Team:Concordia-Montreal/Results
Contents:
1. Construction of Plasmids
2. Characterization of BBa_K2944003
3. Protein Purification and Growth Curve of BBA_K1692032
4. Electronic Device
5. Mobile Application
1. Plasmid Construction
The goal of the biosensor section of this project was to construct two plasmids that would be responsible for detecting a small molecule (Plasmid 1) and produce signal upon detection of that small molecule (Plasmid 2). Throughout this process, multiple steps were required to ensure that our construction would be functional.
The primary step was to successfully amplify our genes of interest with the right sequences to allow for golden gate assembly. For Plasmid 2, the genes of interest that we were working with were Glucose oxidase (GOx) and the Chromoprotein AmilCP.
Due to an inability to order the entire GOx gene (because it was too large), we had to order it as two separate parts. Therefore, each of the two GOx part (designated as GOx part 1 and GOx part 2) were required to be amplified separately before they could be inserted into the entry vector. Specific primers were designed to assemble the two without interfering with the genetic sequence. As seen in Figure 3, both parts were successfully amplified with the appropriate primers, with both sequence being present near the 1.0 kb line of the ladder.
Figure 1: PCR amplification of GOx part 1 and GOx part 2 with overhangs. Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2; GOx part 1 (~1.00kb), Lane 3; GOx part 2 (~0.98 kb).
After confirming that both parts were successfully amplified, they were used in a Golden Gate reaction using the entry vector PYTK001. Assembly reaction was performed and plated (Fig.4). GFP-negative colonies were selected and a colony PCR was performed to confirm the vector assembly as seen in Fig. 5. Gel confirmed entry vector with bands present near the 2.0kb line of the ladder, which is the same size as the complete GOx sequence that was amplified.
Figure 2: GOx-001 vector. Plate demonstrates GOx part 1 and GOx part 2 inserted in PYTK001 entry vector and transformed in E.coli.
Figure 3: GOx-001 cPCR. Lane 1, 22, 23, 31; GeneRuler 1 kb Plus DNA Ladder (Thermo fisher), Lane 2-21, 24-28; GOx-001 (~2.021kb)
After the successful incorporation into the entry vector, the GOx plasmid was ready for a final Golden Gate assembly with the other essential genes. The assembly was verified with cPCR as seen in Fig.6. The cPCR performed with the assemble vector (dubbed CEN6LEU-GOx because of the presence of the CEN6/ARS4 origin of replication, LEU3 auxotrophic marker and Glucose oxidase) utilized a primer that would amplify the promoter (TDH3), GOx and terminator (PGK1). Demonstrating that these three genes were present verified that the vector was assembled in the correct order and contained the gene of interest.
Figure 4: CEN6LEU-GOx cPCR. Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-4; CEN6LEU-GOx amplification (~2.96kb)
For our AmilCP, the gene was extracted from one of the parts provided by the iGEM kit, BBa_K1692032. Once transformed and plasmid prepped, specific primers were used to amplify the amilCP gene and provide the appropriate overhangs. Figure 7 shows a gradient PCR was performed for the specific sequence. However, due to the presence of multiple bands, the gene needed to be gel extracted. Once that was complete, it was ready to be assembled into the entry vector. Entry vector was constructed, with selection of GFP negative colonies (Fig 8), and a cPCR was performed to confirm results as seen in Fig 9. The primers amplified the insert within the entry vector, which was approximately 712 bp. Lane 3 demonstrated a successful assembly and a plasmid preparation was performed to isolate the vector. Unlike the previous insert, the final vector was unable to be completed due to lack of time and troubleshooting.
Figure 5: AmilCP amplifciation with overhangs. Lane 1 and 10; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-9; amilCP (~7.0kb)
Figure 6: AmilCP-001 vector. Plate demonstrates AmilCP inserted in PYTK001 entry vector and transformed in E.coli.
Figure 7: AmilCP-001 cPCR.Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-7; AmilCP (~7.0kb)
As for plasmid 1, the gene of interest that was meant to be assembled was GGV (GAL4-GCR-VP16). The gene of interest was ordered, however due to lack of time, the plasmid was not able to be completed.
2
Characterization by Mass Spectrometry of Part:BBa_K2944003.
Preparation of Standard Curve.pdf
Preparation of Standard Curve Continued.pdf
Preparation of Standard Curve Continued.pdf
Results:
Image built using Prism 6.
There are two superimposed traces: gluconolactone standard and yeast supernatant mixed 50/50 with 40 g/L glucose. The traces are of extracted ion 195.0505 (exact [M-H]- of gluconate. The gluconolactone standard is predominately gluconate upon resuspension in water.
The mass spectrum in the inset is the total ion count of the standard at 0.5 mins between the indicated m/z range. The two masses which are not the exact mass of gluconate are also part of the standard. Their identities are confirmed using the online database mzCloud. Annotated in the figure is we believe these masses correspond to based on the papers referenced below.
Characterization done by Matthew Tiranardi.Thankyou Lauren Narcross for her technical assistance!
References:
Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., … Matz, M. V. (2008). Diversity and evolution of coral fluorescent proteins. PLoS ONE, 3(7). https://doi.org/10.1371/journal.pone.0002680
Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase - An overview. Biotechnology Advances. https://doi.org/10.1016/j.biotechadv.2009.04.003
Lee, M., DeLoache, W., Cervantes, B., & Dueber, J. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975-986. doi: 10.1021/sb500366v
Taylor, V. F., March, R. E., Longerich, H. P., & Stadey, C. J. (2005). A mass spectrometric study of glucose, sucrose, and fructose using an inductively coupled plasma and electrospray ionization. International Journal of Mass Spectrometry, 243(1), 71–84. doi: 10.1016/j.ijms.2005.01.001
Zhang, Z., Gibson, P., Clark, S. B., Tian, G., Zanonato, P. L., & Rao, L. (2007). Lactonization and Protonation of Gluconic Acid: A Thermodynamic and Kinetic Study by Potentiometry, NMR and ESI-MS. Journal of Solution Chemistry, 36(10), 1187–1200. doi: 10.1007/s10953-007-9182-x
3. Protein Purification and Growth Curve
See more here: Part:BBa_K1692032
Figure A circular dichroism spectrum of amilCP indicates that the secondary structure is predominantly composed of β-sheets as indicated by the single minimum around 217nm and the maximum above 195nm. The minimal broadening towards 210nm and the second maximum at 190nm suggests that there are discrete α-helix structures in the proteins but are not a significant feature of this protein. The impact of the β-sheets on the ellipticity of circularly polarized light prevents any smaller features from being characterized. Furthermore, a negative of polarized light followed by a positive rotation of polarized light suggests that the β-sheets are arranged in an anti-parallel fashion throughout the protein.
4. Electronic Device
The objective of the electronic device is to serve as an interface between the biosensor/electrochemistry patch and the user. This objective has been met as all the components on the device are functioning nominally. The electronic device is thus able to produce an iontophoretic current, measure a low current input, measure temperature and transmit the data to an external Bluetooth device. The current design of the electronic device is the product prototype which corresponds to the second iteration of the design. It is shown in Figure 1 and 2.
5. Mobile Application
The objective of the mobile application is to provide an intuitive interface for the user who will be using our product This objective has been met as both front-end and back-end interfaces of the application are working. The mobile application can thus receive data from the electronic device, inform the user of their current condition, refer them to the nearest help locations. Some the screens of the mobile application are shown in Figure 3 and 4.
