Team:McMaster/Engineering
Engineering
What was the experimental workflow and process of our project?For the detailed design process, please refer to the following page: Design
Detection Module
Gibson vs Golden Gate Assembly
Gibson assembly is a cloning method which allows the assembly of multiple linear DNA fragments in a plasmid. Compared to another DNA assembly method, Golden Gate, Gibson assembly is scarless, and doesn’t require specific restriction sites. Furthermore, Gibson assembly can combine multiple fragments at once in a one-pot reaction, while Golden Gate assembly requires precisely positioning the DNA part in the final construction of the plasmid. As such, the Gibson assembly is much more efficient for synthesizing genetic circuits from gene fragments.
In our project, our circuits will express colicin E1 and E9 upon activation of a nitric oxide and autoinducer 3 AND gate.
Created with Biorender.com
GFP & Microplate Reading
Green Fluorescent Protein (GFP) will be fused to the PyeaR promotor and its transcript to understand the activity and regulation of PyeaR. The intensity of GFP expression will be determined using microplate reading to understand the effect of different factors, such as presence of NO and the NarX/L feedback system, on PyeaR activation.
Phosphotransfer Assay
In our circuit design, QseB binds to the flhDC promoter in order to regulate the expression of the final circuit module containing the effector colicin molecules. As such, it is a crucial connector between the AND gate detection module and the effector module. Thus, we plan to study QseC phosphate transfer to QseB, a critical component to this process, by examining QseB phosphorylation status using phosphotransfer assay. Phosphotransfer reactions will be performed, whereupon take autophosphorylated radiolabeled QseC protein is mixed with the response regulator or with the water control so that the proteins can then be resolved on 15% SDS-PAGE and visualized for the presence of γ32P using autoradiography. The link to a more detailed protocol and a sample result can be found below.
If the phosphotransfer assay does not work correctly, the following changes can be made for troubleshooting purposes: The addition of more QseC, The addition of more QseB, The addition of more AI-3, The addition of more Phosphate, and The phosphorylation of QseB with in order to examine if the failure is due to QseB or QseC.
List of Protocols
Green Fluorescent Protein & Microplate Reading
Effector Module
Explanations
Purification
Affinity chromatography is a separation technique that is used to isolate proteins that are highly selective towards an immobilized ligand inside the chromatography column. This purification method is used to isolate the colicins proteins from the cell lysate for use in downstream experiments. Both colicin polypeptides have a His tag attached to the N-terminus which will bind strongly to the Ni2+ metal ion that is immobilized in the chromatography column. Imidazole is used to elute the colicin protein after the rest of the cell lysate has left the column. Imidazole completes with the his-tag and has a higher affinity for the metal resin, therefore it will displace the protein. Colicin protein is collected and processed through gel electrophoresis to verify it’s identity.
If final protein yield is too low: 1) increase size of the his-tag to increase affinity for the metal resin. 2) use higher concentration of imidazole to remove the bound proteins. 3) Hidden His-tag: purify the protein under denaturing conditions. After elution, renature the protein. OR add small linker sequence consisting of glycines to separate the tag from the protein.
Created with Biorender.com
Co-culture experiment
Co-culturing is the biological system which associates with coexisting two or more organisms that grow in contact to one another within a defined media. This experiment is used to determine whether the transformed bacteria is able to kill AIEC with the presence of nitric oxide and autoinducer-3. Using a 12-well plate for plating both the AIEC strain and our engineered E. coli strain. Adjusting the nitric oxide and autoinducer-3 levels to test the killing ability of the colicin produced. It is expected for the colicin isolates from recombinant E. coli to kill the AIEC when an increased amount of nitric oxide and autoinducer-3 is present.
Created with Biorender.com
If the results are not as expected, here are some of the potential considerations for trouble shooting: 1) Scaling up/down molecules/nutrients for the bacteria to produce the expected response; 2)Increasing incubation period for expected bacterial growth; 3)Enforcing population density; 4)Adjusting pH, dilution factor of media, etc.
Crystal Violet Assay
Crystal violet (CV) is a trianiline, cell permeable dye used to quantify biomass in biofilms (Wilson et al., 2017). Although the number of cells can be manually counted, manual counting is limited by complicated 3D films and user bias (Wilson et al., 2017). As such, we will use spectrometry to reliably and accurately estimate the number of E. coli cells (Schiebel et al., 2017, Wilson et al., 2017).
For our project, CV will dye both AIEC and E. coli Nissle, as such a baseline sample containing only Nissle will be used as a comparator. From this experiment, we will determine the amount of AIEC cells present to evaluate the efficiency of our colicin-producing EcN in reducing AIEC population.
Alternative Steps for troubleshooting (Cellular and Biochemical Assay Kits FAQ, n.d.): High variability in results: prepare fresh reagents at appropriate temperatures and avoid air bubbles when mixing; High absorbance: Ensure the concentration of the analyte is within the linear range of the generated standard curve; Ensure adequate washing; Low absorbance: ensure the wavelength is optimized.
Created with Biorender.com
XTT
Because CV stains both living and dead cells (Schiebel et al., 2017), a separate colorimetric XTT assay will be performed to quantify the number of living cells. XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) is a tetrazolium salt that is reduced to formazan by metabolizing, living bacterial cells (Kuhn et al., 2003). AIEC in biofilms will either be exposed to the supernatant of our engineered bacteria and or be co-cultured, and the number of surviving AIEC cells is quantified by the XTT absorption measured. This indicates the efficiency of colicin-production by our modified-Nissle bacteria and the effectiveness of colicin in AIEC in a biofilm state. Similar processes were used before for E. coli cultures and P. aeruginosa supernatant (Lopes et al., 2011), but if there are issues the following steps can be taken.
Alternative Steps for Troubleshooting (ATCC, 2011):
Blanks: Ensure growth medium does not contain reducing agents; Check for contamination and discard.
High absorbance: Check for contamination and discard; Reduce number of cells plated per well.
Low absorbance : Cells not proliferating , ensure proper culture conditions or increase culture time; increase number of cells plated per well.
Created with Biorender.com
Microplate readings
Using crystal violet and the 96 well plates we can quantitatively determine biofilm viability. This is an improvement to the existing biofilm metabolic assays that can have significant error. This is done by deriving the specific growth rate of a bacteria biofilm from a series of metabolic assays using pH indicator phenol red, aiding in the quantification of the relative number of viable bacteria in a biofilm. This is of great importance as it allows us to monitor the condition of the AIEC biofilms that we are targeting. Growth or increase in growth would indicate a mistake in our circuit, and decreased growth and number of viable cells in the biofuel would indicate our circuit’s performance as less viable cells in the biofilm indicate cell death by colicins. A parameter that this method finds is the specific growth rate of the bacteria at a point in time, indicating our circuit's performance at a period of time.
List of Protocols
Buffer Exchange - Removal of Imidazole
Ni-NTAAffinity Chromatography Buffer Preparation
Ni-NTAAffinity Chromatography via AKTA purification system
Plate Reader Calibration and Analysis
Timeline
References
- ATCC. (2011). XTT Cell Proliferation Assay Kit: Instruction Manual. Manasas, VA: ATCC.
- Cellular and biochemical assay kits FAQ. (n.d.). Abcam. https://www.abcam.com/kits/cellular-and-biochemical-assay-kits-faq
- Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., & Ghannoum, M. A. (2003). Uses and limitations of the XTT assay in studies of Candida growth and metabolism. Journal of Clinical Microbiology, 41(1), 506–508. https://doi.org/10.1128/JCM.41.1.506-508.2003
- Lopes, S. P., Machado, I., & Pereira, M. O. (2011). Role of planktonic and sessile extracellular metabolic byproducts on Pseudomonas aeruginosa and Escherichia coli intra and interspecies relationships. Journal of Industrial Microbiology & Biotechnology, 38(1), 133–140. https://doi.org/10.1007/s10295-010-0838-y
- Schiebel, J., Böhm, A., Nitschke, J., Burdukiewicz, M., Weinreich, J., Ali, A., Roggenbuck, D., Rödiger, S., & Schierack, P. (2017). Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes. Applied and Environmental Microbiology, 83(24). https://doi.org/10.1128/AEM.01660-17
- Wilson, C., Lukowicz, R., Merchant, S., Valquier-Flynn, H., Caballero, J., Sandoval, J., Okuom, M., Huber, C., Brooks, T. D., Wilson, E., Clement, B., Wentworth, C. D., & Holmes, A. E. (2017). Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Research & Reviews. Journal of Engineering and Technology, 6(4), http://www.rroij.com/open-access/quantitative-and-qualitative-assessment-methods-for-biofilm-growth-a-minireview-.pdf.