iGEM 2021 — Team McMaster

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:McMaster/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:McMaster/Engineering

[![](https://static.igem.org/mediawiki/2021/8/84/T--McMaster--logo.png)](index.html)

* Project

  [Background & Rationale](https://2021.igem.org/Team%3AMcMaster/BackgroundRationale)
  [Project Description](https://2021.igem.org/Team%3AMcMaster/Description)
  [Design](https://2021.igem.org/Team%3AMcMaster/Design)
  [Engineering](https://2021.igem.org/Team%3AMcMaster/Engineering)
  [Implementation](https://2021.igem.org/Team%3AMcMaster/Implementation)
* [Parts](https://2021.igem.org/Team%3AMcMaster/Parts)
* [Modelling](https://2021.igem.org/Team%3AMcMaster/Model)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3AMcMaster/Human_Practices)
  [Education](https://2021.igem.org/Team%3AMcMaster/Education)
  [Communication](https://2021.igem.org/Team%3AMcMaster/Communication)
  [Inclusivity](https://2021.igem.org/Team%3AMcMaster/Inclusivity)
  [Aging Podcast](https://2021.igem.org/Team%3AMcMaster/Podcast)
* Cooperation

  [Collaboration](https://2021.igem.org/Team%3AMcMaster/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AMcMaster/Partnership)
* [Contribution](https://2021.igem.org/Team%3AMcMaster/Contribution)
* [Team](https://2021.igem.org/Team%3AMcMaster/Attributions)

# Engineering

What was the experimental workflow and process of our project?

[Detection Module](#detection) [Effector Module](#effector) [Timeline](#timeline)

**For the detailed design process, please refer to the following page: [Design](https://2021.igem.org/Team%3AMcMaster/Design)**

## Detection Module

![](https://static.igem.org/mediawiki/2021/5/50/T--McMaster--Flowchart1.png)

**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.

![](https://static.igem.org/mediawiki/2021/b/b4/T--McMaster--Gibson.png)

*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

[Gibson Assembly](https://static.igem.org/mediawiki/2021/9/91/T--McMaster--Gibson_Assembly.pdf)

[Transformation](https://static.igem.org/mediawiki/2021/b/bc/T--McMaster--Transformation.pdf)

[DNA Minipreparation](https://static.igem.org/mediawiki/2021/f/f2/T--McMaster--Miniprep.pdf)

[Restriction Enzyme Digests](https://static.igem.org/mediawiki/2021/4/4a/T--McMaster--Restriction.pdf)

[Gel Electrophoresis](https://static.igem.org/mediawiki/2021/0/0c/T--McMaster--Gel.pdf)

[Green Fluorescent Protein & Microplate Reading](https://static.igem.org/mediawiki/2021/2/26/T--McMaster--GFP.pdf)

[Phosphotransfer Assay](https://static.igem.org/mediawiki/2021/1/12/T--McMaster--Phosphotransfer.pdf)

[Gel Electrophoresis](https://static.igem.org/mediawiki/2021/0/0c/T--McMaster--Gel.pdf)

[Back to Top](#top)

## Effector Module

![](https://static.igem.org/mediawiki/2021/a/ad/T--McMaster--Flowchart2.png)

### 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.

![](https://static.igem.org/mediawiki/2021/9/9d/T--McMaster--His_Tag.png)

*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.

![](https://static.igem.org/mediawiki/2021/e/ee/T--McMaster--Co_Culture.png)

*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.

![](https://static.igem.org/mediawiki/2021/2/22/T--McMaster--CVImage.png)

*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.

![](https://static.igem.org/mediawiki/2021/6/63/T--McMaster--XTTImage.png)

*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

[Bradford Assay](https://static.igem.org/mediawiki/2021/c/cd/T--McMaster--Bradford.pdf)

[Buffer Exchange - Removal of Imidazole](https://static.igem.org/mediawiki/2021/1/11/T--McMaster--Buffer.pdf)

[Crystal Violet Staining](https://static.igem.org/mediawiki/2021/2/2a/T--McMaster--CViolet.pdf)

[Ni-NTAAffinity Chromatography Buffer Preparation](https://static.igem.org/mediawiki/2021/7/75/T--McMaster--NIA1.pdf)

[Ni-NTAAffinity Chromatography via AKTA purification system](https://static.igem.org/mediawiki/2021/b/b0/T--McMaster--NIA2.pdf)

[Plate Reader Calibration and Analysis](https://static.igem.org/mediawiki/2021/1/14/T--McMaster--Plate_Reader.pdf)

[SDS-PAGE](https://static.igem.org/mediawiki/2021/1/13/T--McMaster--SDS.pdf)

[XTT Assay](https://static.igem.org/mediawiki/2021/f/f8/T--McMaster--XTT.pdf)

[Back to Top](#top)

## Timeline

[Back to Top](#top)

## 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.

![](https://static.igem.org/mediawiki/2021/0/0d/T--McMaster--Footer_Logo.png)

McMaster SynBio 2021

![](https://static.igem.org/mediawiki/2021/4/48/T--McMaster--Sponsors3.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:McMaster/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:McMaster/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)