Introduction

During our time in the laboratory, there have been several paradigm shifts and important moments that have defined our experiments. We have adapted the procedures according to the needs of our project, incorporating or removing elements from the assays, optimising protocols, radically changing experimental techniques, or making complete modifications to what we had done up to that point.

In this section, we want to highlight three key moments in the development of our laboratory work over the past months, with the aim of perhaps helping other iGEM teams who may face difficulties in their experimental work, or providing ideas about what might be going wrong if the experimental results are not as expected.

Overview

Scheme of the three most important milestones during the experimental work of our project

Milestone 1: Optimization of Cell-Based Assay

Throughout our work in the laboratory, we have carried out several rounds of assays in HEK293T cells. We were able to optimize the process in some of its experimental and procedural variables after reviewing the results and making various observations throughout the procedure.

Cell Density

The first step for the HEK293T cell assay is seeding a specific cell density in 24- or 48-well plates. In our case, we used 48-well plates. After several trials, we determined that the optimal cell density was 30,000 cells per well.

It is important to note that the cell assay lasts 5 days, so if we seed the cells on day 1, they will go through 3–4 cell division cycles before we measure luciferase levels. Therefore, the initial number of cells seeded must be relatively low to avoid:

• The cells reaching confluence and stopping proliferation
• Renilla luciferase levels becoming so high that they exceed the luminometer's detection limit

Treatment Timing Optimization

After reviewing the literature, we found an article [1] in which, after transfecting the cells, only 4 hours were allowed before starting the theophylline treatment, followed by 20 hours of treatment until luciferase reading. This article reported successful results.

Up to that point, we had been waiting 24 hours before treating the cells with theophylline and selenium to allow recovery from transfection and then 48 hours for the treatments to take effect.

We decided to revert to the 24- and 48-hour timings as they provided more reliable and reproducible results for our experimental conditions.

Selenium Supplementation

Paul Copeland recommended supplementing the culture with a selenium concentration of 50 nM. In the article Factors impacting the aminoglycoside-induced UGA stop codon readthrough in selenoprotein translation [2], they used 50–250 nM Na₂SeO₃ in human HEK293 cell culture with UGA-SECIS reporters, which also improved readthrough efficiency with aminoglycosides.

In our experiments, we tested supplementation with 50 nM and 100 nM. We concluded that selenium supplementation was not necessary under our conditions, as the FBS component of the medium contains variable traces of selenium. Under our experimental conditions, these traces were sufficient so that the selenocysteine amino acid concentration was not limiting.

Theophylline Optimization

Theophylline was a real challenge in the laboratory. We faced two main challenges:

Dilution for Luciferase Assay

In the initial rounds of the cell assay, luciferase values were sometimes so high that they exceeded the luminometer's detection limit. After performing several trials and with the guidance of the technicians and PhD students assisting us, we determined that the optimal dilution under our conditions was 1/60. With this dilution, the luciferase values were reduced and readable by the luminometer.

Milestone 2: The Transition from Cell-Based Assays to Cell-Free System Assays

One of the key moments in our experimental work and in our project overall was the transition from cell-based assays to cell-free system assays. Below, we explain why we made this transition and the considerations involved.

In the various HEK293T cell assays, SECIS control showed good results. However, the aptamer control only performed well in the first round. Despite attempting to replicate the conditions of that initial round in subsequent experiments, we were unsuccessful. At this point, we formulated hypotheses about what might be happening. There were mainly two possibilities:

• Our aptamer control construct does not function.
• Some step in the experimental process is not optimized or ideal for the cells, or there is an additional factor in HEK293T cells that prevents the aptamer control construct from functioning.

To investigate and continue the project without stalling, we tested the aptamer control in a cell-free system. If it worked there, it would indicate that the construct itself was functional and that the issue lay within the experimental process.

The in vitro system worked, confirming that the construct was functional, but something in HEK293T cells prevented the aptamer control from functioning. Our aptamer control construct was designed based on the construct described in Anzalone's article [5], which had been tested in yeast cells. We contacted Anzalone to clarify two main points:

• We wanted to know whether he had tested his construct in HEK293T cells.
• We wanted his opinion on the following hypothesis: theophylline aptamer folding might be highly temperature-dependent. Yeast, which Anzalone used, grows at 30ºC, and the cell-free system is also conducted at 30ºC. In contrast, HEK293T cells grow at 37ºC, and this 7ºC difference might prevent proper aptamer folding.

Anzalone replied that he had tested the construct in HEK293T cells and obtained results similar to ours—it did not function in this context. He considered our hypothesis plausible and noted that frameshift efficiency is inherently low in HEK293T cells.

Since the cell-based experiments did not succeed and the underlying reason could not be addressed in the short term, we shifted focus to the cell-free system. This process uses TnT® Coupled Reticulocyte Lysate Systems from Promega®, catalog #L1170, and involves artificial transcription and translation.

Cell-Free System Considerations

The cell-free assay worked for both the SECIS control and the aptamer control constructs. However, several considerations had to be taken into account:

All Constructs

For all constructs in the cell-free system, two key considerations were essential:

Scheme of how the primer with 49 thymidines produces a PolyA tail in the mRNA

Milestone 3: Back to the Fundamentals

Now that we have obtained some good results in the cell-free system, it is worth mentioning that in the first experiments of this type, the results turned out to be really strange. It was very curious because the pattern of successful results seemed inverted: while in HEK293T cells we obtained good results with the SECIS control construct and poor results with the aptamer control, in the cell-free system the opposite occurred.

Sometimes, when the results look so unusual, it is very likely that one of the fundamental steps is not working as it should. That is why it is important to carefully review everything again. That is what we did, and we discovered that the PCR step, performed prior to the cell-free assay, was not working correctly. Specifically, we found that the reverse primer was not amplifying properly.

Firstly, this primer did not present very specific annealing, since it was not fully complementary to the DNA sequence. In other words, part of the primer (outside of the 49 thymines) was not binding to the DNA. Secondly, the primer was not amplifying our construct, meaning that our construct was excluded from the PCR product.

This happened with the SECIS control plasmid, as well as with the riboswitch plasmids. The aptamer control plasmid, however, did include the construct of interest within the PCR product and did have total complementarity with the reverse primer. This is because the aptamer control is located in a slightly different plasmid compared to the others.

Once we realized this, we designed new primers, ordered them, and repeated the PCR. The resulting gel is the one shown:

Gel of the PCR products created with the new primers

The new primers worked successfully, and with the resulting PCR products we obtained good and consistent results. For us, this was an important lesson: sometimes the problem is simpler than it seems, and we may underestimate certain aspects, such as the primers in a PCR, assuming that they will amplify the desired region without issue.

In addition, this experience taught us the importance of reviewing and double-checking the most fundamental steps of laboratory procedures, since they are like the foundations of a building—if one of them fails, the whole structure collapses.