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.
If you want to read up on the biological concepts behind our project click here.
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.
A new beginning! The iGEM UZurich 2025 Team members were selected by former team members.
Oh Oh Exams.
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.
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 which helped us by answering these questions as well as by critically questioning our plans.
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.
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 invited us to work on our project in their laboratory!
Exams… again! This semester went by so quickly.
Let the fun begin. But safety first: Our wet lab team started with a safety introduction in the laboratory. We are ready for our first steps in the lab.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
DNA hybridization: Finally, we also performed a successful DNA hybridization with double binding for asymmetrical RPA :)
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.
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.
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.
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:
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.