iGEM 2025 — Team uzurich

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Notebook
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![Header Image](https://static.igem.wiki/teams/5686/header/notebook2.webp)
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# Notebook

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[Introduction](#Introduction)
[December](#December)
[January](#January)
[February](#February)
[March](#March)
[April](#April)
[May](#May)
[June](#June)
[July](#July)

[07.07. - 11.07.2025](#July-Week-07-11-07-2025)
[14.07. - 18.07.2025](#July-Week-14-07-18-07-2025)
[21.07. - 25.07.2025](#July-Week-21-07-25-07-2025)
[28.07. - 31.07.2025](#July-Week-28-07-31-07-2025)
[August](#August)

[04.08. - 08.08.2025](#August-Week-04-08-08-08-2025)
[11.08. - 15.08.2025](#August-Week-11-08-15-08-2025)
[18.08. - 22.08.2025](#August-Week-18-08-22-08-2025)
[25.08. - 29.08.2025](#August-Week-25-08-29-08-2025)
[September](#September)

[01.09. - 05.09.2025](#September-Week-01-09-05-09-2025)
[08.09. - 12.09.2025](#September-Week-08-09-12-09-2025)
[15.09. - 19.09.2025](#September-Week-15-09-19-09-2025)
[22.09. - 26.09.2025](#September-Week-22-09-26-09-2025)
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## Introduction

On this page you will find our monthly and weekly progress in the lab. To find detailed documentation, troubleshooting and results of each conducted experiment, open the according tab.
You can find the protocols that formed the basis of our experiments, as well as the reasoning and structure behind our experimental design, [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).
If you want to read up on the biological concepts behind our project click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D).

Exciting eleven months lay behind us. Read through our journey, learn about the project's trajectory and TRACE back the origins of our idea to where it all began.

## December

A new beginning! The iGEM UZurich 2025 Team members were selected by former team members.

## January

Oh Oh Exams.

## February

Time to be creative: An intense and exciting ideation phase resulted in numerous (and sometimes a tiny bit crazy) ideas. One of them will later turn into our beloved project. At this point however, only our very dedicated team member Tatia believed in the vision.

## March

A looot of research to be done. To turn an idea into a project, one must conduct a thorough investigation: What literature already exists on this topic? What difficulties might our project encounter? Is our plan feasible? Luckily, we encountered a lot of [kind scientists, stakeholders, students and former iGEM participants](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D) which helped us by answering these questions as well as by critically questioning our plans.

## April

Down to the final vote. Through her unwavering enthusiasm Tatia has gained a strong fellowship. The votes are counted and the decision is official: Our team wants to improve public health by augmenting STI testing rates. Making diagnostics more accessible and simple to carry out will encourage people to get tested and diagnosed. [Our vision: a simple at-home urine test on a strip, applicable without thermocycling, additional substances or trained personnel.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

## May

Turning dreams into plans. Once we had decided on the project, we dove right back into the literature to figure out the details. How can we achieve our goal? Which enzymes and materials do we need? How can we prove the designed mechanism functions? Who could advise us on these methods? Lots of work and good advice went into the project at this time of the year. Great news: The [wonderful Lukas Pelkmans Lab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D) invited us to work on our project in their laboratory!

## June

Exams… again! This semester went by so quickly.

## July

Let the fun begin. But safety first: Our wet lab team started with a [safety introduction](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety-and-security%27%29%20%7D%7D) in the laboratory. We are ready for our first steps in the lab.

### Week 07.07. - 11.07.2025

**LbCas12a Expression:**
We transformed the vector pDS113 into Rosetta2 cells, picked a colony and grew them over night. The next day we were ready to induce the protein expression with IPTG.
However, after running a gel electrophoresis to confirm expression, we were surprised by an unexpected pattern. The expression level of our protein appeared to be inexplicably low. So back to the start we go. We decided that we will conduct a test expression to verify which E. Coli strain is best suited to express our vector.

[Expression and Purification of LbCas12a protein](https://static.igem.wiki/teams/5686/lab/notebook/expression-and-purification-of-lbcas12a-proteins.pdf)

**PCR:** This week we performed the first PCR amplification and purification for each target gene of the different pathogen sequence. Next week, we plan to run an agarose gel to verify the PCR products.

[1. PCR sequence amplification (11.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/1-pcr-sequence-amplification-11-07-25-benchling.pdf)
[2. PCR Purification (11.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/2-pcr-purification-11-07-25-benchling.pdf)

### Week 14.07 - 18.07.2025

**PCR:** We performed gel electrophoresis for the genes of the different pathogen sequences. It worked successfully for most of them. The goal was to identify which genes could be used for the further progression of our experiments.

[3. Spectrophotometric DNA quantification (14.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/3-spectrophotometric-dna-quantification-14-07-25-benchling.pdf)
[4. Electrophoresis (15.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/4-electrophoresis-15-07-25-benchling.pdf)

### Week 21.07 - 25.07.2025

**LbCas12a Expression:** We conducted a test expression to conclude on the best E.coli strain suited for our vector. With fresh confidence that the initial decision to go with Rosetta2 cells was indeed the right one, we restarted large scale expression of pDS113 in Rosetta2 cells.
Since we were unable to determine the cause of the unsatisfactory gel results, we pursued in parallel the transformation and expression of an alternative vector, named pDS115, as a backup. By doing so, we would avoid further delays in case the second pDS113 expression would result in no yield.

[Expression and Purification of LbCas12a protein](https://static.igem.wiki/teams/5686/lab/notebook/00-for-replacing-expression-and-purification-of-lbcas12a-proteins.pdf)

**RPA:** After several weeks of delay due to waiting for the arrival of the RPA kit, we were finally able to start our first RPA experiments this week. We performed a symmetrical and an asymmetrical one. We also tried our first isothermal RPA without incubation, but unfortunately this first attempt did not work.

By isothermal RPA we actually mean a temperature and incubation time gradient test. Something we unfortunately noticed too late to correct in all our documents. Sorry for this concusion but keep this in mind for this page.

[RPA (21.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/rpa-21-07-25-benchling.pdf)
[Electrophoresis (22.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/electrophoresis-22-07-25-benchling.pdf)
[Isothermal RPA L1 HPV16 (24.07.2025)](https://static.igem.wiki/teams/5686/lab/notebook/1-isothermal-rpa-l1-hpv16-24-07-2025-benchling.pdf)
[3. Spectrophotometric DNA quantification of Asymmetrical RPA (25.07)](https://static.igem.wiki/teams/5686/lab/notebook/3-spectrophotometric-dna-quantification-of-asymmetrical-rpa-25-07-benchling.pdf)
[1. Asymmetrical RPA (25.07.25)](https://static.igem.wiki/teams/5686/lab/notebook/1-asymmetrical-rpa-25-07-25-benchling.pdf)

### Week 28.07.-31.07.2025

**LbCas12a Purification:** We were now ready to purify our protein which we had gained from the induction of the pDS113 vector. For this we performed a nickel pulldown, an overnight dialysis and TEV cleavage, an ion exchange chromatography and a size exclusion chromatography.
By running a gel electrophoresis we observed a double band, indicating two proteins of similar size. We deducted that the TEV cleavage was incomplete, leaving us with a mixed population of cleaved and uncleaved protein. Since this would not affect our future use of the protein we carried on and concentrated and aliquoted the protein. We adapted the protocol to avoid this in the future by adding the use of a MBP-Trap in tandem to the SEC column. A western blot confirmed the presence of the protein.
This process yielded 19.42 mg of protein.

To verify the identity of our protein, we sent a sample to the Functional Genomic Center at the University of Zurich for mass spectrometry. They were able to confirm the identity of the protein as LbCas12a. The proteins were freezed in liquid nitrogen and stored in a -80°C freezer.

In parallel we induced expression of the pDS115 vector with IPTG.

[Expression and Purification of LbCas12a protein](https://static.igem.wiki/teams/5686/lab/notebook/00-for-replacing-expression-and-purification-of-lbcas12a-proteins.pdf)
[Report Functional Genomics Center](https://static.igem.wiki/teams/5686/lab/notebook/report-functional-genomics-center-zurich.pdf)

**RPA:**
This week we tested different approaches with RPA. Unfortunately, both the symmetrical RPA with different dilutions and our first attempt with RPA for the syphilis pathogen sequence failed. The highlight, however, was that our first isothermal RPA was successful, meaning it works without any incubation time.

[Isothermal RPA](https://static.igem.wiki/teams/5686/lab/notebook/isothermal-rpa-benchling.pdf)
[failed Diluted RPA (28-29.07)](https://static.igem.wiki/teams/5686/lab/notebook/failed-diluted-rpa-28-29-07-benchling.pdf)
[RPA Syphilis](https://static.igem.wiki/teams/5686/lab/notebook/rpa-syphilis-benchling.pdf)

## August

### Week 04.08.2025 - 08.08.2025

**LbCas12a Purification:** We continued by purifying the proteins gained by the pDS115 expression. Again a nickel pulldown, an overnight dialysis and TEV cleavage, an ion exchange chromatography and a size exclusion chromatography were conducted. But this time we did add a MBP-trap in parallel to the SEC column to filter only for the correctly cleaved protein. This process yielded 0.3 mg of protein
We concentrated and aliquoted the purified protein. The proteins were freezed in liquid nitrogen and stored in a -80°C freezer.

[Expression and Purification of LbCas12a proteins](https://static.igem.wiki/teams/5686/lab/notebook/00-for-replacing-expression-and-purification-of-lbcas12a-proteins.pdf)

**RPA & PCR:** Unfortunately no good news:)
None of the RPA experiments we performed this week worked. We had to troubleshoot a bit and we asked our advisor for help. Meanwhile, we also performed PCR for HPV16 L1 gene and Syphilis polA and tmpA genes to obtain new PCR products, which was only partially successful.

[PCR sequence amplification (05.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/pcr-sequence-amplification-05-08-25-benchling.pdf)
[failed PCR sequence amplification (06.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/failed-pcr-sequence-amplification-06-08-25-benchling.pdf)
[PCR (07.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/pcr-07-08-25-benchling.pdf)
[Normal RPA (08.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/normal-rpa-8-8-25-benchling.pdf)
[Positive control of 2° RPA kit (05.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/positive-control-of-2-rpa-kit-05-08-25-benchling.pdf)
[RPA (11.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/rpa-11-08-25-benchling.pdf)

**DNA hybridization:** This week we finally started our first DNA hybridization experiment with fluorescence. The first attempt did not work as well as we hoped, that’s why we then adjusted a few parameters. By the end of the week, we were able to show that DNA hybridization to one of our pathogen sequences (HPV16 L1 gene) works, with both normal RPA and asymmetrical RPA.

[1. DNA hybridization with fluorescence](https://static.igem.wiki/teams/5686/lab/notebook/1-dna-hybridization-with-fluorescence-benchling.pdf)
[2. DNA hybridization with fluorescence to test asymmetrical RPA, RPA and PCR](https://static.igem.wiki/teams/5686/lab/notebook/2-dna-hybridization-with-fluorescence-to-test-asymmetrical-rpa-rpa-and-pcr-benchling.pdf)

### Week 11.08.2025 - 15.08.2025

**RPA & PCR:** Since the asymmetrical RPA was not working for a while and we needed more product for the DNA hybridization experiments, we tried doing PCR with RPA primers instead of PCR primers to produce RPA amplicons. Luckily, this worked. The asymmetrical RPA also worked again for the 1:10 and 1:20 forward:reverse primer dilutions by the end of the week. Meanwhile we also ran a PCR for HPV16 L1 gene.

[08.08 PCR](https://static.igem.wiki/teams/5686/lab/notebook/3-08-08-pcr-benchling.pdf)
[PCR to get RPA amplicons (14.08)](https://static.igem.wiki/teams/5686/lab/notebook/pcr-to-get-rpa-amplicons-14-08-benchling.pdf)
[8) RPA (14.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/8-rpa-14-08-25-benchling.pdf)
[10& Asymmetrical RPA L1 HPV16 (15.08.2025)](https://static.igem.wiki/teams/5686/lab/notebook/10-asymmetrical-rpa-l1-hpv16-15-08-2025-benchling.pdf)

**DNA hybridization:** In a next step we proved that DNA hybridization also worked with isothermal asymmetrical RPA. Isothermal means it can function without incubation at 37 degrees Celsius, which is an important criteria for our end product - the test on a paper strip for home-using.

[3. DNA hybridization with fluorescence to test specificity of the probe and isothermal asymmetrical RPA](https://static.igem.wiki/teams/5686/lab/notebook/3-dna-hybridization-with-fluorescence-to-test-specificity-of-the-probe-and-isothermal-asymmetrical-rpa-benchling.pdf)
[4. DNA hybridization with fluorescence to test specifity of the probe and asymmetrical RPA (without incubation)](https://static.igem.wiki/teams/5686/lab/notebook/4-dna-hybridization-with-fluorescence-to-test-specifity-of-the-probe-and-asymmetrical-rpa-without-incubation-benchling.pdf)

### Week 18.08.2025 - 22.08.2025

We started writing our famous WIKI page, that‘s why we performed less experiments in the lab this week.

**RPA:** The aim was to produce new RPA products with different primer dilutions, especially 1:50, for the DNA hybridization experiments. Last week only the 1:10 and 1:20 forward:reverse primer dilution worked. We tried two asymmetrical RPA reactions and even an asymmetrical PCR, but none of them worked. As the highlight of the week, our first normal RPA on the strip experiment worked right away. Asymmetrical RPA on the strip, however, did not work in the first attempt.

[11) Asymmetric + normal RPA (18.08)](https://static.igem.wiki/teams/5686/lab/notebook/11-asymmetric-normal-rpa-18-08-benchling.pdf)
[12) normal RPA on a paper strip (18.08)](https://static.igem.wiki/teams/5686/lab/notebook/12-normal-rpa-on-a-paper-strip-18-08-benchling.pdf)
[13) Asymmetrical RPA on a paper strip (19.08.25)](https://static.igem.wiki/teams/5686/lab/notebook/13-asymmetrical-rpa-on-a-paper-strip-19-08-25-benchling.pdf)
[14) Asymmetrical RPA L1 HPV16 (19.08.2025) 1:50 dilution only](https://static.igem.wiki/teams/5686/lab/notebook/14-asymmetrical-rpa-l1-hpv16-19-08-2025-1-50-dilution-only-benchling.pdf)
[15) Asymmetrical PCR to get RPA amplicons](https://static.igem.wiki/teams/5686/lab/notebook/15-asymmetrical-pcr-to-get-rpa-amplicons-benchling.pdf)

**DNA hybridization:** We designed a new specific probe for the pathogen sequence of HPV16 L1 gene with a different fluorophore. The goal was to test double DNA hybridization together with our first probe from the last two weeks. With two probes carrying different fluorophores, we should be able to see signals at two different wavelengths in the fluorescence readout, confirming double binding. First, we tested and confirmed that the new probe was specific to HPV16 and did not bind to the other five pathogen sequences (using their PCR products). We also confirmed that asymmetrical RPA works with the new probe. However, at the end of the week our first double hybridization experiment did not work as expected, so we will need to rethink our approach for the next steps.

[5. DNA hybridization with fluorescence to test specificity of the probe and asymmetrical RPA](https://static.igem.wiki/teams/5686/lab/notebook/5-dna-hybridization-with-fluorescence-to-test-specificity-of-the-probe-and-asymmetrical-rpa-benchling.pdf)
[6. DNA hybridization with fluorescence to test asymmetrical RPA](https://static.igem.wiki/teams/5686/lab/notebook/6-dna-hybrifization-with-fluorescence-to-test-asymmetrical-rpa-benchling.pdf)
[7. DNA double hybridization test with 2 different fluophores for asymmetrical RPA and normal RPA (first attempt)](https://static.igem.wiki/teams/5686/lab/notebook/7-dna-double-hybridization-test-with-2-different-fluophores-for-aymmetrical-rpa-and-normal-rpa-first-attempt-benchling.pdf)

### Week 25.08.2025 - 29.08.2025

Again, a lot of WIKI writing has to be done.

**PCR:** We performed a PCR to obtain new product, which was successful. We then diluted it to 1 ng and prepared a 10,000x dilution series from that (=0.1 pg), giving us fresh material to use for further experiments.

[5) 29.08 PCR](https://static.igem.wiki/teams/5686/lab/notebook/5-29-08-pcr-benchling.pdf)

**RPA:** Since our asymmetrical RPA product for the HPV16 L1 gene was running low, we produced new ones with the 1:10 and 1:20 dilutions that had worked in previous experiments. Unfortunately, the RPA did not work this time, so we will need to repeat it next week.

[16) Asymmetrical RPA L1 HPV16 (29.08.2025) 1:10 and 1:20 dilutions](https://static.igem.wiki/teams/5686/lab/notebook/16-asymmetrical-rpa-l1-hpv16-29-08-2025-1-10-and-1-20-dilutions-benchling.pdf)

**DNA hybridization:** We troubleshooted with our advisor Cheng-Han on our first double DNA hybridization approach. With new input, we tried a second attempt, but it also failed. Therefore, we ran a third and fourth attempt, this time only with normal RPA products of the L1 gene of HPV16 since our asymmetrical RPA products of the L1 gene were limited and we did not want to waste them. After the third attempt, we suspected that the second fluorophore was the problem. It seemed much more light-sensitive than the first fluorophore, as we could only see a readout for the first one. For the fourth attempt, we prepared a fresh aliquot of the oligo directly from the original stock, as the older aliquots were probably exposed to light too often. It seemed we were right about the problem. This time, the double binding in DNA hybridization with normal RPA was successful - a real milestone for us. Next week, we will perform the same experiment with asymmetrical RPA.

[8. DNA double hybridization test with 2 different fluophores for asymmetrical RPA and normal RPA (second attempt)](https://static.igem.wiki/teams/5686/lab/notebook/8-dna-double-hybridization-test-with-2-different-fluophores-for-aymmetrical-rpa-and-normal-rpa-second-attempt-benchling.pdf)
[9. DNA double hybridization test with 2 different fluophores for normal RPA (third attempt)](https://static.igem.wiki/teams/5686/lab/notebook/9-dna-double-hybridization-test-with-2-different-fluophores-for-normal-rpa-third-attempt-benchling.pdf)
[10. DNA double hybridization test with 2 different fluophores for normal RPA (fourth attempt)](https://static.igem.wiki/teams/5686/lab/notebook/10-dna-double-hybridization-test-with-2-different-fluophores-for-normal-rpa-forth-attempt-benchling.pdf)

## September

### Week 01.09.2025 - 05.09.2025

WIKI, WIKI, WIKI writing… and some experiments were done:

**RPA:** We carried out both symmetrical and asymmetrical RPA because we were running low on product for further experiments (LbCas12a and DNA hybridization). Since the RPA did not work last week, we had to start a new attempt. This time we used new primers and a fresh PCR template to make sure the problem was not caused by them.

[17) Symmetric RPA L1 HPV16 (02.09.25)](https://static.igem.wiki/teams/5686/lab/notebook/17-symmetric-rpa-l1-hpv16-02-09-25-benchling.pdf)
[18) Asymmetrical RPA L1 HPV16 (02.09.2025) for 1:10 dilution](https://static.igem.wiki/teams/5686/lab/notebook/18-asymmetrical-rpa-l1-hpv16-02-09-2025-for-1-10-dilution-benchling.pdf)

**DNA hybridization:** Finally, we also performed a successful DNA hybridization with double binding for asymmetrical RPA :)

[11. DNA double hybridization test with 2 different fluophores for asymmetrical RPA (fifth attempt)](https://static.igem.wiki/teams/5686/lab/notebook/11-dna-double-hybridization-test-with-2-different-fluophores-for-asymmetrical-rpa-fifth-attempt-benchling.pdf)

### Week 08.09.2025 - 12.09.2025

We almost didn’t want to mention it anymore, but there was still a lot of WIKI writing left. Luckily, it finally started to take shape.

**CRISPR-Cas:** After a lot of brainstorming, we developed a protocol to perform a standard LbCas12a assay using a ssDNA reporter labeled with ROX on one end and the quencher BHQ2 on the other. This setup allowed us to test whether we could observe the CRISPR reaction directly by eye through a color change in the tube. In our first attempt, we added too little of the ssDNA reporter, which meant that no visible color change occurred. We therefore repeated the experiment with a higher concentration of the reporter. This time, the color was clearly visible before the reaction started, and after incubation we could observe a shift from blue to violet/pink, indicating successful cleavage.

The reaction worked best at 32 °C and 37 °C, where the color change was visible after only 30 minutes. At room temperature and 25 °C the assay also worked, but required longer incubation times of about 60-90 minutes. Our next step will be to adapt this Cas12a assay to work directly on a paper strip.

[1. LbCas12a Assay (12.09.2025)](https://static.igem.wiki/teams/5686/lab/notebook/1-lbcas12a-assay-12-09-2025-benchling.pdf)
[2. LbCas12a Assay (12.09.2025, the second experiment that day)](https://static.igem.wiki/teams/5686/lab/notebook/2-lbcas12a-assay-12-09-2025-the-second-experiment-that-day-benchling.pdf)

### Week 15.09.2025 - 19.09.2025

**RPA:** We performed a new symmetrical RPA since much of our product had already been used for the LbCas12a experiments. We needed to repeat the standard LbCas12a assay for the dry lab, as they require quantitative measurements. Unfortunately, the first RPA run was not very successful: only the reaction with 1 ng PCR template worked, and we did not obtain enough product to purify samples for the Nanodrop analysis. Therefore, we decided to repeat the symmetrical RPA a second time, this time ensuring that we produced enough material to also purify a portion and measure the DNA concentration with the Nanodrop for quantitative data.

[22) symmetrical RPA (16.09.2025)](https://static.igem.wiki/teams/5686/lab/notebook/20-symmetrical-rpa-16-09-2025-benchling.pdf)
[23) symmetrical RPA (17.09.2025)](https://static.igem.wiki/teams/5686/lab/notebook/21-symmetrical-rpa-17-09-2025-benchling.pdf)

**DNA hybridization:** We carried out our first experiment on a lateral flow strip (Milenia Biotec) to test double DNA hybridization using two probes, one labeled with FAM and the other with Biotin. We applied two different probe concentrations (100 µM and 0.1 pM) and expected results within 5 minutes. The experiment was successful on the very first attempt with the higher concentration (100 µM): clear test lines appeared within 5 minutes for both RPA inputs, 1 ng and 0.0001 ng.

[DNA Hybridization on a Lateral Flow Strip (1)](https://static.igem.wiki/teams/5686/lab/notebook/dna-hybridization-on-a-lateral-flow-strip-1-benchling.pdf)

### Week 22.09.2025 - 26.09.2025

**RPA:** The goal of this experiment was to calculate the overestimation factor when measuring asymmetrical RPA (ratios 1:10, 1:20, and 1:50) on the Nanodrop. As pointed out by Cheng-Han, purification of asymmetrical RPA products does not yield only the single-stranded RPA amplicons but also includes excess primers. This leads to an artificially high concentration measurement. To calculate the overestimation factor, two approaches were suggested:

* Measuring RPA products on the Nanodrop with and without the enzyme pellet.
* Estimating the amplicon concentration by analyzing gel band intensities.

We followed the first suggestion. We prepared standard RPA reactions (with all materials) and control reactions (with all materials except the enzyme pellet), purified both in the same way, and measured concentrations with the Nanodrop.

[Nanodrop overestimation calculation (22.09.2025)](https://static.igem.wiki/teams/5686/lab/notebook/nanodrop-overestimation-calculation-22-09-2025-benchling.pdf)

**CRISPR-Cas:** This week we performed additional LbCas12a experiments to provide the Dry Lab with quantitative measurements for validating their models. In parallel, we also carried out the Cas12a assay on lateral flow strips (Milenia Biotec). The strip experiment worked successfully, confirming our setup. With this, we conclude our Cas experiments, as we are approaching the final phase of our project.

[Quantitative LbCas12a measurements for Dry Lab](https://static.igem.wiki/teams/5686/lab/notebook/quantitative-lbcas12a-measurements-for-drylab-benchling.pdf)
[LbCas12a Assay on a Lateral Flow Strip](https://static.igem.wiki/teams/5686/lab/notebook/lbcas12a-assay-on-a-lateral-flow-strip-benchling.pdf)

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Engineering
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![Header Image](https://static.igem.wiki/teams/5686/header/engineering-and-cycles.webp)
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# Engineering & Cycles

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[Introduction](#Introduction)

[RPA](#RPA)

[Cycle 1 - RPA for CRISPR-Cas Method](#RPA-Cycle-1)
[Cycle 2 - Asymmetrical RPA for DNA-Hybridization Method](#RPA-Cycle-2)
[Cycle 3 - Temperature and time gradient experiments](#RPA-Cycle-3)
[Cycle 4 - RPA on the strip](#RPA-Cycle-4)
[Cycle 5 - RPA sensitivity test](#RPA-Cycle-5)

[DNA Hybridization](#DNA-Hybridization)

[Cycle 1 - Establishing Single-Probe Hybridization](#DNA-Cycle-1)
[Cycle 2 – Room-temperature workflow and probe specificity](#DNA-Cycle-2)
[Cycle 3 - Optimizing asymmetrical RPA primer ratios and inputs](#DNA-Cycle-3)
[Cycle 4 - Achieving double hybridization (two fluorophores)](#DNA-Cycle-4)
[Cycle 5 – DNA Hybridization Detection on a Strip](#DNA-Cycle-5)

[CRISPR-Cas12a](#CRISPR)

[Vector Transformation & Protein Expression](#CRISPR-Vector)

[Cycle 1 - pDS113 in Rosetta2](#CRISPR-Vector-Cycle-1)
[Cycle 2 - Test Expression](#CRISPR-Vector-Cycle-2)
[Cycle 3 - pDS113 in Rosetta2 as well as pDS115 in Mach1](#CRISPR-Vector-Cycle-3)

[Protein Purification](#CRISPR-Purification)

[Cycle 1 - Purification of the pDS113-Derived Protein](#CRISPR-Purification-Cycle-1)
[Cycle 2 - Purification of the pDS115-Derived Protein](#CRISPR-Purification-Cycle-2)

[LbCas12a Assay](#CRISPR-Assay)

[Cycle 1 - Initial Activity Verification of LbCas12a](#CRISPR-Assay-Cycle-1)
[Cycle 2 - Activity Evaluation at Different Temperatures and Incubation Times](#CRISPR-Assay-Cycle-2)
[Cycle 3 – LbCas12a Assay on a Lateral Flow Strip](#CRISPR-Assay-Cycle-3)

[Data for Dry Lab](#Dry-Lab)

[Cycle 1 - Quantitative Data Collection for Dry Lab Models](#Dry-Lab-Cycle-1)

[References](#item-z)
{% endset %}
{% block page\_content %}

## Introduction

Discover our step-by-step development process of RPA, DNA hybridization, and CRISPR-Cas experiments, as well as the purification of LbCas12a. This page documents the entire process - from amplification to detection and ultimately towards the creation of a paper-based test. It also explains the reasoning behind our design decisions, the adjustments we made after each test, and the lessons we learned.
We followed the engineering design cycle **Design → Build → Test → Learn** across multiple rounds of experiments. Each iteration refined our methods and informed the next stage of development. Through this systematic progression, we moved from basic amplification methods to reliable detection, leading to a functional diagnostic system.

## RPA

### Cycle 1 - RPA for CRISPR-Cas Method

#### Design

Even though primers and DNA sequences for all six pathogens were designed on Benchling [(see the page “Experiments and Protocols”)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) and checked on BLAST for specificity, we decided to first experimentally focus on one pathogen. In this way we aimed to establish the whole circuit working for one pathogen and then apply to the rest. We selected HPV16, specifically its L1 gene, because a clinical study [[1]](#ref1) demonstrated that HPV16 can be reliably detected in first-void urine at sufficient concentrations, supporting its suitability for non-invasive diagnostic testing. It is worth mentioning that in this study HPV DNA was extracted from the urine samples prior to detection. Among our three HPV16 target sequences, we selected the L1 gene because it showed the highest amplification yield in our Nanodrop measurement (225.2 ng/μL) and is frequently used in diagnostic assays, supporting both assay reliability and clinical relevance.[(see more on our ”Notebook” page)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).
Our first goal was to test the three RPA primer pairs designed for RPA and identify the most efficient one. A second goal was to determine whether there was a difference between using DNA at a concentration of 1 ng/μL and 0.0001 ng/μL (= 0.1 pg/μL), since patient samples may contain very low concentrations of pathogen DNA, and the sensitivity of our test depends on detecting these low levels.

#### Build

The RPA primers were all designed following the instructions provided by TWIST, as we used the TwistAmp basic kit for our RPA reactions. The designed primers were then run in BLAST to be sure that they were unique for HPV16 and wouldn’t bind to other pathogens, thereby preventing the chances of false-positives results. Find the primers in the [Experiments and Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)page. We ordered the primers from Microsynth and DNA sequences from Twist Bioscience. The kit for RPA was bought from TWIST.

#### Test

We conducted the RPA following the protocols detailed on our [Experiments and Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page. A description of the experiments can be found on the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) page.

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-1.webp)

**Figure 1**: Gel showing RPA amplification of the HPV16 L1 gene with two template concentrations (1 ng and 0.1 pg).

Legend:

1. = 1° Amplicon (1 ng/μL)
2. = 1° Amplicon (0.1 pg/μL)
3. = 1° Amplicon negative control
4. = 2° Amplicon (1 ng/μL)
5. = 2° Amplicon (0.1 pg/μL)
6. = 2° Amplicon negative control
7. = 3° Amplicon (1 ng/μL)
8. = 3° Amplicon (0.1 pg/μL)
9. = 3° Amplicon negative control

#### Learn

The results are very dim because we added too little of our sample, namely only 1 μL out of 50 μL, mistakenly following the same loading volume as for PCR gels. Our instructor Cheng-Han advised us to load at least 10% of the product in the gel, which here would have been 5 μL. We followed his advice in the next experiments.

Based on the gel results, no clear difference was observed between amplifying 1 ng/μL or 0.1 pg/μL, so we decided to perform all subsequent experiments using the lower concentration of 0.1 pg/μL, as this better reflects real-world conditions. According to Table 4 from a study by Baud et al. [[2]](#ref2), HPV DNA concentrations in clinical urine samples can range from only a few dozen to a few hundred copies per microliter, depending on sample processing. Since our longest RPA amplicon is approximately 400 bp, 0.1 pg/µL corresponds to roughly 200–300 copies/µL, making this dilution a realistic approximation of the low DNA concentrations found in clinical urine samples. Additionally, the differences between the amplicons in the gel were minor, so we arbitrarily selected the first amplicon for all subsequent experiments.

One mistake we made was not measuring the Nanodrop concentration after purifying the product. Doing so would have allowed us to detect finer differences in concentration. We did not perform this measurement due to some confusion about the validity of Nanodrop readings for RPA, although we later learned that this concern mainly applies to asymmetrical RPA and can be addressed ([see “Learn” part of Cycle 2 - Asymmetrical RPA for DNA-hybridization method](#RPA-Cycle-2-Learn)).

This experiment served as the foundation for our future work and signifies that the first part of the CRISPR detection method is complete.

### Cycle 2 - Asymmetrical RPA for DNA-Hybridization Method

#### Design & Build

The CRISPR method can directly work with double-stranded RPA amplicons, which were already achieved in the first cycle. However, the DNA-hybridization method requires the amplicons to be single-stranded as we learned in a conversation with our instructor Cheng-Han [(see the Attribution page)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D). Double-stranded DNA would prevent proper hybridization and reduce signal efficiency [[3]](#ref3). That is why we performed asymmetrical RPA, to get single-stranded RPA amplicons. Our goal here was to set up a parameter screening with varying primer concentrations to understand whether the gene sequence had a preference for the forward or reverse primers, and select the optimal ratio. Literature [[4]](#ref4) and discussions with our instructor, led us to test three different ratios, namely 1:10, 1:20 and 1:50 (forward:reverse). We had also considered using only one primer and none of the other, but our instructor then explained that we needed an exponential phase of growth at least initially, which is only possible with both primers working together to elongate the strands. Our hypothesis was that, as stated in the paper [[4]](#ref4), the concentration of 1:20 would work best and there would not be a preference between the forward and reverse DNA strands.
An important consideration to note is that asymmetrical RPA drops the yield compared to regular RPA [[4]](#ref4), which is why after every asymmetrical RPA we performed, we purified the product and loaded 10 μL of it in the gels.

#### Test

We conducted the asymmetrical RPA following the protocols detailed on our [Experiments and Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page. A description of the experiments can be found on the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) page.
Results were checked with agarose gel and NanoDrop.

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-2.webp)

**Figure 2**: Gel showing amplification products from asymmetrical RPA with different forward:reverse primer ratios (1:10, 1:20, 1:50) for the HPV16 L1 gene.

Legend:

1. =10 reverse: 1 forward
2. = 1 reverse : 10 forward
3. = 20 reverse: 1 forward
4. = 50 reverse: 1 forward
5. = 1 reverse: 50 forward
6. = Positive control: RPA with normal conditions
7. = Negative control 1 (10 reverse : 1 forward)
8. = Negative control 2 (1 reverse: 10 forward)

| Well numbers | Content 1 | Concentration (ng/μL) |
| --- | --- | --- |
| 1 | 10 reverse : 1 forward , 0.1 pg/μL | N/A due to technical error |
| 2 | 1 reverse : 10 forward , 0.1 pg/μL | 1265.17 |
| 3 | 20 reverse : 1 forward , 0.1 pg/μL | 1917.39 |
| 4 | 1 reverse : 20 forward ,0.1 pg/μL | 2015.26 |
| 5 | 50 reverse : 1 forward ,0.1 pg/μL | 2050.51 |
| 6 | 1 reverse : 50 forward , 0.1 pg/μL | 1732.99 |
| 7 | Positive control (symmetrical RPA with normal conditions) | 3111.1 |
**Table 1**: Nanodrop results of the amplification products from asymmetrical RPA with different forward:reverse primer ratios (1:10, 1:20, 1:50) for the HPV16 L1 gene. Negative results concentrations were not included due to a mistake done while measuring.

#### Learn

The results were successful for all asymmetrical products. However, there is indeed a difference in the gel between the DNA strands outputs and between the different primer ratios. Diluted forward primers show only one line at the expected location of approximately 400bp but are dimmer than the positive control. While, diluted reverse primers show three bands, which could indicate either there was the formation of DNA secondary structures or dimerization of single-stranded RPA products. To test that we would have to denature them, but due to time constraints we didn’t do that, so we cannot conclude whether one strand performs better than the other. To be safe, we proceeded using only diluted forward primers, which also guided the design of DNA-hybridization primers to target the antisense strand.

Regarding primer ratios, agarose gel brightness and NanoDrop results suggested that 1:20 and 1:50 (forward:reverse) worked better than 1:10, with 1:20 appearing brightest. The first well may have been dimmer due to a technical error, so we could not conclusively determine the best ratio.

It is worth mentioning that the NanoDrop results for asymmetrical RPA amplicons had to be corrected later due to two reasons. First, when using NanoDrop, it was forgotten to switch from the DNA-50 mode to the DNA-33 mode, thus we were measuring ssDNA with the dsDNA function, inflating our results. To correct this, we multiplied the results obtained using DNA-50 by 0.66.Second, excess primers added for amplification were co-purified, further inflating concentrations. Our instructor Cheng-Han, advised us to do an experiment to calculate this overestimation factor by comparing purified asymmetrical RPA amplicons with purified asymmetrical RPA reactions without the enzyme pellet. The results of this experiment showed overestimation factors of 9.57 for 1:10, 15.97 for 1:20 and 26.66 for 1:50 forward:reverse primer ratio ([see the page „Notebook: Nanodrop overestimation calculation (22.09.2025)“](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#September-Week-22-09-26-09-2025-nanodrop-overestimation-calculation)). To obtain more accurate results, we divided the measured concentrations by these factors. However, this experiment could be further refined by accounting for the actual volume recovered during purification, as a lower recovery volume would result in a more concentrated product. Such an implementation was not carried out due to time constraints.

In conclusion, this was our first step toward establishing DNA-hybridization as a detection strategy.

### Cycle 3 - Isothermal RPA

#### Design & Build

The goal is to run a parameter screening for different temperatures because this is crucial information for us, as we want our self-test to operate at room temperature (RT). RPA was accomplished by first testing a range of four temperatures (RT = 23.8°C, 25°C, 32°C, 37°C) and two incubation times (20min and 30min). A master mix was prepared so that the only parameters that changed were indeed the temperature and the incubation time. We conducted the RPA following the protocols detailed on our [Experiments and Protocols: Isothermal RPA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page. A description of the experiments can be found on the [Notebook: Isothermal RPA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) page.

#### Test

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-3.webp)

**Figure 3**: Gel showing initial results at different temperatures and incubation times.

Legend:

1. = Negative control, RT, 30min
2. = RT, 20min
3. = 25°C, 20min
4. = 32°C, 20min
5. = 37°C, 20min
6. = 25°C, 30min
7. = RT, 30min
8. = 32°C, 30min
9. = 37°C, 30min

#### Learn

The results on the gel don’t look clear, that is why we decided to attempt it again, this time refining our parameters. We included a positive control and another incubation time variable, namely 60 minutes. This time around, the gel results were much clearer, except for sample 14, which was the only one prepared using a different stock of DNA. We thus assumed that the DNA aliquot used, had an issue.

What we learned from the agarose gel and NanoDrop results is that: RPA is robust across a wide temperature range (25-37 °C), but when performed at room temperature (~23 °C) it requires a longer incubation time to achieve amplification. At 20 minutes, yields at room temperature were very low, but after 30 minutes they reached levels similar to those obtained at 25-37 °C. This indicates that for our project, RPA can be reliably performed without equipment at ambient conditions, provided the reaction time is adjusted to around 30 minutes.

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-4.webp)

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-5.webp)

**Figure 4 & 5**: Gels showing optimized experiment at different temperatures and incubation times.

1. = Positive control: 39°C, 20min
2. = Negative control: 39°C, 20min, no DNA
3. = RT, 20min
4. = 25°C, 20min
5. = 32°C, 20min
6. = 37°C, 20min
7. = RT, 30min
8. = 25°C, 30min
9. = 32°C, 30min
10. = 37°C, 30min
11. = RT, 60min
12. = 25°C, 60min
13. = 32°C, 60min
14. = 37°C, 60min (first lane of gel on the right)

| Temperature [°C] | Incubation time [min] | Order of gel | DNA amount [ng/μL] |
| --- | --- | --- | --- |
| 39 | 20 | 1 (positive control) | 106 |
| 39 | 20 | 2 (negative control) | 33.4 |
| RT / ~23.8°C | 20 | 3 | 12.9 |
| 25 | 20 | 4 | 38.7 |
| 32 | 20 | 5 | 34.1 |
| 37 | 20 | 6 | 83.5 |
| RT / ~23.8°C | 30 | 7 | 87.3 |
| 25 | 30 | 8 | 75.2 |
| 32 | 30 | 9 | 103.5 |
| 37 | 30 | 10 | 121.4 |
| RT / ~23.8°C | 60 | 11 | 108.3 |
| 25 | 60 | 12 | 96.1 |
| 32 | 60 | 13 | N/A |
| 37 | 60 | 14 | 51.1 |
**Table 2**: Final visualization of RPA products with varying temperature and incubation times.

### Cycle 4 - RPA on the strip

#### Design & Design

After having achieved the first steps for our two detection methods and experimented with different temperatures and incubation times, the next big step was to test RPA on the strip. We are happy to say that for this, we used the protocol “RPA on Paper“, written by the iGEM 2017 Munich team, “CascAID” [(see the Experiments and Protocols page)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D). Our goal was to make this work for symmetrical and asymmetrical RPA, which is necessary for our two detection methods, even though their protocol was only designed for the former. A description of the experiments can be found on the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) page.

#### Test

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-6.webp)

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-7.webp)

**Figure 6 & 7**: Agarose gels showing symmetrical and asymmetrical RPA results directly performed on paper.

Legend left:

1. 2-5 = 1 ng/μL symmetrical RPA
2. 6-9 = 0.1 pg/μL symmetrical RPA

Legend left:

1. 7-10 = 1 ng/μL asymmetrical RPA
2. 11-14 = 0.1 pg/μL asymmetrical RPA

| Concentration of DNA | DNA amount [ng/μL] |
| --- | --- |
| 1 ng/μL | 3.7 |
| 0.1 pg/μL | -0.8 |
**Table 3**: NanoDrop results for symmetrical RPA on the strip. We didn’t measure the amount of the asymmetrical RPA on the strip as it didn’t appear in the gel at all.

#### Learn

Symmetrical RPA on paper strips yielded only a 3-fold increase, which is technically amplification but represents a very low efficiency compared to expectations. This highlights the need for protocol optimization. Upon discussion with our instructor Cheng-Han, we identified several potential issues and troubleshooting strategies.

First, to minimize enzyme degradation, he recommended avoiding repeated lyophilization of the enzyme pellet. Instead, only the primers and rehydration buffer should be freeze-dried directly on the strip, while the enzymes and probe are added afterwards.

Second, harvesting efficiency may be a limiting factor, since our current workflow involves soaking the paper in water and purifying the eluate. To evaluate this, we could compare NanoDrop results from liquid-phase RPA reactions with those from liquid-phase RPA reactions subsequently applied to the strip and then purified.

Finally, to ensure complete wetting of the strip and reduce evaporation losses, it was suggested to load twice the amount of reaction mixture onto the strip and optimize the reaction volumes.

While these insights provide a clear roadmap for optimization in the next **Design → Build → Test → Learn** cycle, we were not able to fully implement them within the available timeframe.

### Cycle 5 - Diluted RPA

#### Design & Build

One challenge for our prototype is the lysis of pathogens, ensuring that our methods can access their DNA and reliably detect even very small amounts. To address this, we tested a broader range of diluted DNA concentrations beyond the already tested 0.1 pg/µL. Specifically, we extended our experiments to 0.01 pg/µL and 1 fg/µL for both symmetrical and asymmetrical RPA. These reactions were performed with diluted DNA templates using our existing stocks of primers and DNA. We conducted the RPA following the protocols detailed on our [Experiments and Protocols: Diluted RPA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page. A description of the experiments can be found on the [Notebook: Failed diluted RPA (28-29.07) page.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#July-Week-28-07-31-07-2025-failed-diluted-RPA).

#### Test

When we ran the reactions, they stopped working altogether. On agarose gels, we observed either smears or no bands at all. In some cases, even the DNA ladder appeared unreliable, which suggests that both the reaction itself and our gel conditions could be contributing to the unclear results.

#### Learn

As a first troubleshooting step, we suspected a degraded DNA aliquot and prepared fresh aliquots from the original purchased tube, but the problem persisted ([Notebook: diluted RPA troubleshooting template (30.07))](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D). Since ordering a new DNA stock would have delayed our project by at least a week, we instead prepared fresh primer aliquots, but results did not improve ([Notebook: diluted RPA troubleshooting primers (31.07)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)). Moreover, opening a new RPA kit ([Notebook: normal RPA (8.8.25)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)) and testing alternative templates (Notebook: failed RPA Syphilis (31.07) and RPA (11.08.25)) also failed, suggesting that the issue was not specific to the target DNA.

We further learned that gel quality control played a role: inconsistent ladders were linked to buffer replacement frequency, so we adopted our instructor’s advice to refresh the gel buffer every three days when running many gels, instead of only once a week. Another lesson was to prepare individual aliquots of nuclease-free water for each experiment to reduce the risk of contamination. As an alternative way to get RPA amplicons for our next applications, namely our two detection methods, we performed a PCR using RPA primers to generate RPA amplicons ([Notebook: PCR to get RPA amplicons (14.08)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)), which the first time produced successful NanoDrop and agarose gel results. However, this method did not work again afterwards.

![agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-8.webp)

**Figure 8**: Gel showing RPA performance with highly diluted DNA templates to test detection sensitivity.

Legend:

1. 5-6 = PCR L1 HPV16 with RPA primers

Finally, after following the advice to mix and tap the aliquots after freeze-thawing, we obtained a smeared but correctly sized band with HPV16, showing that proper template handling was critical for RPA success ([Notebook: RPA (14.08.25)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)). We later realized that a more effective approach would have been to sequence the DNA stock and to purchase a replacement tube as soon as RPA experiments stopped working.

Due to time constraints, we didn’t return to testing highly diluted RPA templates.

## DNA Hybridization

### Cycle 1 - Establishing Single-Probe Hybridization

#### Design

At the beginning, our aim was simple: demonstrate that DNA hybridization works as a detection method for our target pathogen sequence. We designed complementary DNA oligonucleotides, with one serving as the template (RPA or PCR amplicons of the HPV16 L1 gene) and the other carrying a fluorescent label. To explore how sequence length and stability influence sensitivity and specificity in DNA hybridization, we tested three variants: a 15-mer probe with LNA modifications, a 20-mer DNA probe, and a 25-mer DNA probe, each labeled with the fluorophore Dy681.To provide templates, we prepared products from symmetrical RPA (dsDNA), asymmetrical RPA (ssDNA), and PCR. All probes and primers were checked with BLAST to ensure specificity to HPV16.

#### Build

Each probe was rehydrated to 100 µM stock and used at 1:1000 dilution (working ~100 nM). For every reaction we prepared a 10 µL hybridization mix: 1 µL probe, 1 µL 0.5 M NaCl, 7 µL nuclease-free water, and 1 µL template. Templates came from symmetrical RPA (dsDNA; 1 ng input; 1:1 primers), asymmetrical RPA (ssDNA-enriched; 1:50 forward:reverse), and PCR (≈200–210 ng product). Negative controls contained water instead of template.

#### Test

In our first attempt, we combined the probes with symmetrical RPA, asymmetrical RPA, and PCR products. We denatured at 95 °C for 5 min, cooled to 25 °C, and incubated ≥30 min. Products were run on 1.5% agarose with ROTI® Gel Stain and were read on the fluorescence imager.

![agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/img-0622.webp)

**Figure 9**: Gel showing hybridization with 15-/20-/25-mer probes on RPA/PCR templates.

Legend:

1. = Ladder
2. = Asymmetrical RPA and diluted 15-mer LNA oligo + fluorophore (Dy681)
3. = Asymmetrical RPA and diluted 20-mer oligo + fluorophore (Dy681)
4. = Asymmetrical RPA and diluted 25-mer oligo + fluorophore (Dy681)
5. = Symmetrical RPA and diluted 15-mer LNA oligo + fluorophore (Dy681)
6. = Symmetrical RPA and diluted 20-mer oligo + fluorophore (Dy681)
7. = Symmetrical RPA and diluted 25-mer oligo + fluorophore (Dy681)
8. = PCR and diluted 15-mer LNA oligo + fluorophore (Dy681)
9. = PCR and diluted 20-mer oligo + fluorophore (Dy681)
10. = PCR and diluted 25-mer oligo + fluorophore (Dy681)
11. = Negative control for diluted 15-mer LNA oligo + fluorophore (Dy681)
12. = Negative control for diluted 20-mer oligo + fluorophore (Dy681)
13. = Negative control for diluted 25-mer oligo + fluorophore (Dy681)

#### Learn

Single-probe hybridization was successful across all template types. Symmetrical RPA (lanes 5-7) gave the brightest signals, while asymmetrical RPA (lanes 2-4) and PCR (lanes 8-10) showed weaker but still visible bands. Among the probes, the 15-mer with LNA (lanes 2,5 and 8) performed best. Based on these results, we decided to adopt the LNA probe as our standard and move on to testing more challenging conditions such as at room-temperature hybridization and specificity against other pathogens.

### Cycle 2 – Room-temperature workflow and probe specificity

#### Design

For our test to be practical on a paper strip, hybridization needed to work at room temperature without a denaturation step. In addition, we had to prove that our probe was specific to HPV16 and would not cross-react with DNA from other pathogens. We therefore designed an experiment to test asymmetrical RPA products at room temperature and to run hybridization reactions against PCR products of several non-target pathogens (Chlamydia, Gonorrhoea, Syphilis, Trichomonas).

#### Build

For asymmetrical RPA at RT we used 1:50 forward:reverse primers, incubated at ~23 °C for ≥35 min. For specificity, we used PCR products (~200 ng) from the non-targets. Hybridization mixes remained 10 µL with the 15-mer LNA probe at 1:1000, but for the asymmetrical RPA tube we sometimes increased the volume of the DNA to 2 µL and reduced water accordingly (6 µL) to compensate for lower amplification yield.

#### Test

For the pathogen PCR products, we used the standard profile of 95 °C denaturation for 5 min followed by 25 °C annealing for ≥30 min. For the asymmetrical RPA, we ran the reaction at room temperature only, without denaturation. Products were analyzed on 1.5% agarose gels with fluorescence readout and were read on the fluorescence imager.

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/img-0623.webp)

**Figure 10**: Specificity test of the 15-mer LNA probe against non-target pathogen sequences, and hybridization from asymmetrical RPA at RT without denaturation.

Legend:

1. = Ladder
2. = PCR L1 HPV16
3. = PCR 23S Chlamydia
4. = PCR E6 HPV16
5. = PCR 16S Gonorrhoea
6. = PCR polA Syphilis
7. = PCR tmpA Syphilis
8. = PCR TVK3 Trichomonas
9. = PCR 18S Trichomonas
10. = Asymmetrical RPA (with too much fluorescence)

#### Learn

Two important outcomes came from this cycle:

1. The specificity test succeeded - none of the non-target pathogen templates showed fluorescence (lanes 3-9), confirming that our probe binds only to HPV16 L1 (lane 2).
2. In the first attempt, the asymmetrical RPA product at RT did not yield visible signals, likely because the amplification was too weak under these conditions. After redesigning the setup with adjusted probe handling, the second attempt succeeded: fluorescence signals were visible (lane 15), confirming that hybridization can work at room temperature without a denaturation step. In this run, excess fluorophore and a damaged gel limited the image quality, but the positive signal clearly demonstrated feasibility.

### Cycle 3 - Optimizing asymmetrical RPA primer ratios and inputs

#### Design

We hypothesized that the ratio of forward to reverse primers in asymmetrical RPA influences the production of ssDNA and thus the success of hybridization. To increase ssDNA yield, we compared two asymmetrical RPA primer ratios: 1:10 (forward : reverse) and 1:20 (forward : reverse), combined with different template inputs (1 ng/µL and 0.1 pg/µL ).

#### Build

We produced fresh asymmetrical RPA reactions at 1:10 and 1:20. Hybridization kept the 15-mer LNA probe at 1:1000 in a 10 µL mix: 1 µL probe, 1 µL 0.5 M NaCl, 7 µL water, 1 µL template (in some repeats we increased template to 3 µL and reduced water to 5 µL to improve sensitivity). Normal RPA and a no-template control were run in parallel.

#### Test

Thermal profile remained 95 °C 5 min → 25 °C ≥30 min (unless explicitly RT only). Gels were 1.5%, read by fluorescence.

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/img-0624.webp)

**Figure 11**: Comparison of hybridization efficiency with 1:10 and 1:20 forward:reverse primer ratios in asymmetrical RPA.

Legend:

1. = Ladder
2. = Asymmetrical RPA 1:10 (forward:reverse) with 1ng DNA template + diluted oligo with fluorophore (Dy681)
3. = Asymmetrical RPA 1:10 (forward:reverse) with 0.1 pg DNA template + diluted oligo with fluorophore (Dy681)
4. = Asymmetrical RPA 1:20 (forward:reverse) with 1ng DNA template + diluted oligo with fluorophore (Dy681)
5. = Asymmetrical RPA 1:20 (forward:reverse) with 0.1 pg DNA template + diluted oligo with fluorophore (Dy681)
6. = Symmetrical RPA with fluorophore (Dy681)
7. = Negative control without template

#### Learn

The 1:10 (forward:reverse) asymmetrical RPA (lanes 2-3) gave robust hybridization with 1 ng and with 0.1 pg DNA template. The 1:20 (forward:reverse) asymmetrical RPA (lanes 4-5) worked with 1 ng but failed with 0.1 pg DNA template, indicating reduced ssDNA availability. We standardized on asymmetrical RPA 1:10 (forward:reverse) for downstream work.

### Cycle 4 - Achieving double hybridization (two fluorophores)

#### Design

We introduced a second Cy7-labeled probe with a distinct emission wavelength to enable two-site hybridization on the same target, facilitating multiplexing and providing stronger confirmation of true positives. In addition, this probe design allows one site to function as an anchoring probe, potentially biotin-labeled for attachment to the lateral flow strip, while the other can be visualized through a gold nanoparticle-labeled detection probe. To reduce photobleaching and maintain signal intensity, the high-λ channel was read first.

#### Build

Both probes were prepared at 100 µM stocks and used at 1:1000 dilution. For single-template double binding, we used a 10 µL hybridization mix: 1 µL Probe A (low-λ), 1 µL Probe B (high-λ), 1 µL 0.5 M NaCl, 6 µL water, 1 µL template. For low-yield asymmetrical RPA we sometimes used 2 µL template and 5 µL water. Templates were symmetrical RPA 1:1 (forward:reverse) (1 ng/µL) and asymmetrical RPA 1:10 (forward:reverse) (both 1 ng/µL and 0.1 pg/µL). Early runs used working aliquots; after suspected dye degradation we switched to fresh aliquots directly from the original stock and handled them under light protection.

#### Test

We followed the denaturation profile for hybridization (95 °C 5 min → 25 °C ≥30 min) when template allowed. Fluorescence was recorded high-λ first, then low-λ, to minimize bleaching. Gels were 1.5% agarose.

![Two agarose gels](https://static.igem.wiki/teams/5686/lab/engineering/img-0625.webp)

**Figure 12 & 13**: Double hybridization with two probes labeled with different fluorophores (Dy681 & Cy7) on symmetrical RPA products.

Legend:

1. = Ladder
2. = Symmetrical RPA with first diluted oligo with fluorophore (Dy681)
3. = Symmetrical RPA with second diluted oligo with fluorophore (Cy7)
4. = Symmetrical RPA with both diluted oligos with fluorophore (Dy681 & Cy7)
5. = Negative control without template

![Two agarose gels](https://static.igem.wiki/teams/5686/lab/engineering/img-0664.webp)

**Figure 14 & 15**: Double hybridization with two probes labeled with different fluorophores (Dy681 & Cy7) on asymmetrical RPA products.

Legend:

1. = Ladder
2. = Asymmetrical RPA 1:10 (forward:reverse) with 1ng DNA template and both diluted oligos with fluorophores (Dy681 & Cy7)
3. = Asymmetrical RPA 1:10 (forward:reverse) with 0.1 pg DNA template and both diluted oligos with fluorophores (Dy681 & Cy7)
4. = Negative control without template

#### Learn

In the first three double DNA hybridization experiments, only one channel (Dy681) consistently showed signal. The high-λ fluorophore (Cy7) had likely been over-exposed during previous handling. After switching to fresh, light-protected aliquots and adjusting the readout order, double hybridization was successful. On symmetrical RPA, fluorescence was clearly visible in both channels (Figures 12 & 13, lane 4). The same result was then achieved with asymmetrical RPA at a 1:10 forward:reverse ratio using both 1 ng and 0.1 pg inputs (Figures 14 & 15, lanes 2-3). These results demonstrated that two independent probes can bind simultaneously to dsDNA (symmetrical RPA) as well as ssDNA-enriched (asymmetrical RPA) templates under our experimental conditions.

### Cycle 5 – DNA Hybridization on a Strip

#### Design

After establishing reliable single- and double-probe DNA hybridization in gels, the next step was to transfer the method onto a paper strip to evaluate whether hybridization could also be detected in a lateral flow test. We used the Milenia Biotec HybriDetect kit and designed the experiment with two different probes, one labeled with FAM and the other with Biotin, so that double binding could be read out directly on the strip. In this test, anti-FAM antibodies conjugated with gold nanoparticles bind the FAM label and, together with capture of the biotinylated partner at the test line, generate the visible red signal. To systematically test sensitivity, we combined two probe concentrations (100 µM and 0.1 pM) with two symmetrical RPA product concentrations (1 ng/ µL and 0.1 pg/µL).

#### Build

Both FAM- and Biotin-labeled oligonucleotides were rehydrated to 100 µM stock. For annealing, we prepared 20 µL mixes containing 2 µL FAM probe, 2 µL Biotin probe, 2 µL 0.5 M NaCl, 12 µL nuclease-free water, and 2 µL template (symmetrical RPA product). The mixes were denatured at 95 °C for 5 min, cooled to 25 °C, and incubated for ≥30 minutes. Afterwards, the 20 µL hybridization products were pipetted directly onto the sample application pad of the HybriDetect strips. Strips were then placed upright into 100 µL assay buffer and left at room temperature until the bands developed.

#### Test

Within 2-5 minutes, control and test lines appeared depending on the probe and template input.

![Four lateral flow strips: first and third one with two lines, second and fourth one with just second line.](https://static.igem.wiki/teams/5686/lab/engineering/img-5411.webp)

**Figure 16**: Detection of hybridized DNA on lateral flow strips with different probe and template concentrations.

Legend:

1. Symmetrical RPA (1 ng) + 100 µM FAM/Biotin labeled probes → positive signal
2. Symmetrical RPA (1 ng) + 0.1 pM FAM/Biotin labeled probes → no signal
3. Symmetrical RPA (0.1 pg) + 100 µM FAM/Biotin labeled probes → positive signal
4. Symmetrical RPA (0.1 pg) + 0.1 pM FAM/Biotin labeled probes → no signal

#### Learn

This experiment showed that DNA hybridization can successfully be detected on a paper strip, marking an important milestone toward a paper-based diagnostic. At 100 µM probe concentration, valid test results were achieved for both undiluted (1 ng) and diluted (0.1 pg) symmetrical RPA products. At 0.1 pM probe concentration, however, no signal was visible, indicating that the input was too low to be detected on the strip under our current conditions. These results highlight the importance of sufficient probe concentration for reliable readout and provide a clear direction for optimization in future work.

## CRISPR-Cas12a

### Vector Transformation and Protein Expression

We decided to express and purify the Cas protein used in our experiments ourselves. The vectors, materials and necessary equipment were kindly provided to us by the [Jinek Laboratory at the University of Zurich](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D). Christelle Chanez, member of the Jinek Lab, assisted us in the process.

### Cycle 1 - pDS113 in Rosetta2

#### Design

Two Cas12a vectors were available, pDS113 and pDS115. Since pDS115 contained a GFP addition which was not necessary for our experiments, we went ahead with the pDS113 vector. The vector was sent for sequencing before use to make sure it was intact.
From the lab's experience with these specific vectors, we knew that the expression worked well in Rosetta2 competent cells. We therefore chose to conduct the pDS113 expression in Rosetta2 cells.

![Vector Map of pDS113](https://static.igem.wiki/teams/5686/lab/engineering/ec-fig-17-pds113.webp)

![Vector Map of pDS115](https://static.igem.wiki/teams/5686/lab/engineering/ec-fig-18-pds115.webp)

**Figure 17 & 18**: pDS113 and pDS115 vector map

##### Build

We transformed the pDS113 vector into Rosetta2 cells according to the [heat shock protocol](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-heatshock-benchling.pdf).
The following day we observed a number of healthy colonies on the plate, indicating that the transformation was successful and the bacteria had acquired the antibiotic resistance encoded in the vector.

We picked a colony and inoculated our starter culture overnight with 50 µg/mL kanamycin and 33 µg/mL chloramphenicol. The starter culture was then used to inoculate 6L of LB medium with 50 µg/mL kanamycin the next day. We observed the OD600 of our culture with regular measurements and cooled for 30min at 18°C with shaking once OD600 reached 0.61. To be able to test successful expression later, we took a sample of the uninduced culture.
Finally we induced expression with 0.4 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) for overnight expression. The following day we were able to harvest our protein. After spinning down the culture we resuspended the pellet in our lysis buffer.

##### Test

To verify the correct expression of our protein we ran a SDS-PAGE with the uninduced and induced probe. We expected a band at 148 kDa in the induced sample, but not in the uninduced. However, only an extremely faint band was visible in the induced sample. Instead it seemed as if something was retained in the wells of the induced samples.

![SDS-PAGE](https://static.igem.wiki/teams/5686/lab/engineering/ec-fig-19.webp)

**Figure 19**: SDS-PAGE gel LbCas12a pDS113 Rosetta2 uninduced / induced.

##### Learn

These test results could have different explanations. We first went through our protocol to exclude human error. We then concluded that the expression was unexpectedly low. We also considered the issue to be the lysis of the cells. In this case, the additional lysis in the Maximator Cell homogenizer might resolve the problem. Low protein expression might be explained if the chosen bacterial strain, Rosetta2, was not well suited to express this specific vector. We therefore decided to conduct a test expression with different bacterial strains, to investigate how well they express our vector.

### Cycle 2 - Test Expression

#### Design

To determine the best-suited bacterial strain for expressing the pDS113 vector carrying LbCas12a, we conducted a test expression. The following strains were chosen: BL21 Star, BL21 RIL, Rosetta2, T7 Express, and BL21 Arabinose-Induced. All of these strains are compatible with T7-driven expression systems, but differ in codon usage compatibility, protein folding support, and overall expression efficiency.

##### Build

Chemically competent cells of each strain were transformed with the pDS113 vector and plated on LB agar containing the appropriate antibiotic. Single colonies were picked and grown overnight in LB. The following day, cultures were inoculated into fresh medium and grown until mid-log phase. Protein expression was induced with 0.5 mM IPTG, and the cultures were incubated for an additional 3 hours at 37°C. Samples were collected for both induced and uninduced conditions.

##### Test

After expression, cells were harvested by centrifugation and lysed using a standard lysis buffer. The lysates were then incubated with Ni-NTA magnetic beads to test for His-tagged protein binding. Elution fractions were analyzed by SDS-PAGE to evaluate the level of LbCas12a expression. We compared band intensities between strains and between induced and uninduced conditions.

![SDS-PAGE](https://static.igem.wiki/teams/5686/lab/engineering/ec-fig-20.webp)

**Figure 20**: SDS-PAGE gel LbCas12a pDS113 expression in BL21 star, T7 express and Rosetta2

##### Learn

All strains expressed LbCas12a to varying degrees, but Rosetta2 showed the clearest difference between uninduced and induced samples. In Rosetta2 lysates, the LbCas12a band was more intense and more protein was successfully pulled down using the nickel beads, indicating better expression and solubility. This confirmed the initial choice for Rosetta2 as the most suitable host strain for the large-scale protein purification of LbCas12a.

### Cycle 3 - pDS113 in Rosetta2 & pDS115 in Mach1

#### Design

Following the results of the test expression we proceeded with large-scale cultures of Rosetta2 cells.

However, since we were unsure of what caused the unexpected pattern in the initial gel, we wanted to prepare for the case that the large-scale expression with the pDS113 vector would not be successful and we would not be able to obtain sufficient protein for our future experiments. We therefore decided to start the transformation and expression of the alternative vector pDS115 in Mach1 cells, while working in parallel on the large-scale expression and purification of the pDS113 culture.

We also wanted to make sure that the problem does not lie in the cell lysis. Therefore we used a different lysis machine for each culture.

##### Build

Transformation, expression and induction were carried out according to protocol. For the pDS113 culture the Maximator Cell homogenizer was used again to lyse the cells. The pDS115 culture was lysed with the Cell Lyser 1.

##### Test

The different lysis method did not impact the outcome of the uninduced vs induced gels. We proceeded with the purification and did not encounter further indication that the protein expression level was as low as the initial gel indicated.

##### Learn

The unexpected gel pattern therefore remains unexplained, but did not result in any consequences for the success of the protein recovery.

### Protein Purification

### Cycle 1 - Purification of the pDS113-Derived Protein

#### Design

The protein purification was carried out according to protocol, and the vectors provided by the Jinek Lab.

##### Build

The purification process included a Ni-NTA pulldown, an overnight TEV cleavage and dialysis, an ion-exchange chromatography and finally a size-exclusion chromatography.

##### Test

When running the SDS-PAGE gel after the size exclusion chromatography we observed a double band around 148 kDa. We were unable to separate these two protein populations due to their small differences in size. It seemed plausible that the TEV cleavage had been incomplete and the two bands corresponded to the cleaved and uncleaved protein.

Before the protein was used in further experiments a sample was sent to mass spectrometric analysis at the Functional Genomics Center Zurich to confirm our theory. They were able to confirm the identity of the protein as the cleaved LbCas12a and also noted the presence of a second, slightly bigger protein which might correspond to the uncleaved protein. Based on this we assumed that our purification had yielded a mixed population of TEV-cleaved and uncleaved LbCas12a protein.

![SDS-PAGE](https://static.igem.wiki/teams/5686/lab/engineering/ec-fig-21.webp)

**Figure 21**: LbCas12a pDS113 SEC chromatogram and corresponding SDS-PAGE gel

##### Learn

We learned that the TEV-cleavage conducted during the overnight dialysis was insufficient to correctly cleave all of the product. To avoid mixed populations in further purifications we would need to separate the cleaved protein from the uncleaved.

### Cycle 2 - Purification of the pDS115-Derived Protein

#### Design

To avoid the same issue as in the first purification, we applied what we learned and added an additional separation step to the purification. As the uncleaved protein contains a malE fragment, an MBP trap would be able to separate it from the cleaved protein.

##### Build

We followed the same protocol for the purification, but added a MBP Trap in tandem to the size exclusion column.

##### Test

Gels were carried out after Ni-NTA-pulldown, dialysis and size exclusion chromatography. Again a double band was observed in the SDS-PAGE gel after dialysis, but only a single band was visible in the final gel after the SEC and MBP trap chromatography.

![SDS-PAGE](https://static.igem.wiki/teams/5686/lab/engineering/fig-22.webp)

**Figure 22**: LbCas12-eGFP, SDS-PAGE gel after SEC and MBP trap

##### Learn

Our conclusion that the double band represented a mixed population of cleaved and uncleaved LbCas12a protein has been further confirmed by these results. The MBP Trap resolved the problem by filtering for the cleaved protein. Our product now consists of pure cleaved LbCas12a and the protocol has been adapted for future use.

### LbCas12a Assay

### Cycle 1: Initial Activity Verification of LbCas12a

#### Design

To verify the activity of our LbCas12a protein, we used a synthetic single-stranded DNA (ssDNA) reporter labeled with the fluorophore ROX at the 5’ end and the quencher BHQ2 at the 3’ end (ROX-N12-BHQ2). In its intact form, the quencher suppresses the ROX fluorescence. Upon cleavage of the reporter by the activated LbCas12a nuclease, the fluorophore is separated from the quencher, resulting in a measurable and visible fluorescence signal at around 602 nm. This allowed us to visually demonstrate the activity and specificity of our system.

##### Build

The LbCas12a protein, the crRNA and the ssDNA reporter (ROX-N12-BHQ2) were used at a concentration of 100 µM. For a single experiment we used a 50 µL Cas12a mixture: 0.25 µL crRNA, 0.21 µL LbCas12a, 0.1 µL ssDNA reporter, 0.5 µL RNase inhibitor (40 U/ µL), 5 µL NEBuffer 2.1 (10X), 7.5 µL symmetrical RPA product of the HPV16 L1 gene, and 36.44 µL nuclease-free water.

##### Test

To ensure proper mixing, we first combined all of the reagents except the LbCas12a protein and the crRNA in a single tube and vortexed the mixture. Afterwards, we added the Cas12a protein and crRNA and mixed gently by pipetting up and down. Then we incubated the tube. All in all we tested 3 different incubation temperatures (25°C, 30°C and 37°C) as well as different incubation times (25°C for 60 and 90 minutes, 30°C for 30 and 60 minutes and 37°C for 30 minutes).

##### Learn

During this experiment, we noticed that the concentration of the ssDNA reporter was initially too low to allow visual detection of a color change. The reaction mixture was transparent at the start, and no color shift could be observed by eye. However, under UV light, a faint red fluorescence confirmed that the Cas12a reaction had occurred. To test whether the low reporter concentration was the limiting factor, we added 9 µL of additional ROX-labeled reporter (100 µM) and 1 µL NEBuffer 2.1 to one of the previously incubated reactions.

After 30 minutes at 37°C, the mixture turned from the initial blue to a violet. This change was clearly visible without any UV light or other specialized equipment. Repeating the incubation for a further 30 minutes did not result in a stronger color, suggesting the reaction had reached saturation.

Still, the color intensity was not strong enough for robust visual detection. We concluded that increasing the reporter concentration further would improve visibility and consistency of the signal in future assays.

![SDS-PAGE](https://static.igem.wiki/teams/5686/lab/engineering/img-0279.webp)

**Figure 23**: Visible color change in the Cas12a assay using a ROX-BHQ2 reporter at different times points.

Legend:

1. = Before incubation with the new ssDNA reporter concentration
2. = After 30 minutes incubation
3. = After 60 minutes incubation

### Cycle 2 - Activity Evaluation at Different Temperatures and Incubation Times

#### Design

Building on our first assay, where a visible color change was observed only at 37°C, we set out to test whether LbCas12a retains activity at other temperatures, including room temperature. For this we increased the concentration of the ssDNA reporter to improve signal intensity. By systematically varying incubation temperature and time, we aimed to identify the temperature range in which LbCas12a remains active.

##### Build

For this assay, the LbCas12a protein, the crRNA and the ssDNA reporter were all used at a concentration of 100 µM. For each reaction, we used 50 µL of the following Cas12a mixture: 0.25 µL crRNA, 0.21 µL LbCas12a, 8 µL ssDNA reporter, 0.5 µL RNase inhibitor (40 U/ µL), 6 µL NEBuffer 2.1 (10X), 7.5 µL symmetrical RPA product of the HPV 16 L1 gene, and 27.54 µL nuclease-free water.

##### Test

The LbCas12a protein and the crRNA were gently mixed in a separate tube by pipetting (vortexing was avoided to protect enzyme activity). The ssDNA reporter, the RNase inhibitor, the NEBuffer 2.1, the symmetrical RPA product and the nuclease-free water were combined and vortexed briefly. Finally, the two mixtures were put together and mixed gently before incubation.

The reactions were incubated at four different temperatures 37°C (positive control), 32°C, 25°C, and room temperature (~ 23 °C). Each condition was monitored by photographing the tubes at the start, and again after 30, 60, and 90 minutes of incubation.

##### Learn

The reaction tubes which were incubated at 37°C and 32°C showed a clear colour shift after 30 minutes of incubation. After another 30 minutes of incubation, the reaction tubes incubated at 25°C and at room temperatures also showed a change in colour. From this experiment we learned that LbCas12a functions most efficiently at elevated temperatures, producing strong and rapid signals at 37°C and 32°C. Nonetheless, the enzyme also retains activity at 25°C and even at room temperature, although it requires longer incubation times of at least one hour to achieve visible results. This finding suggests that the assay is feasible under ambient conditions, which is particularly valuable for our intended application. In the next cycle, we will focus on getting quantitative results for our dry lab with the help of a plate reader. The plan will also be to integrate a negative control, which was not done here.

| Temperature / Sample | 30 minutes of incubation | 60 minutes of incubation | 90 minutes of incubation |
| --- | --- | --- | --- |
| Room temperature (approx. 23 °C, Tube 5) | - | + | + |
| 25°C (Tube 4) | - | + | + |
| 32°C (Tube 3) | + | + | + |
| 37°C (positive control, Tube 2) | + | + | + |
**Table 4**: Table showing color change in Cas12a assay tubes incubated at different temperatures (23 °C, 25 °C, 32 °C, 37 °C) after 30, 60, and 90 minutes.

![Four pictures of each four Eppendorf tubes with numbers 2-4 and violet liquid content](https://static.igem.wiki/teams/5686/lab/engineering/img-0278.webp)

**Figure 24**: Visible color change in the Cas12a assay using a ROX-BHQ2 reporter at different temperatures and time points.

Legend:

1. Picture 1: Before incubation
2. Picture 2: After 30 minutes of incubation
3. Picture 3: After 60 minutes of incubation
4. Picture 4: After 90 minutes of incubation

### Cycle 3 – LbCas12a Assay on a Lateral Flow Strip

#### Design

After verifying Cas12a activity in tube-based assays, the next step was to demonstrate whether target detection could also be achieved in a lateral flow format. We used the Milenia Biotec HybriDetect kit together with a FAM-/Biotin-labeled ssDNA reporter. To systematically assess sensitivity, we tested two concentrations of reporter (50 nM and 100 µM) in combination with two input amounts of symmetrical RPA product (1 ng and 0.1 pg, equivalent to a 10,000× dilution).

##### Build

We prepared two separate mixes to protect Cas12a activity:

* Tube A: 1 µL FAM-Biotin-labeled ssDNA reporter (0.05 µM = 50 nM), 2 µL NEBuffer 2.1, 2 µL RPA product, 13 µL nuclease-free water (total 18 µL).
* Tube B: 1 µL crRNA (0.5 µM) + 1 µL LbCas12a protein (5 µM).

After vortexing tube A briefly and mixing tube B gently by pipetting, tube B was added to tube A to reach a total volume of 20 µL. The mixtures were incubated at 37 °C for 20 minutes. The full reaction volumes were then applied to the sample pads of lateral flow strips, which were placed upright into 100 µL of assay buffer at room temperature.

##### Test

We prepared four different strips:

* Strip 1: Symmetrical RPA (1 ng input) + 50 nM ssDNA reporter.
* Strip 2: Symmetrical RPA (0.1 pg input) + 50 nM ssDNA reporter.
* Strip 3: Symmetrical RPA (1 ng input) + 100 µM ssDNA reporter.
* Strip 4: Symmetrical RPA (0.1 pg input) + 100 µM ssDNA reporter.

After 2-5 minutes, all strips displayed the control line to confirm validity. The test line was visible on each strip, demonstrating successful Cas12a-mediated target recognition and cleavage.

![Four Milenia HybriDetect strips with two lines on each, intensity of the second line decreasing](https://static.igem.wiki/teams/5686/lab/engineering/img-5493.webp)

**Figure 25**: Cas12a detection on Milenia HybriDetect strips with different reporter and RPA input concentrations.

Legend:

1. Strip 1: Symmetrical RPA (1 ng input) + 50 nM FAM/Biotin labeled ssDNA reporter
2. Strip 2: Symmetrical RPA (0.1 pg input) + 50 nM FAM/Biotin labeled ssDNA reporter
3. Strip 3: Symmetrical RPA (1 ng input) + 100 µM FAM/Biotin labeled ssDNA reporter
4. Strip 4: Symmetrical RPA (0.1 pg input) + 100 µM FAM/Biotin labeled ssDNA reporter

##### Learn

This experiment confirmed that LbCas12a detection can be combined with lateral flow readout. According to literature [[5]](#ref5), 50 nM reporter should be sufficient, and indeed this worked reliably with both high and diluted RPA inputs. However, even at the higher reporter concentration (100 µM), results were equally positive, suggesting robustness of the system. Importantly, clear signals developed within just 2 minutes across all tested setups. This shows that the Cas12a assay is not only functional but also rapid and compatible with a paper-based format, providing a strong proof of concept for its diagnostic potential.

## Data for Dry Lab

### Cycle 1 - Quantitative Data Collection for Dry Lab Models

#### Design

Building on our experience from the LbCas12a assays, we proceeded to collect a range of data points to support the dry lab in testing their models. This experimental cycle consisted of five distinct experiments, all conducted simultaneously due to logistical constraints. Since the Pelkmans lab did not have access to a plate reader, we collaborated with the Plückthun lab to obtain quantitative readouts. The five experiments focused on measuring background fluorescence, kinetic parameters, the fluorescence signal threshold, a calibration factor, and enzyme denaturation characteristics.

#### Build

We conducted five assays, each targeting a specific parameter of the LbCas12a system. All reactions used a 100 μM stock concentration of the ssDNA reporter (ROX-N12-BHQ2). The experiments were assembled as follows:

*Experiment 1: Reporter Concentration Optimization*
Two versions of the Cas12a reaction were prepared, differing in the volume of ssDNA reporter used: 8 μL (positive control) and 4 μL. All other components remained constant.

*Experiment 2: Background Fluorescence*
LbCas12a reactions were assembled without DNA template to measure background fluorescence levels. We performed five such reactions, to enhance the reliability of the results.

*Experiment 3: Fluorescence Threshold Determination*
RPA product was diluted to create a series of samples with the following concentrations: undiluted (1 ng/μL), 1:10, 1:100, 1:1’000, 1:10’000, and 1:100’000. These dilutions were then incorporated into standard Cas12a reaction mixtures.

*Experiment 4: Free Fluorophore Calibration*
Two Cas12a reactions were assembled to generate free fluorophores. One was treated with 2 μL of 0.5 M EDTA directly after the assembly. This was used as a negative control. Then both of them were incubated for 60 minutes at 37 °C. The EDTA was added in order to stop the enzyme activity. After the 60 minutes incubation time, we also added 2 μL of 0.5 M EDTA into the other previously untreated reaction. The reaction where we added the EDTA after the incubation was then used to create a dilution series for the calibration curve generation.

*Experiment 5: Enzyme Denaturation*
Aliquots of LbCas12a enzyme sufficient for four reactions were incubated at elevated temperatures (45 °C and 55 °C) for 10 minutes to induce partial denaturation. Control aliquots remained at standard temperature. All samples were then incorporated into LbCas12a assays.

##### Test

*Experiment 1:*
Reactions with 4 μL and 8 μL of the ssDNA reporter were incubated at 37 °C. Fluorescence readings were taken at 5, 15, and 30 minutes to compare signal intensity and determine the impact of reporter concentration on the detection sensitivity.

*Experiment 2:*
The five no-DNA control reactions were incubated under standard conditions (37°C, 30 minutes), and fluorescence was measured to assess background signal levels.

*Experiment 3:*
All six dilutions of the RPA product were tested under standard reaction conditions (37 °C, fixed reaction volume). Fluorescence output was measured to determine the minimal DNA input needed to surpass background fluorescence, effectively establishing the system’s threshold.

*Experiment 4:*
The reaction treated with EDTA after the incubation (now containing free fluorophores) was serially diluted. The fluorescence was measured across the dilution series to generate a calibration curve, allowing conversion of raw fluorescence values into quantitative output. The other, undiluted reaction was measured as well, providing a reference point.

*Experiment 5:*
Denatured enzyme samples were used in reactions incubated at 37 °C for either 45 or 75 minutes. Control reactions with fresh enzymes followed the same protocol. Fluorescence measurements allowed comparison of enzyme activity post-heat treatment, providing insight into thermal stability and optimal handling conditions.

##### Learn

With these experiments, we were able to determine a range of parameters for our dry lab models.

*Experiment 1:*
In this experiment, our aim was to determine the kinetic parameters of the LbCas12a enzyme. However, due to a misunderstanding between the wet lab and dry lab teams, we only obtained data for two substrate concentrations, which is insufficient for a reliable parameter estimation. To overcome this limitation, we consulted a published study [[4]](#ref4) that reports kinetic values for LbCas12a under various experimental conditions. From these, we selected the conditions most comparable to our own setup and used the corresponding parameters for our reaction model. Further details can be found on our [Reaction Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D#reaction-model) page.

*Experiment 2:*
It was crucial for us to determine the background fluorescence in the absence of target DNA. This was the only way to accurately calculate the fluorescence generated by the reaction itself.

*Experiment 3:*
With the dilution experiment, we were able to determine the experimental threshold that must be exceeded to detect a signal. Based on this, our [Reaction Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D#reaction-model) allowed us to establish the limit of detection (LOD), defined as the minimum number of DNA copies required to generate a detectable signal and thereby indicate the presence of an infection.

*Experiment 4:*
This experiment provided the necessary data to determine a linear calibration factor of 130.73 a.u./nM. Using this factor, we were able to calculate the concentration of cleaved reporter molecules from the measured fluorescence.

*Experiment 5:*
The Dry Lab has chosen a different method than initially intended to predict the test’s shelf life. Consequently, the results of this experiment were not used. For more details, see the [Reaction Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D#reaction-model) page.

## References

1. Van Keer S, Peeters E, Vanden Broeck D, et al. Clinical and analytical evaluation of the RealTime High Risk HPV assay in Colli-Pee collected first-void urine using the VALHUDES protocol. Gynecol Oncol. September, 2021;162(3):575-83. Available from:
   [doi: 10.1016/j.ygyno.2021.06.010](https://doi.org/10.1016/j.ygyno.2021.06.010).
2. Vorsters, A., Van den Bergh, J., Micalessi, I. et al. Optimization of HPV DNA detection in urine by improving collection, storage, and extraction. Eur J Clin Microbiol Infect Dis 33, 2005–2014 (2014). Available from:
   [doi: 10.1007/s10096-014-2147-2](https://doi.org/10.1007/s10096-014-2147-2).
3. Dally, S., et al. Single-Stranded DNA Catalyzes Hybridization of PCR-Products to Microarray Capture Probes. PLOS ONE, 9(7), e102338 (2014). Available from:
   [doi: 10.1371/journal.pone.0102338](https://doi.org/10.1371/journal.pone.0102338).
4. Wang Y, Wang Y, Luo L, et al. Visual detection of Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme. RSC Adv. 2019;9(52):30350-7. Available from:
   [doi: 10.1039/c9ra05709a](https://doi.org/10.1039/c9ra05709a).
5. Zhang C, Li Z, Chen M, et al. Cas12a and lateral flow strip-based test for rapid and ultrasensitive detection of spinal muscular atrophy. Biosensors. May 14, 2021;11(5):154. Available from:
   [doi: 10.3390/bios11050154](https://doi.org/10.3390/bios11050154).
6. Nalefski EA, Kooistra RM, et al. Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets. Nucleic Acids Res. March 13, 2024;52(8):4502-22. Available from:
   [doi: 10.1093/nar/gkae152](https://doi.org/10.1093/nar/gkae152).

{% endblock %}

{% extends "layout-sidebar.html" %}
{% block title %}
Experiments & Protocols
{% endblock %}
{% block headimage %}
![Header Image](https://static.igem.wiki/teams/5686/header/experiments2.webp)
{% endblock %}
{% block header %}

# Experiments & Protocols

{% endblock %}
{% set sidebarcontent %}
[Introduction](#Introduction)
[RPA](#RPA)
[LbCas12a](#LbCas12a)
[DNA Hybridization](#DNA-Hybridization)
[Combination of the amplification and detection](#Combination)
[Multiplexing](#Multiplexing)
[Additional protocols and sequences](#Additional)

[References](#item-z)
{% endset %}
{% block page\_content %}

## Introduction

On this page you can find the reasoning and structure of our experimental workflow.
You can also find all protocols we worked with attached under each section. Have fun :)

## RPA

To be able to detect extremely low amounts of DNA in a sample, the diagnostic tool would need to contain a DNA amplification step in addition to the detection mechanism. Our research showed that Recombinase Polymerase Amplification (RPA) would work best for our purpose. RPA is fast, works at a constant low temperature, and is therefore very well suited for a test that relies on as few resources as possible.
[[1]](#ref1)

We first confirmed that the symmetrical RPA works for our target sequence, HPV16. Following this success, we proceeded to test it with asymmetrical RPA. In contrast to symmetrical RPA which produces double-stranded DNA (dsDNA), the asymmetrical RPA results in single-stranded DNA (ssDNA). ssDNA would later be required for our final diagnostic test, if we were to use DNA hybridization as the detection method. In contrast, symmetrical RPA is used in combination with CRISPR-Cas12a as a detection method, because the CRISPR-Cas12a enzyme binds to dsDNA. The experiments with asymmetrical RPA yielded excellent results as well.

We then demonstrated that RPA also works at room temperature. This is an important feature for future at-home testing, since users cannot easily perform incubations at higher temperatures.

Next, we investigated whether RPA can be performed directly on a paper strip. The symmetrical RPA on the strip experiment worked on the first attempt. Unfortunately, asymmetrical RPA on a strip did not work on our first try and still needs optimization. We now aim to optimize the conditions for asymmetrical RPA on paper.

[Protocol Asymmetrical RPA](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-asymmetric-rpa-benchling.pdf)
[Protocol Symmetrical RPA](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-symmetrical-rpa-benchling.pdf)
[Protocol Purification for single-stranded DNA](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-purification-for-single-stranded-dna-benchling.pdf)
[Protocol RPA on a strip](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-rpa-on-a-paper-strip-benchling.pdf)
[Protocol Nanodrop overestimation calculation](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-nanodrop-overestimation-calculation-benchling.pdf)

## LbCas12a

To assure specificity and a clear test result, we explored the use of Cas12a as a detection mechanism for DNA target sequences.

To do so, we expressed and purified the LbCas12a protein ourselves with the assistance of Christelle Chanez from the [Jinek Laboratory at the University of Zurich](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D). The expression vectors were provided by the Jinek Laboratory.

A test expression was conducted to determine the best suitable E. Coli strain for our vector (pDS113). Then a large-scale expression was conducted in Rosetta2 cells and the protein was purified. In parallel an alternative vector (pDS115) was expressed in Rosetta2 cells and then later purified. See [Engineering and Cycles](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)) for more information on this decision.

CRISPR-Cas12a is among the most widely used and established detection approaches for STI diagnostics
[[2]](#ref2)
. We found many publications describing its use, which is why we initially planned to rely on CRISPR-Cas for our final test. However, as our project progressed, we developed the idea of DNA hybridization, which turned out to be better suited for our circumstances (see below). Still, to be able to compare the novel method to the conventional approach, we decided to test this method as well. We began with a standard Cas12a assay in a reaction tube, where we aimed to detect a clear color change by eye. For this we used the protein derived from the pDS113 vector. After confirming that this setup worked successfully, we also performed additional Cas experiments to provide quantitative measurements for the Dry Lab, helping them verify their models. Finally, we moved on to test CRISPR-Cas on a paper strip using the Milenia Biotec HybriDetect kit. This last Cas experiment also worked and gave us clear results, rounding off our Cas12a tests.

[Protocol LbCas12a (pDS113) Expression and Purification](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-lbcas12a-pds113-expression-and-purification.pdf)
[Protocol LbCas12a (pDS115) Expression and Purification](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-lbcas12a-pds115-expression-and-purification.pdf)
[Protocol Heatshock](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-heatshock-benchling.pdf)
[Protocol Miniprep (Plasmid DNA purification)](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-miniprep-plasmid-dna-purification-benchling.pdf)
[Protocol Test expression (small scale expression)](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-test-expression-small-scale-expression-benchling.pdf)
[Protocol LbCas12a Assay](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-lbcas12a-assay-benchling.pdf)
[Protocol Quantitative LbCas12a Measurements for Dry Lab](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-quantitative-lbcas12a-measurements-for-drylab-benchling.pdf)
[Protocol LbCas12a Assay on a Lateral Flow Strip](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-dna-hybridization-on-a-lateral-flow-strip-benchling.pdf)

## DNA Hybridization

As an alternative detection method for our project we came up with the novel idea of using DNA hybridization. While CRISPR-Cas is very specific, it requires proteins, guide RNAs, and complex reaction conditions. This makes it more expensive and sensitive to temperature and storage changes
[[3]](#ref3)
. In contrast, DNA hybridization only needs a DNA probe, which is very stable and cheap to synthesize
[[4]](#ref4). For these reasons we concluded that DNA hybridization would be the preferred detection method for a paper-based home test, given it is comparably sensitive and specific. To be able to be conclusive on this hypothesis, we proceeded to experiment with both methods. We have shown that single DNA hybridization (DNA hybridization with one binding) works for products of symmetrical, asymmetrical and even isothermal RPA products from the pathogen sequence of HPV16. We also designed a second probe with a different fluorophore to test double hybridization, meaning that two probes bind at the same time, while each of them can be detected at different wavelengths. Whereas the first double DNA hybridization attempt did not work, we successfully confirmed that the new probe is specific. After several more trials, we finally achieved double DNA hybridization using two different oligos, each labeled with a distinct fluorophore, on both symmetrical and asymmetrical RPA products of the HPV16 L1 gene. The detection of both signals marked an important milestone in our project. As a next step, we aimed to transfer the entire setup onto a lateral flow strip, which worked on the first attempt. This brings us one step closer to developing a paper-based home test.

[Protocol DNA hybridization with fluorescence](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-dna-hybridization-with-fluorescence-benchling.pdf)
[Protocol DNA Hybridization on a Lateral Flow Strip](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-dna-hybridization-on-a-lateral-flow-strip-benchling.pdf)

## Combination of the amplification and detection

To ensure compatibility we conducted experiments on the detection of the probes with the product of the RPA experiments as a substrate. We did not have the resources to test the direct interaction of the methods. However, the literature indicates that RPA and CRISPR-Cas proteins do not inhibit each other
[[5]](#ref4)
. There has been no research conducted on the combination of RPA and DNA hybridization, but there is no reason to believe that the presence of the additional protein might interfere with the hybridization. Our idea of the test design separates the amplification and the hybridization spatially, connecting them by microfluidic pathways. This should avoid premature binding of the scarce DNA probe before sufficient amplification ensures a visible band.

## Multiplexing

The experiments described were all conducted with the L1 sequence of HPV16. This proof of principle would need to be extended by repeating the experiments with the additional sequences. However, the different sequences should not influence the outcome of the experiments, as long as the gRNA and hybridization elements are designed accordingly (this includes verifying specificity and secondary structures).

## Additional protocols and sequences

[Sequences](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/wiki-page-for-sequences.pdf)
[Protocol PCR](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-pcr-benchling.pdf)
[Protocol PCR Purification](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-pcr-purification-benchling.pdf)
[Protocol Gel Electrophoresis](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-agarose-gel-electrophoresis-benchling.pdf)
[Protocol Spectrophotometric DNA quantification / NanoDrop](https://static.igem.wiki/teams/5686/lab/experimentsprotocols/protocol-spectrophotometric-dna-quantification-nanodrop-benchling.pdf)

## References

1. Lobato IM, O'Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. TrAC Trends Analyt Chem. October 26, 2017;98:19-35. Available from:
   [doi:10.1016/j.trac.2017.10.015.](https://doi.org/10.1016/j.trac.2017.10.015)
2. Gul I, Raheem MA, Reyad-ul-Ferdous M, et al. CRISPR diagnostics for WHO high-priority sexually transmitted infections. TrAC Trends Anal Chem. January 2025;182:118054. Available from:
   <https://doi.org/10.1016/j.trac.2024.118054>
3. Xu Y, Li Z. CRISPR-Cas systems: Overview, innovations and applications in human disease reserach and gene therapy. Comput Struct Biotechnol J. September 8, 2020;18:2401-2415. Available from:
   [doi:10.1016/j.csbj.2020.08.031.](https://doi.org/10.1016/j.csbj.2020.08.031)
4. Cheung SW. DNA Hybridization. Brenner’s Encyclopedia of Genetics (second Edition). 2013. Available from:
   [https://www.sciencedirect.com/topics/neuroscience/dna-dna-hybridization.](https://www.sciencedirect.com/topics/neuroscience/dna-dna-hybridization)
5. Matthews MC, van der Linden J, Robène I, et al. A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4. Sci Rep. January 19, 2025;15:2436. Available from:
   <https://doi.org/10.1038/s41598-025-85633-8>

{% endblock %}

{% extends "layout-sidebar.html" %}
{% block title %}
Results
{% endblock %}
{% block headimage %}
![Header Image](https://static.igem.wiki/teams/5686/results/img-0664.avif)
{% endblock %}
{% block header %}

# Results

{% endblock %}
{% set sidebarcontent %}
[Wet Lab](#wetlab)

[Overview of Achievements](#wetlab-overview)
[Recombinase Polymerase Amplification (RPA)](#rpa)
[DNA Hybridization](#dna-hybridization)
[CRISPR-Cas Detection](#crispr-cas-detection)
[Dry Lab](#drylab)

[Overview of Achievements](#drylab-overview)
[Development and Validation of a Predictive Kinetic Model](#development)
[Model-Driven Engineering to Overcome System Failure](#model-driven)
[A Comprehensive Framework for the Diagnostic Lifecycle](#comprehensive)
[Expanding the Modeling Scope](#expanding)
[Analysis, Learnings, and Future Directions](#analysis)
[Human Practices](#hp)

[Overview of Achievements](#hp-overview)
[Stakeholder interactions](#stakeholder)
[End User centered Design](#end-user)
[Product](#product)
[Entrepreneurship](#entrepreneurship)

[References](#item-z)
{% endset %}
{% block page\_content %}

## Wet Lab

### Overview of Achievements

* We established RPA as our amplification method and tested three conditions: symmetrical RPA, asymmetrical RPA, and symmetrical RPA under temperature-varied incubation.
* Performed RPA directly on paper strips (both symmetrical and asymmetrical) and observed a modest signal increase, indicating feasibility but requiring further optimization.
* Demonstrated single-probe DNA hybridization on PCR, symmetrical RPA, and asymmetrical RPA products.
* Confirmed probe specificity for DNA hybridization, meaning no cross-reactivity with non-target pathogens.
* Achieved successful double DNA hybridization with two fluorophores, proving simultaneous binding.
* Successful purification of LbCas12a proteins for use in subsequent experiments.
* Developed CRISPR-Cas12a tube assays with visible color change at multiple incubation temperatures.
* Transferred both DNA hybridization and CRISPR-Cas onto lateral flow strips as a step toward a paper-based self test.

### Recombinase Polymerase Amplification (RPA)

As the amplification method for our diagnostic system, we selected Recombinase Polymerase Amplification (RPA) because it operates at low, constant temperatures close to body temperature, eliminating the need for thermal cycling and making it highly suitable for use in portable or at-home settings [[1]](#ref1).

We evaluated five different RPA configurations:

* Symmetrical RPA, producing double-stranded DNA for CRISPR-based detection.
* Asymmetrical RPA, producing single-stranded DNA for DNA hybridization-based detection.
* Temperature and time gradient experiments to evaluate activity across different incubation times and temperatures, including room temperature.
* Symmetrical and asymmetrical RPA performed directly on a strip.
* RPA sensitivity testing.

To simplify development and troubleshoot effectively, we decided to focus on a single pathogen to establish a fully functional workflow before extending the method to others. Using the HPV16 L1 gene as a model sequence, we tested three primer pairs designed by our team (see the [Experiments and Protocols Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)) at two DNA concentrations: 1 ng/µL and 0.1 pg/µL.

![Agarose Gel](https://static.igem.wiki/teams/5686/results/rpa-pic-1.webp)

**Figure 1**: Gel showing RPA amplification of the HPV16 L1 gene with two template concentrations (1 ng/µL and 0.1 pg/µL).

1. = 1° Amplicon (1 ng/μL)
2. = 1° Amplicon (0.1 pg/μL)
3. = 1° Amplicon negative control
4. = 2° Amplicon (1 ng/μL)
5. = 2° Amplicon (0.1 pg/μL)
6. = 2° Amplicon negative control
7. = 3° Amplicon (1 ng/μL)
8. = 3° Amplicon (0.1 pg/μL)
9. = 3° Amplicon negative control

Symmetrical and asymmetrical RPA yielded reliable amplification even at the lowest DNA input, as confirmed by agarose gel electrophoresis and Nanodrop analysis. For asymmetrical RPA, we screened different primer ratios (1:10, 1:20, and 1:50) based on literature reports [[2]](#ref2). Due to technical challenges, we could not conclusively determine the optimal ratio, but the setup provides a clear framework for further optimization. With these two experiments completed, the first steps toward both detection pathways were successfully achieved.

To explore truly equipment-free conditions, we conducted a temperature and incubation time screening. We found that RPA can be reliably performed at room temperature if the reaction time is extended from 20 to 30 minutes. According to Nanodrop measurements, this setup achieved an amplification of approximately one million-fold compared to the input concentration.

| Temperature [°C] | Incubation time [min] | Order of gel | DNA amount [ng/μL] |
| --- | --- | --- | --- |
| 39 | 20 | 1 (positive control) | 106 |
| 39 | 20 | 2 (negative control) | 33.4 |
| RT / ~23.8°C | 20 | 3 | 12.9 |
| 25 | 20 | 4 | 38.7 |
| 32 | 20 | 5 | 34.1 |
| 37 | 20 | 6 | 83.5 |
| RT / ~23.8°C | 30 | 7 | 87.3 |
| 25 | 30 | 8 | 75.2 |
| 32 | 30 | 9 | 103.5 |
| 37 | 30 | 10 | 121.4 |
| RT / ~23.8°C | 60 | 11 | 108.3 |
| 25 | 60 | 12 | 96.1 |
| 32 | 60 | 13 | N/A |
| 37 | 60 | 14 | 51.1 |
**Table 1**: Final visualization of RPA products with varying temperature and incubation times.

We also tested RPA directly on paper strips following the [2017 iGEM Munich team (“CascAID”)](https://2017.igem.org/Team%3AMunich) protocol. While asymmetrical RPA did not yield detectable amplification, symmetrical RPA on strips produced a 3-fold increase, which is technically amplification, but with low efficiency compared to expectations. This highlights the need for further optimization of reaction conditions on paper substrates.

![Strips in dessicator.](https://static.igem.wiki/teams/5686/results/1-rpa-dessicator-fixed.avif)

**Figure 2**: Strips in dessicator to freeze-dry components.

![Paper strip in small pieces.](https://static.igem.wiki/teams/5686/results/2-rpa-paper-strips.webp)

![Paper strip in small pieces.](https://static.igem.wiki/teams/5686/results/3-rpa-strips-cut.webp)

![Paper strip pieces soaked in water in an Eppendorf tube](https://static.igem.wiki/teams/5686/results/4-rpa-purification-fixed.avif)

**Figure 3, 4 & 5**: Puryfing the RPA amplicons.

Legend: Set-up for RPA on the strip.

1. Strips in dessicator to freeze-dry componenets. (Figure 2)
2. Then to purify the RPA amplicons, divide the paper strip in equal pieces then soak them in water. (Figure 3, 4 & 5)

Although multiple input dilution series were initiated to characterize sensitivity more precisely, we were unable to complete them due to time and equipment limitations (see the [Engineering and Cycles Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)).

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-6.webp)

![Agarose Gel](https://static.igem.wiki/teams/5686/lab/engineering/rpa-pic-7.webp)

**Figure 6 & 7**: Agarose gels showing symmetrical and asymmetrical RPA results directly performed on paper.

Legend left:

1. 2-5 = 1 ng/μL symmetrical RPA
2. 6-9 = 0.1 pg/μL symmetrical RPA

Legend left:

1. 7-10 = 1 ng/μL asymmetrical RPA
2. 11-14 = 0.1 pg/μL asymmetrical RPA

From these experiments, several key insights emerged: RPA successfully amplified the HPV16 L1 gene from as little as 0.1 pg/µL input, demonstrating high sensitivity. The reaction proved robust across a wide temperature range, including room temperature, as long as the incubation time was adjusted. Amplification efficiency reached approximately one million-fold compared to the input concentration, confirming strong DNA production even under low-resource conditions. On-paper RPA was shown to be feasible, though further optimization is needed to improve yield.

Looking ahead, we plan to:

* Optimize symmetrical and asymmetrical RPA on lateral flow strips to achieve higher yields by testing alternative freeze-dried components and troubleshooting for more efficient purification.
* Perform asymmetrical RPA at room temperature.
* Conduct room-temperature RPA directly on strips and optimize reaction conditions.
* Repeat experiments with increasingly diluted templates to determine sensitivity limits.
* Extend current experiments to include additional pathogens.

Together, these findings help guide the refinement of our amplification system and support the goal of building a reliable, user-friendly diagnostic test.

### DNA Hybridization

A central aim of our project was to establish and evaluate DNA hybridization as a detection method. In contrast to CRISPR-Cas systems, which depend on proteins, guide RNAs, and carefully controlled reaction conditions, DNA hybridization offers a simpler, more cost-effective, and robust alternative, particularly well suited for paper-based diagnostic tools intended for home use [[3]](#ref3)[[4]](#ref4).

To begin, we tested single-probe hybridization using three different probe lengths: a 15-mer LNA probe, a 20-mer, and a 25-mer, each labeled with the fluorophore Dy681. As templates, we used DNA from three amplification sources: symmetrical RPA (dsDNA, 1 ng/μL input), asymmetrical RPA (ssDNA-enriched, with a 1:50 forward:reverse primer ratio), and PCR products (~200 ng/μL). Each reaction (10 μL total volume) included a probe at a 1:1000 dilution from a 100 μM stock, along with NaCl, nuclease-free water, and the respective DNA template.

After denaturation at 95 °C followed by annealing at 25 °C, we observed visible signals across all template types. Among them, symmetrical RPA produced the strongest hybridization bands, and the 15-mer LNA probe consistently yielded the most robust performance compared to the longer probe designs.

![Agarose gel](https://static.igem.wiki/teams/5686/results/img-0622.webp)

**Figure 8**: Gel showing hybridization with 15-/20-/25-mer probes on RPA/PCR templates.

Legend:

1. = Ladder
2. = Asymmetrical RPA and diluted 15-mer LNA oligo + fluorophore (Dy681)
3. = Asymmetrical RPA and diluted 20-mer oligo + fluorophore (Dy681)
4. = Asymmetrical RPA and diluted 25-mer oligo + fluorophore (Dy681)
5. = Symmetrical RPA and diluted 15-mer LNA oligo + fluorophore (Dy681)
6. = Symmetrical RPA and diluted 20-mer oligo + fluorophore (Dy681)
7. = Symmetrical RPA and diluted 25-mer oligo + fluorophore (Dy681)
8. = PCR and diluted 15-mer LNA oligo + fluorophore (Dy681)
9. = PCR and diluted 20-mer oligo + fluorophore (Dy681)
10. = PCR and diluted 25-mer oligo + fluorophore (Dy681)
11. = Negative control for diluted 15-mer LNA oligo + fluorophore (Dy681)
12. = Negative control for diluted 20-mer oligo + fluorophore (Dy681)
13. = Negative control for diluted 25-mer oligo + fluorophore (Dy681)

Following the initial single-probe tests, we explored room-temperature hybridization without a denaturation step. To confirm specificity, we tested our probe against non-target pathogens including Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Trichomonas vaginalis, and observed no cross-reactivity. This demonstrated the probe's high specificity.

Initially, products from isothermal asymmetrical RPA did not generate any detectable signal. However, after redesigning the workflow, particularly with improved probe handling and preparation, fluorescence signals were successfully observed at room temperature. This confirmed that effective hybridization can occur without prior denaturation, an important step toward simplifying the workflow for at-home testing.

Next, we optimized the primer ratios for asymmetrical RPA to improve ssDNA enrichment. We compared 1:10 and 1:20 forward:reverse primer ratios and found that 1:10 provided reliable amplification across both standard (1 ng/μL) and diluted (0.1 pg/μL) DNA template inputs. In contrast, the 1:20 ratio failed to produce a signal at low template concentrations. Based on this, we standardized the 1:10 ratio for all downstream hybridization experiments.

To further improve detection flexibility and explore the potential for multiplexing, we introduced a second probe labeled with a distinct fluorophore, Cy7, allowing us to test double-probe hybridization. Initial tests only produced signal in one channel, which we traced back to photobleaching of the longer-wavelength dye. By switching to freshly prepared aliquots and protecting them from light exposure, we achieved successful double-probe hybridization.

This was a key milestone: we observed simultaneous binding of both probes on symmetrical RPA (dsDNA) and asymmetrical RPA products (ssDNA-enriched using a 1:10 forward:reverse primer ratio), at both 1 ng/μL and diluted (0.1 pg/μL) template inputs. These results confirmed that double-probe detection is feasible under our reaction conditions and further supports the robustness of our hybridization-based detection strategy.

![Two agarose gels](https://static.igem.wiki/teams/5686/lab/engineering/img-0625.webp)

**Figure 9 & 10**: Double hybridization with two probes labeled with different fluorophores (Dy681 & Cy7) on symmetrical RPA products.

Legend:

1. = Ladder
2. = Symmetrical RPA with first diluted oligo with fluorophore (Dy681)
3. = Symmetrical RPA with second diluted oligo with fluorophore (Cy7)
4. = Symmetrical RPA with both diluted oligos with fluorophore (Dy681 & Cy7)
5. = Negative control without template

![Two agarose gels](https://static.igem.wiki/teams/5686/lab/engineering/img-0664.webp)

**Figure 11 & 12**: Double hybridization with two probes labeled with different fluorophores (Dy681 & Cy7) on asymmetrical RPA products.

Legend:

1. = Ladder
2. = Asymmetrical RPA 1:10 (forward:reverse) with 1ng DNA template and both diluted oligos with fluorophores (Dy681 & Cy7)
3. = Asymmetrical RPA 1:10 (forward:reverse) with 0.1 pg DNA template and both diluted oligos with fluorophores (Dy681 & Cy7)
4. = Negative control without template

Finally, we transferred our detection system onto a lateral flow strip using the Milenia HybriDetect kit. For this setup, we used two labeled oligonucleotides: a FAM-labeled forward primer and a biotin-labeled reverse primer. On the strip, anti-FAM antibodies conjugated with gold nanoparticles are embedded, which bind the amplicons and generate the visible signal. We tested two probe concentrations, 100 µM and 0.1 pM, in combination with two concentrations of symmetrical RPA products: 1 ng/µL (undiluted) and 0.1 pg/µL (a 10,000-fold dilution).

Clear and reliable signals appeared within 2-5 minutes when using the 100 µM labeled probes, both for the undiluted and the diluted RPA products. In contrast, no signal was observed at 0.1 pM probe concentration, likely due to insufficient input for detection on the strip. These results confirmed that our system can produce fast and visible results on lateral flow strips when operated under appropriate input conditions.

![Four lateral flow strips: first and third one with two lines, second and fourth one with just second line.](https://static.igem.wiki/teams/5686/lab/engineering/img-5411.webp)

**Figure 13**: Detection of hybridized DNA on lateral flow strips with different probe and template concentrations.

Legend:

1. Symmetrical RPA (1 ng) + 100 µM FAM/Biotin labeled probes → positive signal
2. Symmetrical RPA (1 ng) + 0.1 pM FAM/Biotin labeled probes → no signal
3. Symmetrical RPA (0.1 pg) + 100 µM FAM/Biotin labeled probes → positive signal
4. Symmetrical RPA (0.1 pg) + 0.1 pM FAM/Biotin labeled probes → no signal

Some of the early double-probe hybridization experiments presented challenges, primarily due to fluorophore bleaching and low amplification efficiency from isothermal RPA. These issues initially prevented the detection of clear signals. However, through repeated testing, we discovered that protecting fluorophores from light exposure, changing the order of readout steps, and increasing the template volume from 1 µL to 2-3 µL significantly improved both signal strength and consistency.

When transferring the system onto lateral flow strips, we found that probe concentration was a critical factor. Using probes at 0.1 pM failed to produce any visible signal, likely because this concentration falls below the detection limit of the strip format. In contrast, higher probe concentrations, such as 100 µM, resulted in clear, reliable signals across both undiluted and highly diluted RPA products. This highlighted the importance of adapting not only the reaction chemistry but also the input parameters to suit the strip-based context.

These findings have shaped our next steps in improving the hybridization workflow. To simplify the readout and make the system more suitable for at-home use, we plan to replace fluorescent detection with alternatives such as gold nanoparticles or enzymatic colorimetric systems, which would allow for easy visual interpretation without special equipment. In parallel, we plan to refine probe designs, testing shorter sequences and introducing additional LNA modifications, to enhance hybridization efficiency and specificity. Optimizing buffer conditions is another important goal, particularly to ensure that hybridization on paper strips is both rapid and reliable under minimal-infrastructure settings.

Overall, while specificity and successful double-probe binding have already been clearly demonstrated in gel-based assays, transferring the method to paper-based strips remains a work in progress. The current strip results are promising but not yet fully consistent, and we consider them preliminary. In line with our commitment to scientific transparency, all such data are clearly marked as ongoing and subject to further optimization.

### CRISPR-Cas Detection

Although CRISPR-Cas12a was initially considered as the main detection method due to its high specificity, the project later shifted toward DNA hybridization as a simpler and potentially more robust alternative for at-home use. Despite this change in focus, we continued to evaluate both methods in parallel to compare their performance under the same conditions.

For the Cas12a-based detection, we used self-purified LbCas12a protein together with a target-specific crRNA and a fluorophore-quencher ssDNA reporter (ROX/BHQ2). In early tests, the reporter concentration was too low to produce a visible signal. After increasing the reporter levels, a clear fluorescence change became visible to the naked eye, especially at 32 °C and 37 °C within 30 minutes, and also at room temperature after longer incubation. These results confirmed that LbCas12a functioned reliably in our setup and provided a basis for direct comparison with the DNA hybridization method.

Tube 2: Incubation at 37 °C
Tube 3: Incubation at 32 °C
Tube 4: Incubation at 25 °C
Tube 5: Incubation at RT (23°C)

![Four pictures of each four Eppendorf tubes with numbers 2-4 and violet liquid content](https://static.igem.wiki/teams/5686/lab/engineering/img-0278.webp)

**Figure 14**: Visible color change in the LbCas12a assay using a ROX-BHQ2 reporter at different temperatures and time points.

Legend:

1. Picture 1: Before incubation
2. Picture 2: After 30 minutes of incubation
3. Picture 3: After 60 minutes of incubation
4. Picture 4: After 90 minutes of incubation

To evaluate the compatibility of our LbCas12a-based assay with paper-based formats, we transferred the reaction onto a lateral flow strip using the HybriDetect kit. We tested two reporter concentrations (50 nM and 100 µM) alongside two RPA product inputs (1 ng/µL and 0.1 pg/µL) to assess performance under varying conditions. In all four combinations, the control line appeared, confirming that the tests ran correctly. A visible test line developed within 2-5 minutes, indicating successful target detection. As expected, the strongest signals were observed with the higher RPA input (1 ng/µL), suggesting that increased template concentration enhances detection clarity. Overall, these results confirmed that LbCas12a-based detection works not only in tube-based reactions but also reliably on lateral flow strips.

![Four Milenia HybriDetect strips with two lines on each, intensity of the second line decreasing](https://static.igem.wiki/teams/5686/lab/engineering/img-5493.webp)

**Figure 15**: Cas12a detection on Milenia HybriDetect strips with different reporter and RPA input concentrations.

Legend:

1. Strip 1: Symmetrical RPA (1 ng input) + 50 nM FAM/Biotin labeled ssDNA reporter
2. Strip 2: Symmetrical RPA (0.1 pg input) + 50 nM FAM/Biotin labeled ssDNA reporter
3. Strip 3: Symmetrical RPA (1 ng input) + 100 µM FAM/Biotin labeled ssDNA reporter
4. Strip 4: Symmetrical RPA (0.1 pg input) + 100 µM FAM/Biotin labeled ssDNA reporter

In the early stages of our Cas12a-based detection experiments, some assays failed to produce a visible signal due to insufficient reporter concentrations. Through systematic troubleshooting, we learned that several factors, particularly the concentration of the reporter, the amount of template DNA, and the incubation temperature, are critical for achieving consistent and reliable detection.

To further improve performance, we plan to pre-assemble the Cas12a-crRNA complexes prior to each reaction and explore alternative reporter designs, including variations in length and fluorophore/quencher chemistry (e.g., FAM-Biotin or FITC-Biotin). If CRISPR-based detection proves too complex for home use in the long term, one alternative strategy could be to position it as a clinic-based solution, while continuing to develop DNA hybridization as the preferred option for at-home testing.

Despite initial setbacks, CRISPR-Cas detection demonstrated reliable performance in tube-based assays, consistently producing a clear and visible color change. The system also worked effectively on lateral flow strips, delivering rapid results under various conditions. Stronger signals were observed with higher input DNA concentrations, but detection was still successful with diluted templates, highlighting the potential of Cas12a as a flexible and sensitive detection method.

## Dry Lab

### Overview of Achievements

* Successfully developed a predictive kinetic model to simulate the entire RPA-CRISPR/Cas diagnostic system.
* Experimentally validated the model's core RPA amplification module against wet lab data.
* Identified a critical influence of cis-cleavage that would cause system failure.
* Proposed a model-driven “two-chamber” hardware design to resolve the predicted system failure.
* Designed a theoretical framework for predicting enzyme stability to guide future work on diagnostic shelf-life.
* Integrated all modules to create a comprehensive, end-to-end simulation of the diagnostic's full life cycle.
* Developed an Epidemiological model estimating public health impact of our diagnostic to contextualize its real-world relevance.
* Initiated development of a model for asymmetrical RPA / DNA hybridization dynamics.

### Development and Validation of a Predictive Kinetic Model

*Successfully developed a predictive kinetic model to simulate the entire RPA-CRISPR/Cas diagnostic system.*

To understand and optimize our diagnostic's performance, we first developed a mechanistic model based on ordinary differential equations (ODEs). This model simulates the two core processes of our system: the amplification of target DNA via Recombinase Polymerase Amplification (RPA) and its subsequent detection by the CRISPR-Cas12a system. By describing the dynamic interactions between every component, the model allowed us to perform virtual experiments, test hypotheses, and analyze system behavior under a wide range of conditions.

![Graph](https://static.igem.wiki/teams/5686/results/graph-rpa-spectrum-for-initial-g.webp)

**Figure 16**: RPA spectrum for different initial G (Gp32) concentrations.

![Graph](https://static.igem.wiki/teams/5686/results/graph-rpa-spectrum-for-initial-d.webp)

**Figure 17**: RPA spectrum for different initial D (template DNA) concentrations.

*Experimentally validated the model's core RPA amplification module against wet lab data.*

A model's predictions are only as valuable as their connection to reality. The most critical step in our work was to validate our model against experimental data. Due to the proprietary composition of commercial RPA kits, we performed a sensitivity analysis to predict a plausible range of DNA yields. Our model predicted a final DNA output between ~45 nM and 2364 nM. Our wet lab team's benchmark experiment yielded a quantified result of 405 nM.

![Bar chart](https://static.igem.wiki/teams/5686/results/rpa-model-vs-lab-log-bar.webp)

**Figure 18**: Bar chart comparing simulated RPA min/max amplification with lab experiment and detection thresholds (log scale).

![Graph](https://static.igem.wiki/teams/5686/results/rpa-model-vs-lab-log-lines.webp)

**Figure 19**: Line plot comparing simulated RPA min/max amplification with lab experiment and detection thresholds (log scale).

**Implications:**

The successful alignment of our experimental data within the model's predicted range is a crucial achievement. It validates the foundational RPA module of our model, confirming that it accurately captures the core amplification dynamics. This validation provides a strong anchor of confidence for all subsequent predictions and insights generated by the integrated model.

### Model-Driven Engineering to Overcome System Failure

*Identified a critical influence of cis-cleavage that would cause system failure.*

With the RPA module validated, we used the CRISPR/Cas12a & RPA integrated model to investigate potential interactions between the amplification and detection steps. The simulation revealed a critical bottleneck: the cis-cleavage activity of the Cas12a enzyme. The model predicted that at low concentrations of amplified DNA, typical during the early phases of the reaction, the Cas12a enzyme would bind and destroy the target DNA faster than RPA could produce it. This creates a "deadlock" effect that prevents the DNA concentration from ever reaching the threshold required for a detectable fluorescent signal.

![Graph showing the predicted signal of the integrated system flatlining at zero over time](https://static.igem.wiki/teams/5686/results/cas-no-signal.webp)

**Figure 20**: RPA-CRISPR simulation showing no detectable signal.

**Implications:**

This in silico discovery was one of our project's most significant findings. It identified a fundamental design flaw that would have led to persistent experimental failure, saving invaluable time and resources. This highlights the power of modeling to proactively identify and diagnose problems that are difficult to foresee at the bench.

*Proposed a model-driven “two-chamber” hardware design to resolve the predicted system failure.*

The insight from our model pointed directly to a rational solution. Since the cis-cleavage issue is most severe at the early time phase of RPA, we could resolve it by separating the two reactions in time. Our model simulations confirmed that allowing the RPA reaction to proceed for several minutes before introducing the CRISPR-Cas12a components would allow a sufficient concentration of amplicons to accumulate, rendering the effects of cis-cleavage negligible.

![Graph](https://static.igem.wiki/teams/5686/results/cas-delay-300s.webp)

**Figure 21**: A 300 s Cas12a delay leads to full reporter cleavage.

**Implications:**

This finding translates directly into a tangible engineering solution: a "two-chamber" hardware design for a future device. This design would physically separate the amplification and detection steps, ensuring the sample incubates in an RPA chamber before flowing into a second chamber containing the CRISPR reagents. This model-driven design provides a de-risked and scientifically grounded blueprint for a robust and functional diagnostic device.

### A Comprehensive Framework for the Diagnostic Life cycle

*Designed a theoretical framework for predicting enzyme stability.*

A functional diagnostic must also be stable over time. To address this, we developed a theoretical framework to model enzyme degradation and predict diagnostic shelf-life. The model is based on principles of protein denaturation kinetics.

The results reveal how storage conditions dramatically affect shelf life. At refrigeration temperatures (e.g., 5°C), enzyme activity declines slowly over many months. Once brought to room temperature (e.g., 22°C), degradation accelerates substantially, but the assay remains viable for weeks of typical use.

![Graph with two lines: Enzyma Activity and Temperature.](https://static.igem.wiki/teams/5686/results/enz-activity.webp)

**Figure 22**: Graph describes activity decay of rate-limiting enzyme (SBB/gp32) over time, taking different temperatures in account.

*Integrated all modules to create a comprehensive, end-to-end simulation.*

To create a truly holistic tool, we integrated the Enzyme Stability Framework with our validated RPA-CRISPR Kinetic Model. This multi-scale simulation can now forecast not only the immediate signal generation of a fresh test but also how its performance alters after months of storage under varying temperatures. The results were critical, particularly insightful for our entrepreneurial plans.

![Graph showing the predicted signal of the integrated system flatlining at zero over time](https://static.igem.wiki/teams/5686/results/heat-map.webp)

**Figure 23**: Heatmap describing different scenarios of cold storage time followed by room temperature storage, leading to different time-to-result capacities.

The heatmap shows that with proper refrigerated storage (e.g., 100 days at 5°C), followed by room-temperature use, the assay reliably crosses the threshold within 30-40 minutes. However, prolonged room-temperature storage (e.g., 200 days at 22°C) leads to substantial activity loss, extending detection times or preventing threshold crossing altogether.

**Implications:**

This integrated analysis provides a powerful, quantitative validation for our initial entrepreneurial strategy of incorporating freeze-drying. The model clearly shows that without a method to preserve enzyme stability, the test's performance would be unreliable for real-world use where a cold chain is not guaranteed. This result is a cornerstone of our business case, proving that lyophilization is not just a desirable feature but an essential requirement for our diagnostic to be effective and accessible globally.

*Developed a predictive model for asymmetrical RPA*

We developed a mechanistic model of asymmetrical RPA that accurately reproduces experimental kinetics reported in five independent papers. The model captures all three biological phases observed during the reaction and maintains perfect mass balance while providing quantitative predictions for assay optimization. This work represents a key step toward our ultimate goal of integrating a DNA hybridization model to generate predictions for our second approach.

![Graph showing the predicted signal of the integrated system flatlining at zero over time](https://static.igem.wiki/teams/5686/model/asymmetricalrpa/arpa-5-complete-overview.avif)

**Figure 23B**: Full Reaction Dynamics including all characteristic phases.

### Expanding the Modeling Scope

*Developed an Epidemiological model estimating public health impact.*

To connect our technical work to its real-world context, we developed an epidemiological model to simulate the spread of an STI in a population. The model compares scenarios with and without the availability of an accessible, rapid diagnostic like ours. The model showed that introducing our test could significantly lower disease prevalence over time by enabling earlier detection and treatment. This finding offers strong public health justification for our project.

![Graph showing the predicted signal of the integrated system flatlining at zero over time](https://static.igem.wiki/teams/5686/results/epidem-tr1.webp)
![Graph showing the predicted signal of the integrated system flatlining at zero over time](https://static.igem.wiki/teams/5686/results/epidem-tr4.webp)

**Figure 24 & 25** : Graph: Projected Chlamydia cases in Switzerland over the next 10 years (in thousands). Top panel (figure 26): Cases assuming current testing rates. Bottom panel (figure 27): Cases assuming annual testing rates of 0.28 for low-risk and 0.6 for high-risk populations.

### Analysis, Learnings, and Future Directions

To summarize our work, our main achievement was the development of a kinetic model that made a critical prediction. By first checking the RPA amplification part of our model against a real lab experiment, we gained the confidence we needed to trust its insights. This trust was essential when the full model predicted that our combined system would fail due to the "cis-cleavage deadlock." This finding was pivotal, as it allowed us to propose a tangible hardware solution, the two-chamber device, before wasting time and resources on experiments that were destined to fail. Furthermore, by integrating the Enzyme stability model with diagnostic system performance, we gained crucial insights regarding the real-world lifecycle of our test and validated our plan to use lyophilization. This step made us more clear in our entrepreneurial strategies.

However, our Dry Lab's effort to tackle all the edges of our project extended far beyond that. Our Epidemiological model was an invaluable global health justification for our project and served for us as a validation of our primary goal to combat STI epidemics with fully user oriented diagnostics.

However, in the spirit of scientific honesty, it's important to be open about the significant challenges we faced. Our journey was not a straight line, and these difficulties taught us a great deal. A major hurdle was that the commercial kits we used were essentially "black boxes," with unknown concentrations of key ingredients. We learned to navigate this uncertainty by using our model to test a wide range of possibilities, a technique called sensitivity analysis. This experience showed us how computational modeling can be a powerful tool for working with the kind of incomplete information that is common in real-world science. We also learned a hard lesson in interdisciplinary communication when an experiment to get numbers for our Cas12a model didn't work out as planned. We had to adapt quickly, relying on data from previously published papers, which taught us to be flexible and resourceful. Finally, we learned about the importance of project management. Our initial ambition to build two complex models at once was greater than the time we had. Making the tough decision to pause work on the second model to focus all our efforts on the main one was a critical lesson in prioritizing depth and validation over breadth.

## Human Practices

### Overview of Achievements

* We integrated feedback from over 20 stakeholders across ethical, regulatory, clinical, societal, entrepreneurial, and scientific fields, leading to critical changes in our project’s vision, including important adjustments to our technical approach and design based on scientific recommendations, and modifications to the panel of STIs we aim to test for.
* Considered regulatory and entrepreneurial insights to align with medical device standards and potential market strategies.
* Surveyed 417 people on their preferences and expectations for an STI self-test.
* Designed our product page: a mock-up of what a future website for our self-test should look like.
* Collaborated with Dry Lab to assess the impact of higher testing rates on STI cases.
* Developed a business case to ensure real-world implementation of our product is sustainable.
* Conducted awareness campaigns about sexual health on social media and at schools.
* Promoted education on synthetic biology through events at the university and high-school visits.
* Successfully designed and held school workshops, performing PCR and gel electrophoresis.

### Stakeholder interactions

Throughout our project, we engaged with over [20 stakeholders](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)) from diverse fields, whose feedback helped us refine our approaches and ensure our project addresses real-world needs.

### End User Centered Design

To understand public testing behavior and the demand for STI diagnostic tools, we conducted a 25-question [survey](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27survey%27%29%20%7D%7D)), collecting 417 responses over one month. The results confirmed our design approach, provided insights into current testing rates, allowed us to predict the potential impact of our product, and helped analyze our target market for entrepreneurial opportunities.

### Product

During our interviews, we soon realized that the test itself is only half of the work done. People need proper guidance as soon as they open the package containing the test until they have received appropriate treatment. We therefore decided to build a [product page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27product%27%29%20%7D%7D)), displaying the content of our future webpage, guiding people throughout the whole process. We made sure the page is easy to read, displaying only the most important information, while still providing enough knowledge on STIs, our test and what to do with a test result. A lot of this work is based on an interview with a health psychologist from the university of Zurich, [Walter Bierbauer](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27stakeholder-bierbauer%27%29%20%7D%7D)). Make sure to read his interview to see how his input was integrated into the product page.

### Entrepreneurship

We developed a comprehensive [business case](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27entrepreneurship%27%29%20%7D%7D)) to translate our diagnostic innovation into a viable product. Our startup concept addresses an unmet need in accessible, affordable STI testing. We defined our minimum viable product (MVP), created a business model canvas, performed market and competition analyses, and established a financial and scaling strategy. We identified key partners and regulatory pathways to ensure market readiness. Our approach demonstrates both feasibility and sustainability.

## References

1. Lobato IM, O'Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. TrAC Trends Analyt Chem. October 26, 2017;98:19-35. Available from:
   [doi: 10.1016/j.trac.2017.10.015](https://doi.org/10.1016/j.trac.2017.10.015).
2. Wang Y, Wang Y, Luo L, et al. Visual detection of Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme. RSC Adv. 2019;9(52):30350-7. Available from:
   [doi: 10.1039/c9ra05709a](https://doi.org/10.1039/c9ra05709a).
3. Xu Y, Li Z. CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. September 8, 2020;18:2401-2415. Available from:
   [doi: 10.1016/j.csbj.2020.08.031](https://doi.org/10.1016/j.csbj.2020.08.031).
4. Cheung SW. DNA Hybridization. Brenner’s Encyclopedia of Genetics (Second Edition). 2013. Available from:
   <https://www.sciencedirect.com/topics/neuroscience/dna-dna-hybridization>.

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