iGEM 2022 — Team city-of-london-uk


# Engineering Success

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Our design cycle for bacterial lysis was greatly important to our project. Being the first, and most novel step, we had to make sure that the hardware worked every time and was compatible with the biological requirements so that the rest of the triage could take place. The most fundamental aspect of this was specifically how the heating of the chamber was to be sufficient enough to lyse bacterial samples. For this, as we detail below, we had to change the shape of the lysis chamber in order to maximise even temperature distribution. Similarly we changed the very nature of how heating would occur - from chemically to electrically. Once we had established the mechanism of heating, and how this would be incorporated into our mask, we tested the conditions that would be achieved by the chamber on bacterial samples of Lactobacillus Casei; successfully showing that our solution would indeed perform as required. The information below goes through this entire process.

## Our Preliminary Hardware Design Cycle

The first and foremost concern we had when considering constructing such a mask was one of energy. Simply put, could we store enough energy on the mask to safely heat the sample for an hour at 65 degrees.
To start with, we considered an exothermic chemical reaction (such as that of calcium oxide and water) within the mask as a means of generating the heat required. The benefits of such an approach were its ease of use, high energy density and relatively low cost. However, it also posed many challenges we considered too tough to pursue. Firstly, there were the biological implications of having such a reaction occur at the same time as lysis, and more importantly the issue of maintaining a constant temperature for an extended period of time. Therefore, the chemical solution to this problem was soon abandoned
The other more obvious approach (and the one we have opted for) was to electrically heat the sample. While we did initially have concerns regarding the energy required for this, some rough ideal capacity calculations indicated that a consumer button cell was theoretically up to the task, and we went about developing a model to confirm this (see model). Our testing, detailed in the hardware section itself, showed beyond doubt that we could achieve 65 degrees celsius for over 80 minutes. This then allowed us to progress into the biological aspect of our testing.

## Our Biological Experimental Design Cycle

In order to procure the necessary samples to show the occurrence of cellular lysis by heat, several changes were made to our initial gram-staining protocol. Instead of air fixing our bacteria to the slide as instructed, through trial and error, we concluded that heat-fixing the sample using the bunsen flame would provide a far more discernible sample under the microscope. While the baseline image at room temperature taken before heating shows the presence of single cells and even chain formation after 40 minutes of heating - by 80 mins of incubation, change in morphology with disruption of the chain formation and increased debris indicated increased bacterial lysis.

<img style="max-width: 100%;" src="https://static.igem.wiki/teams/4508/wiki/site-res/prolonged-heating-of-l-casei-at-65-c-promotes-lysis-of-bacteria.png"/>

Unfortunately this year, our labs underwent extensive renovation work to promote STEM subjects to younger students, and to commit to sustainability of projects like this in the future. Consequently, for a long period of time during the summer and autumn our team was unable to make use of its full potential and as a result, our access to BSL-2 organisms like the intended mycobacterium smegmatis was curtailed. Further, the UK guidelines were particularly vague about the usage of M. Smegmatis and thus we had to spend a significant period trying to clarify the laws with the legislature. This resulted in successful clarification and permittance to use the bacteria given specific conditions stipulated by the CLEAPSS organisation; these were unable to be fulfilled within the remaining time due to budget and locational restraints intrinsic to being a school situated in the centre of London. This meant we had to rely upon bacterial strains of the lactobacillus family. We were fully aware, however, that these would not be perfect model organisms as they have a far higher heat shock potential than the ideal model organism, Mycobacterium Smegmatis. This invariably resulted in procuring data that would be less positive than if we had used a different strain. However, the fact that we were able to demonstrate the promotion of lysis in L. Casei, in such a short period of time, clearly showed that M. Smegmatis (and, invariably, M. Tuberculosis) would be lysed in a shorter period of time. It is easily stipulated from our results that had we used M. smegmatis, heat lysis would have certainly occurred after 80 minutes at 65C; further validating our hypothesis of one-hour lysis.

# Mycobacterium tuberculosis (M.tuberculosis) vs Lactobacillus Casei (L.Casei)

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_M.tuberculosis_ is a BSL-3 (Figure 1) airborne pathogen as there is a high risk of being infected, potentially resulting in serious repercussions. In fact, the chances of being infected by M.tuberculosis are 'three to nine times higher' compared to handling other clinical specimens<sup>[1]</sup>.

Figure 1; Definitions of various Biosafety Levels<sup>[2]</sup>

<img style="max-width: 100%;" src="https://static.igem.wiki/teams/4508/wiki/labwork/lab-work-pyramid.png"/>

Therefore, we used _Lactobacillus casei_ (BSL-1) instead of _M. tuberculosis_ in the laboratory, as it is safe to use and has similar features to the M. tuberculosis bacterium such as the gram-positive cell wall.<sup>[3]</sup> _L. casei_ is a suitable substitute for _M. tuberculosis_ as it is gram-positive, much safer to use and will require the same conditions for thermal lysis.

## Using L. casei

Our intended use for L. casei is to conduct tests to show and record the successful lysis of its cell wall using heat, and to use microscopy to confirm the successful lysis of the cell.

## How?

### Equipment

- Bunsen burner

- 12 jars of MRS Broth

- Sterile test tubes

- L. casei

- Incubator

### Protocol

1.  Flame the neck of the jar containing MRS broth and transfer to sterile test tubes

2.  Prepare inoculating loops for transfer of bacteria to broth jar

3.  Transfer L. casei culture from jar for isolation in MRS Broth

4.  Replace caps, but do not screw on tightly

5.  Incubate at 25°C for 3 days.

# Prolonged heating of L.Casei at 65 C promotes lysis of bacteria

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<img style="max-width: 100%;" src="https://static.igem.wiki/teams/4508/wiki/site-res/prolonged-heating-of-l-casei-at-65-c-promotes-lysis-of-bacteria.png"/>

The baseline image at room temperature, taken before heating, shows the presence of only single cells. After heating the sample for 20 minutes, chains of L.Casei can be seen to have formed. This effect of increased chain formation and growth has been previously reported at high temperatures<sup>4</sup>. However, by 80 mins of incubation, change in morphology with disruption of the chain formation and increased debris indicates increased bacterial lysis.

## Methodology

1. Heat water bath to 65 C

2. Remove L. casei jars from incubator

3. Gram-stain sample from jar to create a negative control slide (see gram-staining methodology below)

4. Transfer vials of L. casei into water bath

5. Let vials sit for 20 mins, then remove and take a swab to procure a slide for gram-staining

6. Use swab to transfer bacteria in vial to slide

7. Repeat each time for periods at 40 mins, 60 mins and 80 mins

8. Inspect slides for changes

## Gram-staining methodology

1. Dip swab into bacterial culture then smear thoroughly onto microscope slide

2. Heat dry the slide by passing over Bunsen flame using forceps-this will also heat-fix the bacterial smear to the slide

3. Wait for slide to cool down

4. Switch off Bunsen flame

5. Suspend slide over sink using slide racks then flood slide with crystal violet solution

6. Wait for 1 min

7. Pour off excess stain, then wash with bottle of deionised water

8. Flood slide with Gram’s iodine solution and wait for 1 min

9. Tilt slide using forceps, then wash down with a few drops of ethanol until run-off is clear

10. Wash with deionised water

11. Pour off water, then flood slide with Safranin counterstain, leave for 15 seconds

12. Wash away with deionised water and wait for slide to dry

13. Place slide under microscope for inspection

# RPA and Lateral Flow

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## RPA

Part of our initial plan was to test RPA alongside lysis to prove that our selectively designed primers are correctly optimised, however, due to the renovations in some of our labs and delay in fundraising, we were unable to carry out the RPA in time for the Wiki freeze. Our successful fundraising has permitted us to carry out the testing of this component of our project, so we have opted to go ahead with the wet lab to obtain results before the Jamboree in Paris.

## LFA Output

Due to the aforementioned issues encountered by our team, we were unable to access the laboratories to test the final stage of our system. The results below have been provided by similar research conducted by the Wyss Institute. They too, used the Cas12 enzyme in conjunction with lateral flow assay technology - detecting RPA amplicons using gRNA/Cas complexes, resulting in the collateral cleavage of FAM-Biotin probes.<sup>5</sup>

Using gRNAs modelled by Benchling, we are confident in our ability to procure successful results.

The figures below show the sensitivity of the LFA sensor.

<img style="max-width: 100%;" src="https://static.igem.wiki/teams/4508/wiki/site-res/rpa-and-lateral-flow-img1.png">

<img style="max-width: 100%;" src="https://static.igem.wiki/teams/4508/wiki/site-res/rpa-and-lateral-flow-img2.png">

## References

<sup>[1]</sup> Philippe Herman, M. F.-D. B. B. V. V. K. P. C. D. D. T. M. S. M. W. R. S. &. W. M., 2006. Biosafety Recommendations for the Contained Use of Mycobacterium tuberculosis Complex Isolates in Industrialized Countries.. [Online]

Available at: <https://www.biosafety.be/sites/default/files/mtub_final_dl.pdf>

[Accessed 23 July 2022]

<sup>[2]</sup> GenoFAB, n.d. AN OVERVIEW OF BIOSAFETY LEVELS. [Online]

Available at: <https://blog.genofab.com/overview-of-biosafety-levels>

[Accessed 23 July 2022]

<sup>[3]</sup>  Hill, D., Sugrue, I., Tobin, C., Hill, C., Stanton, C. and Ross, R., 2018. The Lactobacillus casei Group: History and Health Related Applications. Frontiers in Microbiology, [online]

Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160870/#__ffn_sectitle>

[Accessed 20 September 2022].

<sup>[4]</sup> Novitsky, T., Chan, M., Himes, R. and Akagi, J., 1974. Effect of Temperature on the Growth and Cell Wall Chemistry of a Facultative Thermophilic Bacillus. _Journal of Bacteriology_, 117(2), pp.858-865.

<sup>[5]</sup> Nguyen, P., Soenksen, L., Donghia, N., Angenent-Mari, N., de Puig, H., Huang, A., Lee, R., Slomovic, S., Galbersanini, T., Lansberry, G., Sallum, H., Zhao, E., Niemi, J. and Collins, J., 2021. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nature Biotechnology, 39(11), pp.1366-1374.