iGEM 2025 — Team ucalgary

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{% block title %}Notebook{% endblock %}
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![Notebook](https://static.igem.wiki/teams/5980/header/notebook.webp)
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![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Wet Lab](#notebook-wet)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Dry Lab](#notebook-dry)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[General](#notebook-general)

# WET LAB

In this section you'll be able to review the wet-lab notebook.
It includes **ALL** of our results and methods used to accomplish our goals.
In this file we have documented all the work we've done in the lab every single day since we started working on the project.
We have divided the notebook into **two parts.**

This browser does not support PDFs. Please download the PDF to view it:
[Download PDF](https://static.igem.wiki/teams/5980/protocols/igem-2025-wet-lab-notebook-part-1.pdf).

This browser does not support PDFs. Please download the PDF to view it:
[Download PDF](https://static.igem.wiki/teams/5980/protocols/igem-2025-wet-lab-notebook-part-2-1.pdf).

# DRY LAB

This browser does not support PDFs. Please download the PDF to view it:
[Download PDF](https://static.igem.wiki/teams/5980/protocols/dry-lab-notebookook.pdf).

# GENERAL

This browser does not support PDFs. Please download the PDF to view it:
[Download PDF](https://static.igem.wiki/teams/5980/protocols/ruminotes.pdf).

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{% block title %}Engineering Success{% endblock %}
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![Engineering](https://static.igem.wiki/teams/5980/header/engineering.webp)
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![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[RumiVec](#engineering-rumivec)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Cycle 1](#engineering-1)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Cycle 2](#engineering-2)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Cycle 3: RumiVec](#engineering-3)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[References](#engineering-references)

# RUMIVEC

The main goal for RumiVec was to develop a composite part that could allow for production of RNA bacteriophages in E. coli cells
in order to validate viral nucleic acid biosensors without using higher risk pathogens.
For this, the sequence coding for the phage genome must contain a modular region
to allow for insertion of any control sequence to be used for testing. This system will be used in future steps to validate the Rumino biosensor
by inserting the Influenza target sequence into RumiVec and then using its products for our test samples.

# CYCLE 1

## Design

The first step was deciding what type of RNA phage we'll produce.
The most well-studied single-stranded RNA phages are from the Leviviridae family,
more specifically the MS2 and QB species [1].
Given that the MS2 phage has a smaller, simpler genome than QB
and can also produce higher yields when produced by bacteria [1],
our group decided to assemble a genetic device for the production of MS2 phages.

In this initial iteration, the idea was to build a construct containing the genome of the wild-type MS2 phage,
followed by a cassette containing a reporter which could be swapped out using Type IIS restriction enzymes.
Replacement of the reporter with an insert sequence would allow for easy screening of colonies
that were successfully transformed with the construct containing the insert.
This construct would require two promoters:
an inducible promoter for expression of an RNA molecule corresponding to the MS2 genome and the cassette;
and a different promoter for expression of only the reporter.
This way, the reporter protein can be produced without expression of viral proteins,
which would result in death of the host cell.

We also realized that the wild-type MS2 genome is not codon-optimized for E. coli cells.
However, some of the genes in the genome overlap,
which means that codon optimizing might disrupt some proteins,
specifically the Lysis and Replicase proteins.
Due to this, we decided to test two constructs in parallel:
a codon-optimized construct and a wild-type construct.

For the reporter, the team decided to use the LacZα peptide
which would allow for blue-white screening to find bacterial colonies
successfully transformed with either the construct with the reporter (blue colonies)
or an insert sequence (white colonies) using X-Gal plates.
More specifically, we combined a constitutive promoter (BBa\_J23100)
with a strong RBS + CDS (BBa\_K329005) flanked by SapI restriction sites
to generate our cassette.

To ensure that the construct could be used as a standard part,
the sequences were screened for any restriction sites used for Type IIS or BioBricks assembly
and removed to meet compatibility requirements.

| Illegal Site | Position | Original Sequence | Modified Sequence |
| --- | --- | --- | --- |
| XbaI | 1303 | TCT AGA | TCT CGT |
| EcoRI | 1629 | G AAT TC | G AAC TC |
| BsaI | 185 | GAG ACC | GAA ACC |
| BsaI | 1687 | GGT CTC | GGT CTG |
| BsaI | 2279 | GA GAC C | GA GAA C |

After the construct was ordered from Twist Bioscience within a pET Kan vector,
we analyzed it using the NEBridge Golden Gate Assembly tool
to verify the sequence for use in Type IIS assembly.
This showed us an extra SapI site in the vector backbone that was overlooked,
as well as a wrong orientation on the cut sites flanking the reporter.

To fix this error, we intended on using NEB HiFi assembly
to correct the orientation and swap the SapI sites for BsaI sites.
For this, we designed two pairs of primers using Primer3Plus to correct the sequences.
The first pair was meant to amplify the regions outside of the reporter,
while the second pair was meant to amplify the reporter.
The reporter primers contained overhangs that replaced the SapI sites
and also added compatible extensions between it and the backbone.

Backbone Forward Primer

![Backbone Forward Primer Figure](https://static.igem.wiki/teams/5980/engineering/backbone-forward-primer-1st-construct.webp)

Backbone Reverse Primer

![Backbone Reverse Primer Figure](https://static.igem.wiki/teams/5980/engineering/backbone-rev-primer-first-construct.webp)

Reporter Forward Primer

![Reporter Forward Primer Figure](https://static.igem.wiki/teams/5980/engineering/reporter-forward-primer-1st-construct.webp)

Reporter Reverse Primer

![Reporter Reverse Primer Figure](https://static.igem.wiki/teams/5980/engineering/reporter-rev-primer-1st-construct.webp)

For the insert sequence, a short 35bp sequence termed Shuffle Sequence 1 (SS1) was designed
with a GC% similar to that of HPAI.
Spacers were then added to flank the sequence
to make it long enough for PCR and detection with Agarose Gel Electrophoresis.
BsaI sites were also added to the ends of this sequence
for Type IIS assembly with the construct.
Similarly, primers were also designed to amplify this sequence
using the Benchling primer design tool.

## Build

The constructs were assembled using Benchling and then ordered through Twist Bioscience.
The idea was to obtain the parts necessary for HiFi assembly
to build the correct construct using the sequences we had.

![Fixed Construct Schema](https://static.igem.wiki/teams/5980/engineering/	assembly-method.webp)

**Figure 1.** Proposed scheme for assembly of fixed construct containing BsaI sites instead of SapI,
and oriented to be inside of the reporter.
PCR products would be assembled using NEB HiFi Assembly.

## Test

During this first cycle, only the PCR of the reporter seemed to work.
Amplification of the vector backbone seemed to result in streaking,
and SS1 didn't generate a product.
This roadblock prevented us from achieving our goals,
and at this point we decided to close this cycle and design new constructs.

![Agorose Gel Electrophoresis](https://static.igem.wiki/teams/5980/engineering/	first-pcrs.webp)

**Figure 2.** Agarose Gel Electrophoresis (1%) of different PCR products.
Only a faint band was observed for amplification of the reporter (LacZα),
while SS1 had none to low yield,
and the backbone had non-specific amplification as observed by the streaking.

## Learn

Based on the data collected from the PCR procedures, we concluded that these initial constructs had too many problems
to be optimally used for future steps. The desired sequences for HiFi assembly couldn't be obtained,
meaning we could not use these constructs to change the reporter for an insert sequence using Type IIS assembly,
which is one of the main goals for our system. From this cycle we identified the main problems with our initial constructs,
which were taken into consideration for the next cycles; mainly wrong orientation of restriction sites,
and use of restriction sites that were also found in the plasmid backbone.

# CYCLE 2

## Design

The same approach for designing the new constructs as in the first cycle was taken here,
but with extra care to ensure that the BsaI sites were in the proper orientation
and none of the other parts used contained a restriction site of this type.
The overhangs for these sites were chosen based on the prefixes and suffixes
for a transcriptional unit outlined by the iGEM registry
to increase standardization of these constructs.

New primers were also designed for the constructs.
These primers bind to the regions outside the BsaI sites,
meaning that they'll amplify the sequence inside the cassette,
whether it be a reporter or an insert.
This provides an alternative screening method
which can be used to confirm for correct Type IIS assembly
as long as the insert is a different length as the reporter (>365bp).
Unfortunately, for this cycle this condition was not considered
and the insert used was the exact same length as the reporter,
which wouldn't allow us to differentiate the products in an agarose gel.

## Build

Just as last time, the constructs were built on Benchling and then ordered as a clonal gene from Twist Bioscience.
The constructs with an insert instead of a reporter were obtained using Type IIS Assembly
with the NEB Golden Gate Cloning Kit directly on the constructs and the SS1 sequence.
These reactions were then used to transform NEB5α cells,
a strain suitable for Blue-White screening.

## Test

The transformed cells were plated on X-gal plates for blue-white screening.
While the construct using the WT MS2 sequence showed the expected results for the screening,
the codon-optimized construct showed white colonies from cells transformed with the reporter construct.

![Agar Plate picture](https://static.igem.wiki/teams/5980/engineering/pms2-300-400-bw-screen.webp)

**Figure 3.** X-Gal LB Agar plate of NEB5α cells transformed with the MS2 construct containing the LacZα reporter.
Plate on the left was the construct with the wild-type MS2 sequences,
plate on the right is the construct with codon-optimized MS2 sequences.
Both constructs were inserted into a pET Kan plasmid.

Based on this screening, only the WT construct was used for the next steps.
Following blue-white screening, the plasmid was purified from blue colonies
and then the reporter was swapped with SS1 using Type IIS assembly as previously described.
The modified plasmid underwent a second round of blue-white screening
from which white colonies were selected and then used to transform BL21(DE3) cells.
These cells were then induced with IPTG for expression of the MS2 phages
and then the cultures were analyzed under a Bleach Agarose Gel [n]
to assess size and integrity of the genome of the phages produced.

![Bleach Gel cells](https://static.igem.wiki/teams/5980/engineering/	bleachgel.webp)

**Figure 4.** Bleach Gel of BL21(DE3) cells transformed with the WT MS2 construct and induced with IPTG.
The left lane represents standards,
the middle and right lanes are each a different colony of transformed cells.

## Learn

The streaking in the Bleach Gel indicates either a degraded sample,
or a highly heterogenous sample (as in many different RNA molecules
were packaged into the phages instead of the single transcript).
When we did literature research into possible issues,
we realized that including all the proteins of the MS2 phages into a single system
leads to poor results due to nonspecific RNA packaging,
consistent with what was observed in our data [2].
Based on previously published work, to develop an MS2-producing system,
only the Coat Protein is strictly required for phage assembly [2].

# CYCLE 3: RUMIVEC

## Design

Based on the results from the bleach gel of the last iteration,
we changed our constructs to include only the Coat Protein (Cp) and the Maturation Protease (Mat),
as these are the only ones required for structural assembly of the MS2 phage [2].
Although the Mat protein is not strictly required, including it will drastically increase stability of the phages
and has a protective effect for the RNA genome [2].
Similarly, we changed the insert sequence we were testing
to ensure that it was of different length as the reporter
to make sure that the assembly could be screened using PCR and Agarose Gel Electrophoresis.

We also learned that the ratio between Cp and Mat proteins is crucial
for accurate assembly of the MS2 phage with the correct RNA molecule [2].
Due to this, during the design of the new construct
we needed to make sure that the RBS chosen for each protein
had similar translation rates as the wild-type versions.
The RBS were chosen based on the translation initiation rates (TIRs)
predicted by the SalisLab Operon/RBS Calculator [3].
The wild-type (WT) untranslated region upstream of the Mat CDS was used as the RBS
as it had similar TIRs as the WT transcript.
However, the WT RBS for the Cp CDS in RumiVec had a TIR significantly lower than expected.
Although the Operon Calculator has an option to design RBS,
it always gives a sequence that maximizes TIR.
Regardless, this option was chosen and then the sequence obtained
was randomly mutated through different iterations
to obtain an RBS with similar TIR as the WT transcript.

![RSB Tir Graphs](https://static.igem.wiki/teams/5980/engineering/rbs-tir-graphs-1.webp)

**Figure 5.** A.) Predicted Translation Initiation Rates of: the wild-type (WT) RBS of either the Maturation Protease (Mat) or Coat Protein (CP)
with the wild-type sequence (WT RBS); the WT RBS of the two proteins within our construct (WT RBS in RumiVec);
and the Custom RBS generated by SalisLab Operon Calculator inside our construct (Custom RBS).
B.) TIRs of the different RBS sequences tested for the Cp CDS. Iteration 6 was chosen as the sequence for the construct.

To ensure that the phages produced by our system were mostly homogenous
with the transcript produced by RumiVec, we designed the construct to include two modified pac sequences,
which are recognized by the Cp for packaging the transcript into the phage capsid.
This sequence was obtained from a study published by Wei, et al.,
which has a much higher affinity and packaging efficiency for RNA molecules by the Cp
compared to the wild-type sequence.
To ensure that the transcript produced from RumiVec is efficiently encapsulated into phage particles,
the predicted secondary structures of the sequence were analyzed using Vienna's RNAFold
to ensure that the necessary stem loop forms on the respective pac sites.
The resulting model predicts with high-confidence the formation of the secondary structure
in the pac sites required for adequate interactions with the Coat protein,
meaning that RumiVec will be packaged into the phage progeny at high efficiency.

![RNA Fold of two pac sites](https://static.igem.wiki/teams/5980/engineering/	rna-fold-pac-signal-1.webp)

**Figure 6.** RNA Fold of the two pac sites (red arrows) in the transcript expressed by RumiVec.

The CDS for the proteins were designed by inputting the corresponding amino acid sequences
obtained from the phage entry in NCBI (NC\_001417.2) [4] into the IDT codon-optimization tool.

A new insert sequence, named Shuffle Sequence 2 (SS2), was also designed
which has a greater length than the reporter cassette.
This change allows for screening cloned plasmids using colony PCR,
meaning we weren't restricted to blue-white screening
for confirmation of our reactions for this last cycle.

For colony PCR, new primers needed to be designed for the construct.
Given that the region downstream of the reporter remained unchanged,
the same reverse primer as the one used in the last iteration could be used.
The forward primer was designed using Benchling
and then the pair was verified with Primer3Plus
for the constructs including either the reporter or insert sequences.

![Primer 3 Plus figure](https://static.igem.wiki/teams/5980/engineering/primer3plus-construct-and-reporter.webp)

**Figure 7.** Primer3Plus using the newly designed primers on the construct with the reporter.
The predicted PCR product has a length of 362 bp and no issues were identified with the primers.

![Primer 3plus figure SSD insert](https://static.igem.wiki/teams/5980/engineering/primer3plus-construct-and-ss2-insert.webp)

**Figure 8.** Primer3Plus using the newly designed primers on the construct with the SS2 insert sequence.
The predicted PCR product has a length of 362 bp and no issues were identified with the primers.

## Build

Following the same procedures as the previous cycles,
the constructs were built on Benchling and then ordered as a clonal gene from Twist Bioscience.
The constructs with an insert instead of a reporter were obtained using Type IIS Assembly
with the NEB Golden Gate Cloning Kit directly on the construct and the **SS2** sequence.
These reactions were then used to transform NEB5α cells,
a strain suitable for Blue-White screening.

## Test

The transformed NEB5α cells were plated on LB agar plates and incubated.
Two colonies from the transformation were then streaked onto fresh X-Gal plates
and incubated again for Blue-White screening.

![X-Gal LB Agar plate](https://static.igem.wiki/teams/5980/engineering/pms2-500-bluewhite-screening.webp)

**Figure 9.** X-Gal LB Agar plate of NEB5α cells transformed with RumiVec containing either the reporter (a) or the SS2 insert sequence (b, c).
No blue colonies were observed in any of the plates.

The same colonies used to streak the plates were also used for Colony PCR.
The reporter-construct was included as a template control
to compare amplification results and confirm successful insert integration.

![Agarose gel of different samples](https://static.igem.wiki/teams/5980/engineering/colony-pcr-1.webp)

**Figure 10.** Agarose Gel of different samples: Golden-Gate assembly reaction mixture (GG Assembly);
Colony PCR from Colony 1 and 2 of NEB5α cells transformed using the Golden Gate reaction (GG Colony 1/2);
Colony PCR from a colony transformed with the reporter-construct plasmid (pMS2\_500 Transformant);
and PCR on the reporter-construct plasmid itself (pMS2\_500).

## Learn

Our results from Colony PCR show that this is a valuable technique
that can serve as a quick screening method
for any insert/reporter combination using the same primer pair.
This is a simple procedure which can quickly give results
without needing any reagent other than the ones for PCR.
The data from our experiments shows us that the reporter/cassette
can be easily swapped out with any desired sequence
as long as it has compatible ends for Type IIS assembly,
indicating that we met the goal of generating a modular system.

Based on the results obtained from blue-white screening from this iteration,
as well as our experience overall with this technique,
we believe that a different reporter other than LacZα
could serve a better role for our purposes.
Blue-white screening seems to be unreliable and poorly replicable,
which is why a new reporter is likely needed.

# REFERENCES

[1] Bauman, V. R., Markushevich, V. N., Kish, A. Z., & Gren, E. I.a (1976).
Sravnitel'naia fiziologiia replikatsii RNK-soderzhashchikh bakteriofagov MS2 i Q beta
[Comparative physiology of the replication of RNA-containing bacteriophages MS2 and Q beta].
Mikrobiologiia, 45(4), 685-689. https://pubmed.ncbi.nlm.nih.gov/979687/

[2] Mikel, P., Vasickova, P., & Kralik, P. (2015).
Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens.
Food and Environmental Virology, 7(2), 96-111. Advance online publication. https://doi.org/10.1007/s12560-015-9188-2

[3] Salis, H. M., Mirsky, E. A., & Voigt, C. A. (2009).
Automated design of synthetic ribosome binding sites to control protein expression.
Nature Biotechnology, 27(10), 946-950. https://doi.org/10.1038/nbt.1568

[4] GenBank. National Center for Biotechnology Information. [cited 2025 Sep 28].
Available from: https://www.ncbi.nlm.nih.gov/nuccore/NC001417

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![Protocols](https://static.igem.wiki/teams/5980/header/protocols.webp)
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![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Chemistry](#protocols-chemistry)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[RumiVec](#protocols-rumivec)

In this page you can view the detailed protocols of the key experiments for our project.

# CHEMISTRY

In this section we documented the main protocols performed to successfully functionalize glass surfaces
and develop a functional biosensor. Of special relevance we'd like to highlight our protocol to immobilize DNA oligos on glass surfaces,
which can be used by other teams to assemble DNA-based synthetic devices.
This protocol was verified on different glass substrates (slides, vials, capillaries),
showing the versatility of the scheme for different purposes.

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/dna-immobilization-on-capillary-tubes-done.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/functionalization-of-dna-with-primary-amines.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/ninhydrin-protocol-done-1.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/toehold-mediated-strand-displacement-reactions-on-solid-surfaces.pdf).

# RUMIVEC

The protocols used for the key experiments to test the RumiVec system are described in this section.

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/agarose-gel-electrophoresis-done.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/colony-pcr-done.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/blue-white-screening.pdf).

This browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5980/protocols/golden-gate-assembly-done.pdf).

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[Download PDF](https://static.igem.wiki/teams/5980/protocols/heatshock-transformation-done.pdf).

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![Results](https://static.igem.wiki/teams/5980/header/results.webp)
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![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Glass Functionalization](#results-glass)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[DNA Immobilization](#results-dna)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Toehold Mediated Strand Displacements](#results-toehold)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Modifying Wettability](#results-wet)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[Conclusions](#results-conclusions)

![Virus Bullet Point](https://static.igem.wiki/teams/5980/header/virus.webp)
[References](#results-references)

To develop our proof of concept we needed to accomplish four main goals:

1. Chemically modify the surface of capillary tubes
2. Immobilize DNA strands on the surface of capillary tubes
3. Verify toe-hold mediated strand displacement reactions (TMSDR) on glass surfaces
4. Modify the wettability of the inside of a capillary tube in order to observe a visible change in water rise

A detailed description of the methods can be found in the [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) section of the wiki.

# GLASS FUNCTIONALIZATION

In order to prepare a TMSDR in a capillary tube, the substrate strand needs to be immobilized on glass.
Modifying the surface with a chemical group that can be used to covalently bind the DNA to the surface is integral.
The chemical 3-Aminopropyltriethoxysilane (APTES) is often used to functionalize these surfaces with primary amines
for crosslinking other reagents to glass [1].
To optimize the reaction, the choice of solvent and temperature were tested
as they are of special relevance to the reaction kinetics and monolayer quality [1].
Different solvents interact differently with water.
The nature of these interactions will dictate the yield and speed of the reaction
as water can either inhibit or promote the reaction at different concentrations [1].

Success of the reaction can be assessed through ninhydrin assays, water contact angle (WCA) measurements, and fluorescence assays.
Ninhydrin is a crystalline compound that reacts with the amine groups of APTES
to form a product known as Ruhemann's purple (RP), which absorbs light at 570 nm [2].
WCAs serve as an indicator of the wettability of the glass surface,
which is significantly altered due to APTES functionalization [1].
Fluorescein isothiocyanate (FITC) is a fluorophore that can be used to label primary amine,
meaning that fluorescence would confirm the presence of these groups.

![Functionalisation of glass surfaces figure](https://static.igem.wiki/teams/5980/results/verification-scheme.webp)

**Figure 1.** Methods employed for verification of amine functionalization on glass surfaces.

The reaction was tested with anhydrous toluene, ethanol, and a mixture of toluene and ethanol in a 9:1 ratio.
Similarly, a second group of tests was done with the reaction at room temperature, 50°C or 70°C.
All the reactions were done overnight.
The products of the ninhydrin test showed significant absorption of light at 570nm indicating a successful reaction.

![Ninhyrdin absorbance tests](https://static.igem.wiki/teams/5980/results/	absorbanceninhydrin.webp)

**Figure 2.** Ninhydrin tests absorbance values of glass slides functionalized with APTES in Ethanol (top),
Toluene (bottom) and Ethanol+Toluene (middle).

Likewise, the contact angles for all the groups showed a significant difference from the negative control:

![Water droplets on glass slides](https://static.igem.wiki/teams/5980/results/contact-angle-of-droplets.webp)

**Figure 3.** Water droplets on reacted (left) and unreacted (right) glass slides.

![WCA Temperature Graph](https://static.igem.wiki/teams/5980/results/wca-bar-graph-temperature.webp)

**Figure 4.** Mean measurements of water contact angle for glass slides reacted with APTES under different temperatures.
All experimental groups had a significantly higher contact angle than the negative control (n=5, M=25.40°, SD=7.92).
The 50°C (n=5, M=39.33°, SD=6.66°) and 70°C (n=5, M=36.90°, SD=3.13°) were both significantly lower
than the room temperature (n=5, M=55.40°, SD=4.04°) group.

![WCA Solvent Graph](https://static.igem.wiki/teams/5980/results/wca-bar-graph-solvent-choice.webp)

**Figure 5.** Mean measurements of water contact angle for glass slides reacted with APTES under different anhydrous solvents.
All experimental groups were significantly higher from the unreacted glass slide group (n=5, M=25.4°, SD=7.93°).
The ethanol (n=5, M=64.1°, SD=4.62°), toluene (n=5, M=59.0°, SD=8.72°), and 9:1 toluene/ethanol (n=5, M=61.3°, SD = 4.97°)

The measured WCA are consistent with what is expected from aminated surfaces based on current literature [1].
Although all the reactions were successful, the different solvent groups were not found to be significantly different from each other.
Similarly, incubation at 50°C or 70°C did not produce any significant differences either.
Surprisingly, incubation at room temperature seemed to produce significantly more functionalization than any other temperature.
However, this is likely due to significant physioadsorption to the glass rather than covalent immobilization as reported by other studies [1].

Based on our results, and the scheme described by Sun et al. [3],
we decided to modify the glass surfaces at 60°C for 1 hour using a 10% APTES solution in ethanol.
The reaction was first tested inside scintillation vials, then glass slides, and finally capillary tubes
to ensure that if there were any problems at any point, it was not due to the reaction itself.
FITC fluorescence was tested using a UV-lamp (365 nm) in a dark room using PBS Buffer as a visualizing solvent.
All the experimental groups exhibited visible fluorescence while the controls were mostly dark:

![Fluorescence of glass materials](https://static.igem.wiki/teams/5980/results/fluorescence-of-different-glass-materials.webp)

**Figure 6.** Fluorescence of glass vials, slides, and capillary tubes visualized under a UV-lamp.
Substrates on the left were reacted with APTES (10%) for 1 hour at 60°C and then with FITC in DMF under the same conditions.
Substrates on the right are unreacted. PBS buffer was used as the medium for the images.

By successfully functionalizing capillary tubes with primary amines,
we checked off one of our main goals of the project
and could move to the next steps of assembling our sensor.

# DNA IMMOBILIZATION

The team decided to follow the same immobilization strategy as Sun et al. [3].
First, capillary tubes were reacted with APTES to functionalize the surface with primary amines.
Next, the surface was reacted with Glutaraldehyde to get amine-reactive groups (aldehydes) on the inside of a capillary.
Finally, DNA oligonucleotides modified with a primary amine group on the 3' end (purchased from IDT),
were reacted onto the glass to finally immobilize the substrate strand on our capillary tubes.

These series of reactions were verified using DNA strands modified with fluorescent molecules
which were then used to visualize the surface of a modified capillary tube under a confocal microscope.
The data shows a statistically significant signal for the reacted capillary tube when compared to the control
(n = 3, M = 1.31 A.U, SD = 0.22, p = 0.016 versus control, Student's t-test).

![Confocal microscopy Imaging](https://static.igem.wiki/teams/5980/results/confocal-image-of-capillary-1.webp)

**Figure 7.** Confocal microscopy imaging and average count for 560 ± 94 nm fluorescence of capillary tubes functionalized
(n=3, M=1.31 A.U, SD=0.22) and unreacted (n=3, M=0.24, SD=0.09) with DNA strands modified with Cy3.
Capillary tubes are highlighted in the red box.

As part of our work, we also tested a modified version of a method described by Wickramathilaka & Tao
to prepare oligonucleotides [4] functionalized on the 5' end with primary amines.
Primary amines are one of if not the most useful group for chemical crosslinking of biological agents [1],
which means functionalizing DNA strands with such a group can prove incredibly useful
for any synthetic biology projects involving any modifications of oligonucleotides.
To verify this reaction, FITC was reacted with the modified DNA mixture
after which it was purified with an NEB Monarch® PCR & DNA Cleanup Kit.
The purified DNA was then analyzed with a fluorometer,
which showed a much higher signal when compared to controls.

![Fluorescence spectra](https://static.igem.wiki/teams/5980/results/	fluorescnce-spectra-of-dna-reaction.webp)

**Figure 8.** Fluorescence spectra (λex = 495 nm) of samples with DNA strands functionalized with FITC,
purified through a silica column (left); and samples containing all the components of the functionalization reaction except DNA,
purified through a silica column (right).

Two graphs on the right represent a control, two on the right represent the purified DNA solutions.

We also tested if these modified DNA molecules could be immobilized onto glass surfaces for use in biosensors.
For this, double stranded DNA was functionalized with primary amines,
then reacted with glutaraldehyde-functionalized capillary tubes, and then reacted with FITC.
When visualized under a UV Lamp, fluorescence was observed with the naked eye, indicating a successful reaction.

![Fluorescence spectra](https://static.igem.wiki/teams/5980/results/oligo-functionalized-capillary-fluorescence.webp)

**Figure 9.** Fluorescence of glass capillary tube functionalized with FITC-modified DNA strands Under a UV Lamp.

We hope that with this procedure, future teams working on similar projects
can easily immobilize oligonucleotides onto glass surfaces using minimal reagents
and without the need to purchase costly custom oligos.

# TOEHOLD MEDIATED STRAND DISPLACEMENTS

TMSDs are the sensing mechanism of our proposed device,
so it is crucial to ensure that we can successfully run this process on glass surfaces.
For this purpose, we needed to integrate everything that we have accomplished up to this point into a functional biosensor.
The glutaraldehyde-functionalized glass slides were used to immobilize the substrate strands and set the platform for the TMSDs to occur.
The substrate strand was modified in the 5' end with the Cy3 fluorophore and in the 3' end with a primary amine.
The 3' end was chosen as the attachment point as previous studies show that the TMSD is much more efficient
when the toehold is in the 5' end of the substrate strand [4].
The incumbent strand was modified in the 3' end with Cy5,
a dye capable of acting as a Fluorescence Resonance Energy Transfer (FRET) acceptor for Cy3.
By acting as a FRET acceptor, we can monitor when the substrate and incumbent strands are hybridized together.
The target strand was modified in the 3' end with 6-FAM, a fluorophore similar to Cy3.

![Toehod mediated strand displacement figure](https://static.igem.wiki/teams/5980/results/capillary-tube-modification-scheme.webp)

**Figure 10.** Reaction scheme for toehold-mediated strand displacements on glass surfaces.

Three slides were prepared and modified up to different steps of the reaction scheme.
The first slide was functionalized with the substrate and then imaged with the microscope.
The second slide was hybridized with the incumbent strand and then imaged.
The final slide was treated with 100 nM of the target strand, and then imaged.

![Confocal microscope imaging](https://static.igem.wiki/teams/5980/results/	substrateimaging.webp)

**Figure 11.** Confocal microscope imaging (λex = 470/635 nm) of glass slides functionalized with a DNA Substrate modified with Cy3.
The left image corresponds to a channel measuring fluorescence of light >635 nm (Cy5 emission)
and the right image corresponds to a channel measuring fluorescence of light in the 560±94 nm range (Cy3/6-FAM emission).

![Confocal microscope imaging](https://static.igem.wiki/teams/5980/results/substrateincumbentimaging.webp)

**Figure 12.** Confocal microscope imaging (λex = 470/635 nm) of glass slides functionalized with a DNA Substrate modified with Cy3
and hybridized with a DNA incumbent strand modified with Cy5.
The left image corresponds to a channel measuring fluorescence of light >635 nm (Cy5 emission)
and the right image corresponds to a channel measuring fluorescence of light in the 560±94 nm range (Cy3/6-FAM emission).

![Confocal microscope imaging](https://static.igem.wiki/teams/5980/results/displacedcompleximaging.webp)

**Figure 13.** Confocal microscope imaging (λex = 470/635 nm) of glass slides functionalized with a DNA Substrate modified with Cy3
and hybridized with a DNA incumbent strand modified with Cy5.
The left image corresponds to a channel measuring fluorescence of light >635 nm (Cy5 emission)
and the right image corresponds to a channel measuring fluorescence of light in the 560±94 nm range (Cy3/6-FAM emission).
6-FAM not included in the diagram for simplicity.

These results showed that the target sequence is capable of displacing the incumbent strand from the complex at a concentration of 100 nM,
a process that can be observed within minutes.

# MODIFYING WETTABILITY

Our entire proposed project depends on producing a big enough change in wettability to observe a signal easy to observe.
To test how much we can modify the wettability of a capillary tube -and by proxy the water rise due to capillary action-
a chemical called Octadecyltrichlorosilane (OTS) was reacted with the surface of different tubes
and then the heights of the water from the surface were measured.
OTS is highly reactive towards glass surfaces and produces a highly hydrophobic layer as a result [5].
This was confirmed with our experiments which showed that when dipped in water,
no liquid would even go into the capillary.

![Glass capillary tubes dipped in water](https://static.igem.wiki/teams/5980/results/wettability-modification.webp)

**Figure 14.** Glass capillary tubes dipped in water.
The left image shows a capillary functionalized with OTS, the right image shows an unreacted capillary.
Water rise was only observed in the unreacted tube, while water was suppressed for the functionalized tube.

# CONCLUSIONS

Our team achieved strong results developing a proof-of-concept biosensor.
We functionalized glass surfaces with DNA oligos and altered capillary tube wettability enough to observe a visible signal.
This allowed us to assemble a working biosensor and establish methods for advancing the project.
Next, we aim to generate a wettability change as a signal for detecting specific DNA sequences.
To do this, we'll modify oligos with hydrophobic groups now feasible thanks to our successful DNA modification tests.
We'll replace fluorophores with hydrophobic tags in the TMSD system, test sensitivity, and optimize detection limits for point-of-care use.
These results mark a promising beginning for Rumino's journey ahead.

# REFERENCES

[1] Sypabekova, M., Hagemann, A., Rho, D., & Kim, S. (2022).
Review: 3-Aminopropyltriethoxysilane (APTES) Deposition Methods on Oxide Surfaces in Solution and Vapor Phases for Biosensing Applications.
Biosensors, 13(1), 36. DOI: 10.3390/bios13010036

[2] Stauß, A. C., Fuchs, C., Jansen, P., Repert, S., Alcock, K., Ludewig, S., & Rozhon, W. (2024).
The Ninhydrin Reaction Revisited: Optimisation and Application for Quantification of Free Amino Acids.
Molecules (Basel, Switzerland), 29(14), 3262. DOI: 10.3390/molecules29143262

[3] Sun, Y., Liu, K., Zhang, H., Zhao, Y., Wen, J., Zhao, M., Li, X., & Li, Z. (2025).
A tube-based biosensor for DNA and RNA detection.
Science Advances, 11(18), eadu2271. DOI: 10.1126/sciadv.adu2271

[4] Wickramathilaka, M. P., & Tao, B. Y. (2019).
Characterization of covalent crosslinking strategies for synthesizing DNA-based bioconjugates.
Journal of Biological Engineering, 13, 63. DOI: 10.1186/s13036-019-0191-2

[5] McGovern, M. E., Kallury, K. M. R., & Thompson, M. (1994).
Role of Solvent on the Silanization of Glass with Octadecyltrichlorosilane.
Langmuir, 10(10), 3607-3614. DOI: 10.1021/la00022a038

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