iGEM 2021 — Team NYU_New_York

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# Team:NYU New York/Notebook

# Notebook

## March

Devised Project

## April

Began planning

## May

Submitted Final Report (research plan) to our PI

## September

Recruitment efforts and team teaching and regrouping

September 17th: Collaboration with Lucas from the City of London UK Team.

## October

IDT orders arrived and begin device construction

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# Team:NYU New York/Engineering

# Engineering Success

### The team went through the engineering process with both the cell circuit aspect and the hardware aspect of the project.

### Cell Circuit Design

For the cell circuit aspect, much of the testing was automated by the NUPACK software. This allowed for the proper sequence of the PB2 gene to be chosen to allow for the lowest energy, and therefore ensure stability. The cell circuitry aspect also had some workshops during team meetings in order to determine what the best indicator would be for the diagnostic test. GFP was proposed at first since it is commonly used. However, with further research and discussion, the team decided against it since that indicator requires UV light, which is not feasible for home tests. After a careful literature review the team found that X-gal would be a better indicator due to its rapid response to β-galactosidase and the lack of need for laboratory equipment. -----Once this was proposed, another iteration was created in order to keep the cellular system working quickly in a cell free environment. The proposed solution is that the two subunits that make up β-galactosidase, alpha and omega, which in the presence of each other will spontaneously combine, can be used to create β-galactosidase. Producing this smaller molecule will make the design much more efficient. The toehold switch will have the alpha subunit’s RNA in the reporter gene section and the solution located within the portable device will contain the omega subunit. Once exposed to influenza A, the toehold switch will unravel, allowing for the production of the alpha subunit. Once the button on top of the test is pressed the solution will fall on top of the cell free system. This will allow for the omega and alpha subunits to combine, cleave the X-gal and produce a blue tint.

### Hardware Design

For the hardware aspect, the first design only had a viewing window in the bottom which contained the cell free system, as seen in Figure 1. Upon team discussions, it was determined that a second viewing window would be useful to see the solution before mixing with the cell free system, shown in Figure 3. This will serve as a control for the color before exposure to the saliva to aid users in identifying their result. Having a clear control and eliminating room for customer error aligns with feedback received from pharmacists that the test should be easy to use and the result should be easily identifiable. Input from pharmacists and surveys developed for customer feedback indicated that the test must be affordable to reach the general population. This was also taken into consideration. The cell free system allows for easier transport of the system, the small and simple design creates low manufacturing prices, and the genetic components can be grown cheaply.

![](https://static.igem.org/mediawiki/2021/e/e1/T--NYU_New_York--hardwareforImplementation.jpeg)

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# Team:NYU New York/Experiments

# Experiments

For this device to be a functional at home test, the response system must consist of an indicator that allows the user to read results directly from the test itself, without the need for results to be sent to a lab for a diagnosis. A colorimetric indicator was the ideal option for ease of use, and the properties of the color changing dye X-gal, including its fast response time and easily distinguishable color change, made it the best choice. X-gal is a commercially available dye, and has a molecular structure that includes two molecules, a galactose molecule bonded to an indole. When a molecule called ꞵ-galactosidase is in the presence of X-gal, this bond is cleaved and activates a color change from white to bright blue in the dye.

To produce this color change in the testing device for an exclusively positive influenza result, ꞵ-galactosidase has to be present in the test only when influenza is present. ꞵ-galactosidase is encoded by the LacZ gene, a subunit of the lac operon that controls transport and metabolism of lactose in E. coli and other bacterium, so the addition of the LacZ gene sequence to the toehold switch was the ideal choice for an indicator that would activate X-gal in the presence of influenza. LacZ has complex properties that induce expression of the gene only under specific conditions, and is a rather long sequence, so an option that would negate the need to include the entire LacZ sequence in the toehold switch and ensure its expression into ꞵ-galactosidase was ideal, especially for an at-home test where environmental conditions cannot be guaranteed. Alpha-complementation properties of the LacZ gene were utilized, wherein the subunits of LacZ, LacZα and LacZΩ, spontaneously assemble into a working molecule of ꞵ-galactosidase when in the presence of one another. The longer sequence subunit, LacZΩ (iGEM Part BBa\_I732006), would be pre prepared and placed in the testing device, and the smaller subunit, LacZα (iGEM Part BBa\_K891003), would be included in the toehold switch. The LacZΩ subunit can be pre prepared via in vivo translation by the methods outlined by Berg, Squires, and Squires, or by cloning the LacZΩ fragment sequence into the PET28A vector (iGEM Part BBa\_K2141000) and producing it from there.

With this design, the presence of influenza viral RNA activates the toehold switch, which allows the LacZ to create the necessary alpha subunit. For LacZα to actually assemble into a working subunit, and so it can then assemble with LacZΩ to form X-gal activating ꞵ-galactosidase, a cell free expression system (CFE) must be included to express the alpha subunit. The CFE of choice is the iGEM part BBa\_I719015, which allows for expression of any RNA sequence that includes the T7 promoter (iGEM Part BBa\_I712074), which is the main reason it was essential to include the T7 promoter in our toehold switch. The CFE is then transferred to paper to be inserted into the physical testing device by adding the CFE solution to paper and flash freezing in liquid nitrogen.

Other options for a CFE include pre prepared systems manufactured by private companies, or a custom built CFE, which involves producing a lysate from specific E.coli strains, and adding an energy mix, an amino acid mix, some necessary salts, a molecular crowder, and a DNA template. This method was not the ideal choice because of the exhaustive laboratory work required to produce a custom CFE, however a general recipe for a custom built 100μL CFE is as follows: 33μL of lysate at 30mg/mL, 7.1μL of 14x energy mix, 10μL of 10x amino acid mix, 4μL of magnesium glutamate at 100mM, 2.5μL potassium glutamate at 3M, 5μL of molecular crowder PEG8000 at 400g/L, 1-10μL of plasmid DNA template at 100nM, and autoclaved water added to complete the 100μL volume.
Final assembly of the testing device is to insert the CFE, already transferred to paper and flash frozen, into the device along with the prepared toehold switch, assembled LacZΩ subunit, and X-gal dye. A visible change from white to blue in the window signifies to the user a positive influenza result, and a lack of color change signifies a negative result.

Citations

1. Moosmann, P., & Rusconi, S. (1996). Alpha Complementation of LacZ in Mammalian Cells. Nucleic Acids Research, 24(6), 1171–1172. https://doi.org/10.1093/nar/24.6.1171

2. Berg, K. L., Squires, C. L., & Squires, C. (1987). In vivo translation of a region within the rrnB 16S rRNA gene of Escherichia coli. Journal of Bacteriology, 169(4), 1691–1701. https://doi.org/10.1128/jb.169.4.1691-1701.1987

3. Ma, D., Shen, L., Wu, K., Diehnelt, C. W., & Green, A. A. (2018). Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synthetic Biology, 3(1). https://doi.org/10.1093/synbio/ysy018

4. Cell-free gene expression | Nature Reviews Methods Primers. (n.d.). Retrieved October 21, 2021, from https://www.nature.com/articles/s43586-021-00046-x

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# Team:NYU New York/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)