Contribution
Contribution
We fully acknowledge that our project wouldn't be possible without the generosity of the iGEM teams and other scientists who came before us and chose to share their work with the public. We truly believe that access to resources and knowledge can lead to great things, as more and more people from different fields come together to build on the work of others.
That is why, throughout our iGEM journey, we made sure to consider how we could make our work valuable to future teams and the synthetic biology community. We experienced a few notable successes and learning opportunities (because the word "failure" doesn't exist in our dictionary). We hope these experiences will offer useful insights and help make future projects a bit easier for others.
Our team focused on developing a tool for detecting Prymnesium parvum, but we didn't stop there! We set out to create a versatile tool for future iGEM teams interested in using the SHERLOCK platform to create detection systems, regardless of the target they want to identify.
With that vision in mind we designed, built and tested SynLOCK – the crRNA Synthesis System for SHERLOCK. SynLOCK simplifies and standardizes the process of generating custom crRNAs, allowing users to create crRNAs with a 28-nucleotide spacer sequence tailored to their specific needs. This makes SynLOCK a versatile and user-friendly platform for developing various detection tools that are compatible with simple Lateral Flow Assay tests and can be used directly in the field.
We invite you to learn more about our system on the Part Page of its key component – the SynLOCK Cassette – and discover how to customize it to your needs!
The PrymChip is a versatile, 3D-printed device designed to facilitate the excitation and detection of fluorescence signals. It consists of three easily customizable boxes, which can be printed using fused deposition modeling (FDM) technology with e.g. PETG filaments. These boxes were created in AutoCAD, making them accessible for anyone familiar with these programs to modify, whether in size or shape, to suit specific experimental requirements. All components are available for download on our Hardware page as STL and G-code files. The purple toolbox is especially modifiable, allowing users to add tools or print additional holders for customized experimental setups.
The PrymChip is an excellent resource for future iGEM teams, offering a flexible platform with enormous potential for further development and adaptation for environmental assays. More information about the PrymChip can be found on our Hardware page.
The entire system is designed to be highly adaptable. For example, the electronic components responsible for signal excitation can be swapped out for different light sources, such as lasers instead of LED diodes. The diodes’ colors can also be adjusted, depending on experimental needs. Additionally, the filter in the black detection box is replaceable with filters of varying colors, making it a highly customizable base for a fluorescence detection device.
The detection process itself leverages smartphone photography to capture images of the detection chamber. A Python script is used to analyze these images, employing a machine learning algorithm trained on a set of photos to identify fluorescence intensity. This script can be easily modified by swapping out the original training photos with new ones, tailoring the algorithm to specific research needs. This makes PrymChip a universally applicable tool that can be adjusted for any research purpose.
The PrymChip is designed with future improvements in mind. Some possible upgrades include:
This tool offers an excellent starting point for iGEM teams looking to innovate, and we hope to see many exciting applications and modifications in the future.
The LwaCas13a protein is a useful member of the Cas protein family. Upon binding to the target sequence via the crRNA molecule, it can cleave single-stranded RNA probes present in the solution. Our detection method for Prymnesium parvum utilizes this activity. The protein is, therefore, a key and universal element of the test.
Due to the multitude of applications, optimizing the protocol for obtaining and purifying the protein is of great importance. For this reason, we decided to focus on its optimization to make future iGEM teams’ work easier.
Our purification protocol was based on the protocol of Kellner et al. [1]. The first optimization step was to change the resin used for purification by affinity chromatography. The protein sequence encoded both the Strep-Tag and the His-Tag.
While the Strep-tag yields higher purity, the His-tag enables purification on a more cost-effective, high-capacity resin. Given the necessity for a multi-step purification process due to tag cleavage, we concluded that incorporating a His-tag purification procedure into our protocol would be the best approach.
The second modification we introduced was the elimination of tag cleavage by SUMO protease. Although protocols [1,2] described the digestion procedure, we demonstrated that it is unnecessary. The protein with the uncleaved tag retains its activity, structure (confirmed by Circular Dichroism), and stability (confirmed by Differential Scanning Fluorimetry). Ommiting this step reduces purification costs and accelerates the process by at least one day.
We are excited to share our discoveries and modifications with the iGEM community by publishing our data on the Part Registry. We hope you find them helpful, and if you have any questions, please feel free to reach out to us!
Prymnesium parvum is a non-model organism. There are no specific protocols describing the culture procedure of this species. Based on our own optimization of culture conditions and the guidelines provided by Dr. Ewa Górecka at the University of Szczecin, we developed a method for maintaining Prymnesium parvum under laboratory conditions. We hope this method will assist future teams working with this non-model organism.
We maintained our cultures using the F/2 medium. F/2 is a widely utilised medium specifically designed for cultivating coastal marine algae, although it also works well for freshwater species.
Prior to preparing the medium, we made stock solutions for each of the medium ingredients. Table 1. shows the ingredients and their final concentration.
Table 1. F/2 medium ingredients.
|
Ingredient name |
Final concentration [mg/L] |
|
ZnSO4 · 7 H2O |
22 |
|
CoCl2 · 6 H2O |
10 |
|
MnCl2 · 4 H2O |
180 |
|
Na2Mo4 · 2 H2O |
6 |
|
NaNO3 |
75 |
|
NaH2PO4 · 2 H2O |
56.5 |
|
Na2EDTA |
4160 |
|
FeCl3 · 6 H2O |
3150 |
|
CuSO4 · 5 H2O |
10 |
Vitamins: We enriched the F/2 medium with vitamins B1, B7, and B12. The stock solutions for these vitamins were prepared separately and added directly to the newly created cultures so that they do not degrade during the sterilisation process. The final concentrations of the vitamins in the cultures are shown in Table 2.
Table 2. Final concentration of vitamin solutions.
|
Vitamin |
Concentration [mg/L] |
|
B1 |
100 |
|
B7 |
0.5 |
|
B12 |
0.5 |
Each freshly prepared batch of medium was sterilised using a microwave autoclave.
Sea salt: At the beginning of our work with Prymnesium parvum, we utilized a sea salt concentration at 16 g/L, which is a commonly recommended value. After consultation with Dr. Ewa Górecka, we reduced the concentration of added salt to 3 g/L, following her advice.
pH: We monitored the pH value of the medium with pH-meter and maintained it at the level of approximately 8.
Initially, we passaged our cultures every two weeks. However, subsequent observations indicated that this frequency was not necessary. Accordingly, we adjusted the passage frequency to every three to four weeks.
We stored the cultures in a growth chamber at 22°C, with lighting of 100 photon fluence rate, and a 14 h/10 h light/dark cycle. These conditions are summarized in Table 3.
Table 3. Conditions of the growth chamber.
|
Temperature |
22°C |
|
Lightning |
100 photon fluence rate |
|
Light/dark cycle |
14 h/10 h |
We used three different DNA isolation methods
The first two methods utilize spin columns, which bind DNA released after tissue lysis. The third method is based on binding of the DNA to dipsticks made from cellulose paper.
Isolation with the NEB kit yielded a little bit higher DNA concentration than the isolation with the QIAGEN kit. The NEB isolation kit also turned out to be more convenient, as it did not require such a long (5-7h) initial incubation step, as the QIAGEN kit did.
We tested a nucleic acid purification method described in the publication “Nucleic acid purification from plants, animals, and microbes in under 30 seconds,” which is designed for use outside the laboratory setting [4]. Our objective was to adapt this method for field-based nucleic acid isolation within the PrymChip. The technique involves preparing cellulose dipsticks coated with wax for nucleic acid purification. The dipsticks we created are presented in Figure 1.
Figure 1. The dipsticks made out of Wattman paper.
The procedure involves placing a biological sample in a tube containing a cell lysis buffer with ball bearings, followed by shaking the tube for a few seconds. The dipstick is then inserted into the tube to bind nucleic acids, followed by washing with a wash buffer and finally dipping into a tube containing reagents for the amplification reaction. A schematic representation of this method is shown in Figure 2.
Figure 2. Equipment for the in-field isolation with instructions. In Eppendorf tubes lysis buffer (number 1) and wash buffer (number 2) are presented.
Despite our efforts, we were unable to isolate DNA. However, we highlight that this cost-effective field-based method for nucleic acid purification exists and could be further optimized by future iGEM teams for environmental testing purposes.
[1] Kellner, M. J.; Koob, J.; Gootenberg, J. S.; Abudayyeh, O. O.; Zhang, F. SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases. Nat. Protoc. 2019, 14 (10), 2986–3012. https://doi.org/10.1038/s41596-019-0210-2.
[2] Abudayyeh, O. O.; Gootenberg, J. S.; Konermann, S.; Joung, J.; Slaymaker, I. M.; Cox, D. B. T.; Shmakov, S.; Makarova, K. S.; Semenova, E.; Minakhin, L.; Severinov, K.; Regev, A.; Lander, E. S.; Koonin, E. V.; Zhang, F. C2c2 Is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector. Science 2016, 353 (6299), aaf5573. https://doi.org/10.1126/science.aaf5573.
[3] Lichty, J. J.; Malecki, J. L.; Agnew, H. D.; Michelson-Horowitz, D. J.; Tan, S. Comparison of Affinity Tags for Protein Purification. Protein Expr. Purif. 2005, 41 (1), 98–105. https://doi.org/10.1016/j.pep.2005.01.019
[4] Zou Y, Mason MG, Wang Y, Wee E, Turni C, Blackall PJ, et al. (2017) Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol 15(11): e2003916.
