Team:GreatBay SZ/Engineering

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  • Proof Of Concept
  • Engineering Success
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  • Hardware
  • Proposed Implementation
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Index

• Introduction
• Microscopic examination Optimization
• Endospore Staining method
• Methylene Blue Staining
• Cas12a False-positive Result Optimization
• RPA
• Probe
• Cas12a Detection Efficiency Optimization
• Conclusion
• Reference

Engineering Success

Introduction

Behind the successes mentioned above, we encountered various problems at different stages. At first, we realized that some planned experiments did not work. This includes the fact that the ideal spore structure was not visible under the microscope, false-positive results for Cas12a detection which was caused by the sequence after RPA and ssDNA probe over-addition, and problems regarding the Cas12a detection efficiency. These problems forced us to start new engineering cycles (design -> build -> test -> learn -> design... ), eventually promoting the development of our project. This page will show you exactly how we designed and improved our experiments to reach our goal.

Microscopic examination Optimization

Endospore Staining method

In order to observe the results of spores production, Endospore Staining method was used. According to a report by Coluccio AE et.al.[1], S.cerevisiae forms four haploid spores through meiosis under nutrient-deficient conditions. These four haploid spores were then wrapped in an ascospore. Hence, the observation of ascospores were assumed to contain several tiny haploid spores. However, the dyeing result was initially not satisfactory. Although yeast and ascospores was clearly distinguished in the staining result, the internal structure of ascospores was not visible (fig.1). In other words, we could only see the ascospores, and not the haploid spores inside.

【fig.1】Endospore Staining result; Red are S.cerevisiae; Green are spores.

We assumed that this may be due to the prolonged dyeing time of malachite green as well as the influence of saffron counter dye. Hence, in order to clearly observe the haploid spores, we shortened the baking time of malachite green and removed the steps of counterstaining. After inspection, punctate haploid spores can be clearly seen in the ascospores (fig.2), which means the improvement is successful.

【fig.2】Improved Endospore Staining result; The ones with dark green dots are ascospores, where the dark green dots are punctate haploid spores. The ones without dark green dots are vegetative cells.

Methylene Blue Staining

Simultaneously, in order to better distinguish the two forms of spores (ascospores: diploid yeast for sexual reproduction through meiosis; vegetative cells: haploid yeast for asexual reproduction through mitosis) we used the methylene blue staining method. Mentioned in a report by Bruzaite et.al., the three forms of spores can be observed during microscopic examination, namely transparent, dark blue and light blue (Transparent are living vegetative cells; Dark blue are dead vegetative cells; Light blue are mature ascomytic cells). However, during the microscopic examination, we found that the spores sometimes cannot be observed (fig.3). Only the dark blue ones are shown on the graph, which are the dead vegetative cells. This led to our assumption that the dyeing time was too long, causing cell death.

【fig.3】 Methylene Blue Staining result; Dark Blue are dead vegetative cells; The tiny white dots are spores; Others are bacteria.

Thus, we sped up the dyeing process and took a look at one sample in order to avoid sample retention. At the same time, we noticed that if the loading order was reversed, the dye would disperse the sample. In terms of the coverslip, it is necessary to avoid creating bubbles, otherwise it will affect the observation results. After inspection, we got the ideal image (fig.4). Therefore the improvement was successful.

【fig.4】 Improved Methylene Blue Staining result; Transparent are living vegetative cells; Dark Blue are dead vegetative cells; Light Blue are mature ascospores; The tiny white dots are spores.

Cas12a False-positive Result Optimization

RPA

Prior to conducting Cas12a detection, we used RPA to amplify the target sequence with the intention of achieving minimum detection amount. At first, we tested the target sequence directly after RPA. Unfortunately, it was stumped by a false positive result. The test line of the treatment group test strip showed a clear visible line. We believed that this was caused by configurating the system in the same experimental environment, where an extremely small amount of the target sequence may have floated in the air. This amount would not have reached the minimum detection amount, which would not have affected the result. However, because of the addition of a step in the detection process that requires incubation at 37 degrees, it stimulates the RPA system again to amplified the target sequence in air. Therefore, the concentration of the target sequence in the control group reached the minimum detection amount, resulting in a false positive result (fig. 5).

【fig.5】 Cas12a detection false positive result using test strips; The left test strips is control group; The right test strips is experiment of BBa_K3859003. The first line is the test line; The second line is the control line.

As a result, we increased the temperature to 80 degrees after amplification to inactivate the enzyme. This reduced the chances of the results being affected during the detection process. We also configured the experimental group and the treatment group in two independent experimental areas respectively to avoid the target sequence in the environment leading to treatment group contamination. False positive results did not occur (fig.6), which verifies that the improvement was successful.

【fig.6】Cas12a detection result using test strips; The left test strips is control group; The right test strips is experiment of BBa_K3859003. The first line is the test line; The second line is the control line.

Probe

The Cas12a detection technique we used is referenced from a report by Li, SY. et al. The Cas12a protein recognizes the target sequence and stimulates its property of cutting ssDNA arbitrarily in the environment. Based on this feature, probes were added to release signals which can be detected to observe the results.

Referring to Team OSA 2020, they use Cas12a system to detect heavy metals. One of the parts used is BBa_K3696010 (HEX_N12_BHQ1), which is a type of reporter DNA with HEX fluorophore and BHQ1 fluorescence quencher. They use it as a fluorescent probe, so the results can be observed by probing the fluorescence values. We also used this part and made some improvements. We additionally tried using test-strip probes. While we found that even after eliminating the effect of RPA on the assay (which mentioned above), there were still false-positive results (fig.7). We suspect that this may be due to the high concentration of probe.

【fig.7】False-positive result of Cas12a detection result with test-strip probes; The left test strips is control group; The right test strips is experiment of BBa_K3859004. The first line is the test line; The second line is the control line.

In order to avoid false positives, we diluted both probes. The concentration of probe OSA 2020 used is 5uM. So we tried to dilute both probes to 0.33uM or 0.033uM respectively. Both methods presented the ideal detection results. The test strips did not show false positives (fig.8A and fig.8B), and the fluorescence detection was not affected by the change in concentration (fig.8C and fig.8D). Besides, the time of both tests was shortened from 60 min to 20 min, which facilitated practical use. A reduction in concentration means less amount is used. This leads to a decrease in cost of use and makes the product more competitive in the market.

【fig.8A】Result of Cas12a Detection with 0.33uM test-strip probes; The left test strips is control group; The right test strips is experiment of BBa_K3859004. The first line is the test line; The second line is the control line.

【fig.8B】Result of Cas12a Detection with 0.033uM test-strip probes; The left test strips is control group; The right test strips is experiment of BBa_K3859004. The first line is the test line; The second line is the control line.

【fig.8C】Result of Cas12a Detection with 0.33uM fluorescent probes;

【fig.8D】Result of Cas12a Detection with 0.033uM fluorescent probes;

Cas12a Detection Efficiency Optimization

During the detection process, Cas12a protein will form a complex with crRNA. Under the guidance of crRNA, Cas12a protein is able to cleave the target sequence

Referring to the 2018 team, TJU_China, they used LbCas12a to achieve High-throughput disease detection. LbCas12a, or Part BBa_K2644102, will cut 24 base pairs of DNA downstream of the PAM sequence. Similarly, we used LbCas12a for detection. After successful reproduction (fig.9), we attempted to achieve more efficient detection.

【fig.9】Cas12a detection result by using 24nt guide sequence; The left test strips is control group; The right test strips is experiment of BBa_K3859005. The first line is the test line; The second line is the control line.

Since the length of the guide seuqence directly affects the cleavage check point of Cas12a protein, we decided to improve the cleavage check point of LbCas12a. According to the report by Zetsche et al.[4], it is mentioned that the guide sequence of a generally mature crRNA is between 20 and 24nt. Thus, we tried to use the smallest guide sequence length, that is, shorten the guide sequence to 20 base pairs. This way, the cleavage check point of Cas12a protein will be located at the 20th base pair downstream of the PAM sequence. It has been proved that this improvement not only reduces the cost but also shortens the detection time (fig.10). Thus, more efficient detection is achieved.

【fig.10】Cas12a detection result by using 20nt guide sequence; The left test strips is control group; The right test strips is experiment of BBa_K3859005. The first line is the test line; The second line is the control line.

Conclusion

Throughout the experiment, we went through these four main engineering cycles above. We solved the problem of medium contamination by adding Kanamycin antibiotics. We improved the steps of Endospore Staining and Methylene Blue Staining to observe the ideal microscopic results. We've also inactivated the RPA enzyme prior to detection and reduced the concentration of probes used to avoid chances of false positives. Finally, we completed the ARTAG production and inspection functions successfully. If you want to view the exact experimental theory and results, click here.

Reference

• [1]Coluccio AE, Rodriguez RK, Kernan MJ, Neiman AM. (2008). The Yeast Spore Wall Enables Spores to Survive Passage through the Digestive Tract ofDrosophila. PLoS ONE 3(8): e2873. https://doi.org/10.1371/journal.pone.0002873
• [2]Bruzaite, Ingrida, Juste Rozene, Inga Morkvenaite-Vilkonciene, and Arunas Ramanavicius. (2020). Towards Microorganism-Based Biofuel Cells: The Viability of Saccharomyces cerevisiae Modified by Multiwalled Carbon Nanotubes. Nanomaterials 10, no. 5: 954. https://doi.org/10.3390/nano10050954
• [3]Li, SY., Cheng, QX., Wang, JM. et al. (2018). CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov 4, 20. https://doi.org/10.1038/s41421-018-0028-z
• [4]Zetsche et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell (2015), http://dx.doi.org/10.1016/j.cell.2015.09.038