 chemically competent E. Coli Cells. GoldBio. (n.d.). Retrieved October 21, 2021, from https://www.goldbio.com/product/14439/bl21-chemically-competent-e-coli-cells.
Chino, A., Watanabe, K., & Moriya, H. (n.d.). Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe. PLOS ONE. Retrieved October 21, 2021, from https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0009652.
Integrated DNA Technologies ǀ IDT. Integrated DNA Technologies. (n.d.). Retrieved October 21, 2021, from https://www.idtdna.com/pages.
IPE quick - sigmaaldrich.com. (n.d.). Retrieved October 21, 2021, from https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/208/756/ipe-quick-manual-ouc.pdf.
Lakna. (2018, April 6). How to read a plasmid map. Pediaa.Com. Retrieved October 21, 2021, from https://pediaa.com/how-to-read-a-plasmid-map/.
Nanodrop Lite interpretation of nucleic acid 260-280 ratios. (n.d.). Retrieved October 21, 2021, from https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf.
NE;, L. (n.d.). Protein purification: An overview. Methods in molecular biology (Clifton, N.J.). Retrieved October 21, 2021, from https://pubmed.ncbi.nlm.nih.gov/24648062/.
PBS - phosphate-buffered saline (10x) ph 7.4, RNase-free. Thermo Fisher Scientific - US. (n.d.). Retrieved October 21, 2021, from https://www.thermofisher.com/order/catalog/product/AM9624#/AM9624.
Primers/Catalog. (n.d.). Retrieved October 21, 2021, from http://parts.igem.org/Primers/Catalog.
SIB Swiss Institute of Bioinformatics. Expasy. (n.d.). Retrieved October 21, 2021, from https://www.expasy.org/.
Sticky ends. Oxford Reference. (n.d.). Retrieved October 21, 2021, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100532864.
Studier, F. W., & Moffatt, B. A. (2004, October 22). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology. Retrieved October 21, 2021, from https://www.sciencedirect.com/science/article/abs/pii/0022283686903852.