Our contribution for future iGEM teams
We contributed - novel measurements of existing aptamers not reported
in literature (measurement, results), including
raw data suitable for analysis by other teams and researchers results - ITC
results present first documented measurement of KD of the
P4G03 aptamer with that methodology, only other value
encountered in literature review was calculated using fluoresence,
corroborating the tens-nanomolar KD range -
Circular Dichroism data represents first documented cross reactivity
experiments for P4G03 aptamer, indicating specificity for
progesterone over other physiologically relevant, similar hormones DHT
and cholesterol, providing the first documented evidence known to us for
P4G03’s specificity to progesterone over similar hormones -
clear protocols and troubleshooting logs from experimentation (measurement, experiments)
that will enable other teams to use our methodologies more easily -
significant human
practices research that could be useful to other teams working in
the area of women’s health and firefighter health - downloadable
anonymized survey results from >550 individuals relevant to women’s
health - recorded insights from interviews with, observations of
firefighters - and hardware
designs/renderings/CAD files that could be useful to other teams - CAD
files for fluoresence sensor housing - diagrams and creation process for
microfluidic chip mask, injector - identified miniaturizable
potentiostat design useful for bio-impedimetric aptamers
Creating an aptamer sensor required us to validate that an aptamer would have sufficient specificity for our target molecule. To do this we performed a fluorophore-quencher affinity assay with our first aptamer, and isothermal calorimetry and circular dichroism with our second aptamer, to verify that the aptamers we studied had the binding affinity and specificity reported in the literature. In some cases, we only replicated an existing protocol, in other cases we provided significant adaptations. Our validation / extension of these protocols will be useful to future iGEM teams working with aptamers. The measurements (see results, measurements pages) that we obtained will be useful to future iGEM teams working with the PFOA_JYP_21 and P4G032 aptamers we used.
When we were still developing a carcinogen sensor for firefighters, we performed a fluorophore-quencher affinity assay with the PFOA aptamer (PFOA_JYP_2) that we had selected for use. Our measurement of the KD of PFOA_JYP_2 provided an external validation of the aptamer, and of the fluorophore-quencher protocol. Although we did not develop this protocol, we referenced the paper we sourced the protocol from. We also documented the steps we took to replicate the experiment, our results and the code we wrote to analyze the data. This protocol is advantageous because it provides a cheap, easy-to-access way to measure binding affinity, requiring only basic lab equipment like a plate reader. Future iGEM teams could use our documentation to better understand this process and evaluate their aptamers in a similar way.
For isothermal titration calorimetry (ITC), we developed a protocol that allows water-insoluble target molecules to be used. In ITC it is essential that the buffers of the aptamer and target molecule (in our case, progesterone) be identical. We met with Dr. Jason Kenealy to identify the best way to perform ITC and realized that the protocol that we desired to follow did not allow for equivalent buffers for nonpolar solvents. We had to modify the procedure to allow our nonpolar target molecule to be solubilized, while keeping the buffers equivalent. We did this by troubleshooting the amount of ethanol to be used in the target and aptamer buffers and found that 0.15% ethanol is best. Our final protocol that we developed is in the experiments page here, and may be useful to any additional iGEM teams seeking to validate KD values for aptamers specific to nonpolar molecules such as hormones.
For Circular Dichroism (CD), we created a detailed protocol for testing how aptamers purchased from IDT fold and bind in solution. We made the protocol in conjunction with Tanner Blocker from Dr Dixon Woodbury’s lab who work with CD but have never worked with aptamers before. We ran three rounds of CD experiments to measure how the progesterone-binding aptamer P4G03 changes structure in response to different concentrations of progesterone and to compare it with similar hormones like DHT and cholesterol. From these tests, we learned that in solution, the aptamer undergoes increasing conformational change up to a 20 : 1 progesterone-to-aptamer ratio, but then decreases at 40 : 1, suggesting the structure becomes oversaturated or aggregated at high ligand levels. Through troubleshooting, we discovered how important aptamer freshness, solvent consistency, and mixing technique are important for clear CD results. Our protocols, data, and graphs are shared on our wiki so future iGEM teams can replicate this process or adapt it to validate their own aptamers. CD spectrophotometers themselves are expensive, but if future iGEM teams have access to a device, our protocols present a flexible, low-cost, and accessible method that any iGEM team can use. Teams can easily substitute their own aptamer for ours—whether their project involves hormone detection, drug screening, environmental sensing, or metabolite monitoring—and still apply the same process to confirm folding and specificity in solution.
We experienced some technical and logistical hurdles that detracted from our available time to experiment, so we wanted to emphasize some points of advice that will be useful to future teams:
In the first step of actually creating the sensor we discovered from literature that an aptamer must have specific modifications. These modifications were on the 5-prime end of the nucleotide sequence a redox reporter must be added, and the most common redox reporter is methylene blue which is the reporter that our team used. Another modification required for the nucleotide sequence is a thiol attached on the 3-prime end of the nucleotide with a 6-carbon chain. These modifications allow for the aptamer sequence to be attached to a gold electrode.2, 3
We wanted a 6-carbon linker, but one of the suppliers we spent time negotiating with was only able to supply a 3-carbon linker, and we only found out this information in the final ordering process. We could have saved valuable time if we had asked about their modification capabilities earlier.
In the process of building this sensor one of the main tasks that we had to troubleshoot was discovering that there must be a substance (thiolated carbon chains) put between the aptamers on the gold electrode. The aptamers are much larger in size than the gold atoms covering the electrode and the large mass of the aptamers prevent the aptamers from being able to tightly pack on the gold. A small molecule is required to fill in these gaps between the aptamers. An important detail is that the molecule selected to fill these gaps must be equivalent in length to the carbon chain attached to the thiol modification. For our project that meant that our gold electrode required a molecule that was a 6-carbon chain, 6-mercaptohexanol, to be equivalent to the 6-carbon chain on the aptamer.2, 3
When we used thiolated carbon chains that were too short (2-mercaptaethanol), we were able to get an electrical signal from progesterone solution but observed a signal drop when we flushed the electrodes that didn’t return, even after we added more progesterone solution. With one of our lab advisors with experience in AC voltammetry, we hypothesized that this drop was due to the aptamer being washed away as the 2-mercaptaethanol was too short to protect the aptamer from being stripped away.
We are still actively in the process of obtaining useful measurements and refining our protocol for AC voltammetry.
Our hardware designs will provide replicable options or inspiration
for future teams. See our hardware page for
schematics and some downloadable .stl files. These files
could be used directly or edited by another team, the schematics and
documented process descriptions will also enable other teams to develop
similar hardware more easily.
This quantitative fluorescence sensor design with microfluidic chip insert will be useful to future teams seeking modular hardware for fluorescence measurement.
In designing the electronics for PFOA sensor, we borrowed the basic electronic design from last year’s BYU IGEM project. We swapped out the type of LED and added another LED; this was to change the wavelength of the light and provide more light to activate the aptamers. Most of the troubleshooting was spent during the wiring of the electronics. The housing was designed in SolidWorks. The housing allows for the microfluidic chip to slide in and out of the housing. The concept was inspired by Li et al.4, but instead of utilizing a smartphone as the visual measurement device, we incorporated a photodiode, allowing for the device to potentially be made smaller and more precise in future iterations.
An error with the housing was the amount of infill used when it was
printed. The low infill allowed a lot of light to pass through the
housing. If used by another team, we recommend increasing the amount of
infill to block out more light. .stl files available on the
hardware page.
In conjunction with the sensor housing above, we designed a polydimethylsiloxane (PMDS) microfluidic chip and built a corresponding chromium mask to allow for measurement of PFOA. Although we didn’t end up using the chip, the specific fabrication details and diagrams on the hardware page could be useful for future iGEM teams wishing to use our exact fluoresence sensor design. Our concept and design process could also inspire teams wishing to develop their own customized microfluidic chips.
We developed a prototype applicator device that will be capable of applying the team’s future progesterone monitor to a user’s forearm or other area of the body, analagous to how other applicators place glucose monitors on the skin’s surface. This design is unique from other, similar patented applicators. This design was submitted as part of the provisional patent submitted by our team, but the included renders on our hardware page could serve as an inspiration or starting place for future iGEM teams who want to create their own mechanisms.
Our human practices resesarch will provide guidance to future teams seeking to impact womens’ health, including in the areas of early pregnancy and miscarriage.
Our team has focused on how we can improve the process of using
alternating current voltammetry to create an aptamer sensor. Our team
also wanted to make a sensor that would actually solve a problem in the
local community. We went to our local community and asked what was
important to them to measure. After personal interviews, we discovered
our local community wanted better hormone monitoring, specifically
monitoring of hormones during pregnancy and ovulation [see human
practices]. We have had over 500 survey responses from women indicating
interest in a continuous monitor of progesterone and breaking down the
reasons for their interest. These survey responses were collected in
accordance with the appropriate BYU IRB rules for surveys distributed by
a BYU club or class (no IRB required, but certain conditions must be
met). We provide links to download the anonymized .csv
survey results on our human
practices page.
We have discovered that women want the ability to have more self-advocacy in their healthcare. Women want devices that can be used to better understand and even take control of their personal health without exclusive reliance on a doctor. Based on our research, we believe that people want more power when they go to the doctor. They want the ability to know what is going on with their body and they want that data. People want doctors to teach them how to draw conclusions about their health and what they can do to better their own health. People want more correct information on their health and the ability to have their own freedom of decisions in their healthcare. Future iGEM teams can use our research in this underexplored sector to help them identify new human practices questions to ask and new problems to solve through synthetic biology that will make an impact in people’s lives.
Park, J., Yang, K.-A., Choi, Y. & Choe, J. K. Novel ssDNA aptamer-based fluorescence sensor for perfluorooctanoic acid detection in water. Environment International 158, 107000 (2022).
Jiménez, G. C. et al. Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone. ACS Publications https://pubs.acs.org/doi/pdf/10.1021/ac503639s (2015) doi:10.1021/ac503639s.
Xiao, Y., Lai, R. Y. & Plaxco, K. W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc 2, 2875–2880 (2007).
Li, Z., Zhang, S., Yu, T., Dai, Z. & Wei, Q. Aptamer-Based Fluorescent Sensor Array for Multiplexed Detection of Cyanotoxins on a Smartphone. Anal. Chem. 91, 10448–10457 (2019).