Engineering
Engineering
Our project aimed to develop an effective and sensitive tool for detecting
Prymnesium parvum blooms. Throughout our journey, we closely embraced the engineering principles
that lie at the heart of iGEM as well as synthetic biology as a scientific discipline.
We applied the engineering cycle to every aspect of our work, even in tasks that might not traditionally be seen as
engineering.
This framework guided us in designing our hardware, the PrymChip, as well as our
biological parts. It also shaped our
protein purification efforts, SHERLOCK reaction design and the optimization of
growth conditions for our Prymnesium parvum cultures.
We believe that this comprehensive approach played a crucial role in our project's success.
This page provides an overview of the Design-Build-Test-Learn (DBTL) cycles performed throughout our project.
Goal: The goal of this cycle was to establish a Cas13 purification protocol that would be successful in obtaining pure Cas13, while also being cost-effective and time-efficient.
We based our approach on the protocol suggested by Kellner et al. [1], given the widespread use and study of
huLwCas13a.
For protein expression, we selected the pC013 (huLwCas13a) [2] and pR0306 (CcaCas13b) [3]
plasmids from
Addgene, which use the pET system for high-yield production.
This plasmid includes sequences for purification tags (6xHis, TwinStrep) and a
SUMO tag to enhance solubility.
The SUMO tag also allows for tag removal, yielding the native form of the protein after cleavage.
We decided to express the protein in the Rosetta™ 2(DE3)pLysS strain, which supports the pET
system and
produces T7 polymerase and T7 lysozyme.
The latter suppresses protein expression until induction with IPTG.
Overview of the initial protocol for Cas13 production and purification:
Accurate purification of Cas13 was crucial because the Bradford assay measures total protein content, which can include non-Cas13 proteins and skew concentration readings. This could result in adding insufficient Cas13, reducing test sensitivity and causing potential false negatives. To enhance cost-effectiveness, we substituted StrepTag affinity chromatography with Immobilized Metal Affinity Chromatography (IMAC) in Kellner's protocol.
After additional consideration, we noticed that in many protocols, the 6xHis-TwinStrep-SUMO tag at the N-terminus of the protein is cleaved off after purification [1], [4], [5]. However, we found no specific data in the literature justifying the removal of this tag, which requires the use of the relatively costly SUMO protease. We decided to investigate this further and check whether tag removal is truly necessary.
We compared SUMO-digested and undigested protein fractions after IMAC using:
Due to budget constraints, we initially focused on huLwCas13a, postponing ordering synthetic DNA for CcaCas13b
until huLwCas13a's activity was confirmed.
As a result, finally, we decided to apply the redesigned Cas13 purification protocol:
We carried out protein expression and purification according to the revised protocol (also included under “Protein Purification” in the “Experiments” page):
Step 1 – Rosetta 2(DE3)pLysS transformation with pC013 [2] and pR0306 [3] plasmids.
As a result of transformation we observed colonies on both plated Petri dishes:
Figure 1. Single colony grown after transformation of Rosetta 2(DE3)pLysS cells with pC013.
Figure 2. Colonies grown after transformation of Rosetta 2(DE3)pLysS cells with pR0306.
Step 2 – Bacterial cultures and Cas proteins expression.
We used them later to prepare large scale cultures (in total 9 L) that were induced with IPTG overnight.
Step 3 – Harvesting bacteria.
The following day, we harvested the bacteria through centrifugation, obtaining 95.18 g of bacterial pellet for Cas13a and 96.7 g for Cas13b.
Step 4 – Sonication.
A portion of the bacterial pellet for both proteins was disintegrated using sonication. Following centrifugation, the supernatant was prepared for the first step of purification – Immobilized Metal Affinity Chromatography (IMAC).
Step 5 – IMAC.
We performed SDS-PAGE to investigate the results of IMAC:
Figure 3. SDS-PAGE gel for huLwCas13a after IMAC purification.
Figure 4. SDS-PAGE gel for CcaCas13b after IMAC purification.
Wells key: unind. – uninduced bacterial culture, ind. – culture after 18h IPTG induction, sonic. – supernatant after sonication and centrifugation, prec. – sediment after sonication and centrifugation, FT – flow-through from sample application on His-resin column, wash – flow-through from column washing before elution; E1, E2, E3… – fractions eluted with 150 mM imidazole buffer, *E1, *E2, *E3 – fractions eluted with 300 mM imidazole buffer
*Note: Some electropherograms show bands shifted downward from their expected positions, likely due to well overloading. To confirm the proteins' sequences, mass spectrometry would be ideal, but budget constraints prevented us from performing this analysis.
Expression and IMAC Conclusions:
The expression step was effective for both Cas proteins as after induction the bands of a mass corresponding to the produced protein were observed (for Cas13a – 155 kDa, for Cas13a – 160 kDa).
IMAC was effective in partially purifying both proteins – percentage content of contaminants in the solution decreased (compared to wells ind. and sonic). Although they are still present in significant amounts. Moreover, fractions were enriched with the produced protein (thicker bands corresponding to the produced protein – marked by arrows).
Step 6 – SUMO digestion
The SUMO digestion step was conducted, as planned, on half of the collected IMAC fractions.
SUMO digested and undigested pooled fractions were used in the second purification protocol step – IEC.
Step 7 – IEC
SDS-PAGE gels showing results of IEC:
Figure 5. SDS-PAGE gel for huLwCas13a – samples after IEC.
Cas13a with the tag is indicated by the violet arrow, Cas13a without the tag – blue arrow. The purple rectangle marks two bands that correspond to the cleaved 6xHis-TwinStrep-SUMO tag.
Wells key: entry – pooled fractions after IMAC, sample appl. – flow-through from sample application on the column, wash – flow-through from column washing, E1, E2, E3… – eluted fractions. Samples with * correspond to samples after SUMO protease digestion.
Figure 6. SDS-PAGE gel for CcaCas13b – samples after IEC.
Cas13b with the tag indicated by yellow arrow, without by orange arrow.
Wells key: eluted fractions of Cas13b (left picture for SUMO protease undigested, right for digested) E6, E8, E10... – eluted fractions; flow-through – from sample application on the column; wash – flow-through from column washing. The area where a band for the digested tag was expected is marked with black circles. For the undigested protein there is one band in this area, while for the digested protein, there are two. Therefore, the additional band is likely the tag.
IEC Conclusions: For Cas13a and Cas13b (both SUMO protease digested and undigested samples) reduction in protein contaminants, approximately 2-fold, can be observed, although they still remain in significant amounts. Concentration of Cas13a and Cas13b in the collected fractions is visible. After IEC and concentration of pooled fractions to less than 0.5 ml the final purification step – SEC – could be conducted.
Step 8 – SEC
Figure 7. SDS-PAGE gel for huLwCas13a – samples after SEC.
The protein with the tag is marked by the violet arrow, while the protein without the tag is marked by the blue arrow.
Wells are labeled as follows: E1, E2, E3, etc., representing eluted fractions. Samples marked with * are those that underwent SUMO protease digestion.
Figure 8. SDS-PAGE gel for Cas13b – samples after SEC*.
The protein with the tag is marked by the yellow arrow, while the protein without the tag is marked by the orange arrow. Wells are labeled as follows: E1, E2, E3, etc., representing eluted fractions.
SEC Conclusions: After the 3-step purification process, we obtained pure huLwCas13a and CcaCas13b proteins, as confirmed by SDS-PAGE (Figures 7 and 8). As expected, a single band was observed for the undigested proteins. In SUMO-digested fractions, two bands were observed, indicating incomplete tag cleavage.
As planned, we conducted a comparative analysis of tagged and untagged proteins to test their functionality. Expected results included:
Nevertheless, we hoped to not observe significant differences between both digested and undigested
proteins.
CD Results:
Figure 9. Dependence of mean residue molar ellipticity for huLwCas13a with and without the tag in the far-UV range.
Figure 10. Dependence of mean residue molar ellipticity for CcaCas13b with and without a tag in the far-UV range.
Table 1. Proportions of various secondary structure types determined from Circular Dichroism spectra for huLwCas13a.
| huLwCas13a | Mean proportions of secondary structure types determined using algorithms from the CDPro package ± standard deviation | |||
|---|---|---|---|---|
| α-helix | β-sheet | β-turn | unordered | |
| undigested | 64.5 ± 2.1 | 3.8 ± 0.9 | 10.2 ± 0.7 | 20.9 ± 2.5 |
| digested | 58.1 ± 10.2 | 4.7 ± 6.3 | 18.2 ± 9.9 | 19.5 ± 7.2 |
Table 2. Proportions of various secondary structure types determined from Circular Dichroism spectra for CcaCas13b.
| CcaCas13b | Mean proportions of secondary structure types determined using algorithms from the CDPro package ± standard deviation | |||
|---|---|---|---|---|
| α-helix | β-sheet | β-turn | unordered | |
| undigested | 76.8 ± 11.1 | 1.5 ± 2 | 6.9 ± 4.3 | 15.2 ± 10.5 |
| digested | 80.4 ± 7.4 | 0.5 ± 2 | 4.4 ± 3.3 | 14.7 ± 7.8 |
CD Conclusion: The spectra in Figures 9 and 10 overlap significantly, and the contribution of individual secondary structures to the protein structure (Tables 1 and 2) does not differ substantially. This indicates (contrary to our expectations) that the tag does not have a significant impact on the structure of Cas13a and Cas13b.
NanoDSF Results:
Figure 11. Dependence of the first derivative of the fluorescence intensity change from temperature for huLwCas13a.
Figure 12. Dependence of the first derivative of the fluorescence intensity change from
temperature for
CcaCas13b.
Key: D – sample digested using SUMO protease, UD – sample undigested
NanoDSF Conclusion: Contrary to our expectations, NanoDSF results indicated no statistically significant difference between tagged and untagged proteins in thermal stability.
Activity tests in SHERLOCK Results:
Figure 13. A comparison of the activity of huLwCas13a with and without 6xHis-TwinStrep-SUMO tag.
Figure key:
Activity tests in SHERLOCK Conclusion: Contrary to our expectations the Cas13a protein, both digested and undigested by SUMO protease, showed similar levels of activity.
Based on our tests, we made several adjustments to the IMAC protocol:
Moreover, we have drawn the following conclusions regarding the entire protocol:
Overall, this DBTL cycle and the engineering framework applied throughout this procedure enabled us to obtain pure and active Cas13 for future experiments.
[1] Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR
nucleases. Nat Protoc. 2019 Oct;14(10):2986–3012. doi: 10.1038/s41596-019-0210-2.
[2] Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM,
Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F.
Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 13. doi: 10.1126/science.aam9321. PMID:
28408723.
[3] Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid
detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi:
10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508.
[4] An B, et al. Rapid and sensitive detection of Salmonella spp. using CRISPR-Cas13a combined with recombinase
polymerase amplification. Front Microbiol. 2021 Oct. doi: 10.3389/fmicb.2021.732426.
[5] Khan H, et al. CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2. Chinese
Chem Lett. 2019 Dec;30(12):2201–2204. doi: 10.1016/j.cclet.2019.10.032.
Goals:
The goal of this iteration was to design and test initial versions of RPA primers and PrymcrRNAs.
In our test, we targeted the ITS2 (Internal Transcribed Spacer 2) sequence of Prymnesium parvum for specific detection. The ITS2 region, located between the 5.8S and nuclear large subunit rRNA genes [1], is commonly used for species identification due to its variability between species and conservation within a species. This makes it an ideal target for species-specific primers, including those used to detect Prymnesium parvum [2]. We aimed to leverage these features using our crRNA molecules.
Gal RPA Primers:
The first RPA primer pair we designed was based on the primers created by Galluzzi et al. [1], with a T7 promoter appended to the 5' end of the forward primer. This modification allows the sequence amplified by RPA (Recombinase Polymerase Amplification) to be transcribed in vitro into RNA, which is necessary since Cas13a only detects RNA. Since the GalF and GalR primers were proven effective in PCR in previous experiments, we chose them as a reliable starting point for designing our RPA primers.
Figure 1. Binding of Gal primers to the ITS2 region of the Prymnesium parvum genome.
PrymcrRNA1 and PrymcrRNA2:
In order to design crRNAs that would bind to a sequence in the genome of Prymnesium parvum, we needed to start from scratch. To establish a starting sequence, we performed PCR on DNA isolated from Prymnesium parvum using the standard Galluzzi [1] primers. We sent the PCR product for sequencing and received high-quality reads. However, the product was only about 130 bp long, which was too short for designing multiple crRNA molecules. Since we anticipated that some crRNAs might not be functional, we aimed to design two initial crRNA sequences to begin with.
At the time of us designing the crRNAs, there were no available Prymnesium sequences originating from the Oder River. Since we needed a reference template for designing crRNAs and primers, we had to find an alternative Prymnesium sequence representation.
Therefore, online nucleotide BLAST was performed for forward and reverse sequencing products revealing. The search results indicated several high-scoring matches. The top results included Prymnesium strains:
Given the high sequence similarity to our samples (PCR products), the production of type-B prymnesins, and the geographical proximity to the Oder River, we chose KAC39 as the Prymnesium template. This selection provides a reference for designing crRNAs and primers for our project.
KAC39 sequence was downloaded from NCBI:
MK091113.1 Prymnesium parvum strain KAC39 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence
and annotated based on KJ756812.1.
For crRNA design, we opted to use our own sequencing data to ensure complete accuracy with the Oder strain of Prymnesium parvum. Based on this data, we designed two crRNAs, termed PrymcrRNA1 and PrymcrRNA2. The binding sites for these crRNAs are shown in Figure 2.
Figure 2. Binding sites of PrymcrRNA1 and PrymcrRNA2 to the Prymnesium genome.
Figure 3. Overview – binding of both PrymcrRNAs as well as the Gal primers to the Prymnesium genome.
Gal primers:
We ordered the primers from a manufacturer as the designed DNA sequence. Sequences can be accessed on their Part Registry page.
PrymcrRNAs:
We ordered the crRNAs from a manufacturer as the designed DNA sequence. Sequences can be accessed on their Part Registry page.
We conducted the SHERLOCK reactions using both fluorescence and LFA readouts, following the protocols detailed on our “Experiments” page. Each experiment was carried out in multiple replicates, and comprehensive descriptions of all experiments can be found on the “Notebooks” page. Here, representative results from these experiments have been included.
Figure 4. SHERLOCK fluorescence readout result 05/06/2024*.
Note: The DNA templates used are: “prymDNA” for Prymnesium parvum DNA and “synDNA” for synthetic DNA from Kellner et al. [1]. The crRNAs include “PrymCrRNA1” and “PrymCrRNA2,” designed by us for Prymnesium parvum detection, along with “syncrRNA” from Kellner et al. [1]. The sample “synDNA1 syncrRNA” serves as a positive control, while “synDNA1 prymcrRNA1” and “synDNA1 prymcrRNA2” act as negative controls due to mismatches between the DNA template and crRNAs.
Figure 5: SHERLOCK fluorescence readout result 24/06/2024*.
In this test, the “negative control + prymcrRNA1” produced a fluorescence signal. In this sample, water was added to an RPA mix containing Gal primers.
Figure 6. LFA test result 08/07/2024*.
LFA strips**:
*The dates refer to dates of experiments as included in the “Notebook” page. Please refer to the appropriate Lab Notebooks (SHERLOCK Lab for fluorescence measurements, LFA Lab for LFA readouts) for a more detailed description of the performed experiments.
**The description of the samples has been simplified for clarity. For a comprehensive overview of their contents, please refer to the LFA Lab documentation included in the “Notebooks”.
In our experiments, we confirmed that PrymcrRNA2 effectively detects Prymnesium parvum DNA when paired with Gal primers.
However, we did not achieve the same success with PrymcrRNA1, even though it produced positive results for samples containing Prymnesium DNA. This issue arose because we also obtained positive results in the negative controls (Figure 5 and 6). In multiple tests using both fluorescence and LFA readouts, we found that PrymcrRNA1 combined with Gal primers led to false positives (Figure 5), as the negative controls also showed positive results. This also clarifies why the negative controls in Figure 4 were truly negative—because syn RPA primers were used in the RPA reaction. This highlighted the critical importance of including negative controls in our experiments.
We initially concluded that the Gal primer pair and PrymcrRNA1 combination might be incompatible, so we decided to temporarily exclude it from experiments. Consequently, we had to design new RPA primers – we aimed to source at least 2 new pairs. Further research was needed to determine whether PrymcrRNA1 is functional. To investigate this, we planned to test it with the different, newly designed pairs of primers. We also decided to later investigate further to determine whether the issue lay with the forward or reverse primer, and whether one of the Gal primers could still be used in future experiments.
Our hypothesis was that one or both of the Gal primers might inadvertently serve as a target for PrymcrRNA1. We suspect this could be the forward primer due to its proximity to the PrymcrRNA1 binding site (Figure 3).
Goal: To design new RPA primer pairs based on the KAC39 sequence for Prymnesium parvum in order to optimize our SHERLOCK test.
We designed 2 new RPA primer pairs:
1. Mod (ModF, ModR): we based their sequences on the Luo et al. publication [2]. For ModF, we
used
the PR-RPA-4-F primer from the publication, and we appended the T7 promoter to its sequence. For ModR, we used a
modified version of the PR-RPA-4-R primer, changing an A to a G to better align with the Prymnesium
parvum
DNA fragment sequenced by us.
Figure 7. Fragment of the ITS1-5.8S-ITS2 region of the Prymnesium parvum genome showing the simulated binding of the primer suggested by Luo et al. (below) and the primer modified by us – ModR (above).
Figure 8. Binding of Mod primers to the ITS2 region of the Prymnesium parvum genome.
2. Alt – their sequences were designed using Primer-BLAST, following the guidelines provided in the TwistAmp® DNA Amplification Kits Assay Design Manual.
Figure 9. Binding of Alt primers to the ITS2 region of the Prymnesium parvum genome.
We ordered the primers as DNA parts with the designed sequence, which can be accessed on their Part Registry page.
Figure 10. SHERLOCK fluorescence readout result 16/07/2024. Results for the 530 nM concentration of Prymnesium DNA.
Figure 11. SHERLOCK fluorescence readout result 16/07/2024. Results for the 200 fM concentration of Prymnesium DNA.
Figure 12. LFA test result 15/07/2024.
LFA strips**:
The results of both readouts – fluorescence and LFA – were once again consistent with each other.
We confirmed that both pairs of our newly designed primers successfully allowed amplification using the RPA method. Importantly, no false positives were observed in the negative controls, indicating that both PrymcrRNA molecules are potentially compatible with the primers. We also confirmed that PrymcrRNA1 is functional, as it successfully detects Prymnesium DNA. Contrary to our initial assumptions, we were able to design two functional crRNA molecules from the start, without needing a trial-and-error approach.
We successfully detected Prymnesium DNA using the following new primer-crRNA combinations: Mod-PrymcrRNA1, Alt-PrymcrRNA1, and Mod-PrymcrRNA2. However, we were unable to consistently detect Prymnesium DNA with the Alt-PrymcrRNA2 pair. Additionally, we further confirmed the functionality of the Gal-PrymcrRNA2 combination.
The most effective primer-PrymcrRNA pair was Mod-PrymcrRNA1, which achieved high fluorescence intensity for both 530 nM and 200 fM samples. No other combination successfully detected the 200 fM target concentration, and this pair also produced the strongest T-line in the LFA readout.
We considered the possibility of mixing the primers to see if any combination could be more effective than Mod-PrymcrRNA1. However, since this experiment focused on system optimization rather than confirming component functionality, this approach was tested in the next cycle.
We successfully concluded this cycle, having identified four functional primer-crRNA combinations (Mod-PrymcrRNA1, Alt-PrymcrRNA1, Mod-PrymcrRNA2, and Gal-PrymcrRNA2) for detecting Prymnesium parvum using the SHERLOCK system.
Goal:To identify the most effective primer-crRNA combination from all possible combinations.
As described in Iteration 2, we wanted to determine if mixing forward and reverse primers from different sets would enable to achieve a lower limit of detection than the previously established 200 fM. We also explored whether combining the two PrymcrRNAs in equimolar amounts would enhance effectiveness.
To evaluate this, we assessed eight primer combinations using fluorescence readout:
Each combination was tested with both PrymcrRNA1 and PrymcrRNA2, as well as with negative and positive controls, resulting in a total of 41 conditions being assessed.
Sequences can be accessed on their Part Registry page, as described in Iteration 1-2. The procedure was
performed according to the established protocol.
Figure 13. SHERLOCK fluorescence readout result 17/07/2024. Results for the 480 nM concentration of Prymnesium DNA*.
Figure 14. SHERLOCK fluorescence readout result 17/07/2024. Results for the 200 fM concentration of Prymnesium DNA*.
*The dates refer to dates of experiments as included in the “Notebook” page. Please refer to the appropriate Lab Notebooks (SHERLOCK Lab for fluorescence measurements, LFA Lab for LFA readouts) for a more detailed description of the performed experiments.
The most effective combination appears to be ModF + GalR + PrymcrRNA1, as it produced high signals for both 480 nM and 200 fM target DNA concentrations. This result was confirmed in two separate repetitions.
We also discovered that it's not just one Gal primer, but likely the combination of both, that inadvertently activates PrymcrRNA1. When each Gal primer was paired with a different primer, the results were consistent. Since our primary goal was to engineer the most effective combination of primers and crRNAs, and we successfully achieved that, we decided not to investigate this issue further.
Following the success with mixing the primers, we considered that mixing equimolar amounts of the crRNAs might also enhance sensitivity, as suggested by a paper [3]. We planned to explore this idea further in the next iteration using an LFA readout, while also validating the results obtained so far with LFA.
We aimed to evaluate the effectiveness of the selected primer combinations in the LFA procedure based on the fluorescence readout tests from Iteration 2. The specific combinations tested were: GalF + ModR, ModF + GalR, ModF + ModR.
Additionally, we investigated whether combining the two PrymcrRNAs at a 1:1 ratio
(PrymcrRNA1:PrymcrRNA2 = 1:1)
would improve detection sensitivity.
Sequences can be accessed on their Part Registry page, as described in Iteration 1 and 2. The procedure
was
performed according to the established protocol.
Figure 15. LFA test result 18/07/2024**.
LFA test strips:
†PrymcrRNA1/2 indicates the equimolar mixture of PrymcrRNA1 and PrymcrRNA2.
**The description of the samples has been simplified for clarity. For a comprehensive overview of their contents, please refer to the LFA Lab documentation included in the “Notebooks”.
Combining these results with ones from the previous iteration, it can be stated that the most optimal primer-crRNA combination for our test is ModF + GalR + PrymcrRNA1. We decided to use this combination for all subsequent tests, and it later underwent precise Limit of Detection (LOD) determination (as described in the “Measurement” page).
Combining the two PrymcrRNAs did not enhance specificity, as samples with only PrymcrRNA1 showed more intense T-lines.
The negative control for the GalF + ModR primer combination yielded a positive result. As a result, all other findings using this primer pair were not considered. However, the negative controls for the ModF + GalR and ModF + ModR combinations were negative, indicating that the results from these combinations are reliable and can be interpreted with confidence.
[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008 Feb;46(2):261-6. doi:10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.
[2] Luo N, Huang H, Jiang H. Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick. Front Mar Sci. 2022 Oct. doi:10.3389/fmars.2022.1032847.
[3] Fozouni P, Son S, Derby MDDL, Knott GJ, Gray CN, D’Ambrosio MV, Zhao C, et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell. 2021 Jan;184(2):323-333.e9. doi:10.1016/j.cell.2020.12.001.
Goals: Engineer a Standardized System for Customizable crRNA Synthesis for the SHERLOCK Platform.
After recognizing the need for a user-friendly crRNA synthesis system, we designed our initial SynLOCK Cassette. We imagined our system as a set of elements that could accept the custom crRNA spacer of a user’s choice.
The initial SynLOCK Cassette design (shown in Figure 1.) includes the T7 promoter for in vitro transcription, the DR loop for huLwCas13a binding to the crRNA molecule, SapI sites for cloning the custom spacer sequence, and a BbsI site for vector linearization prior to in vitro transcription.
Figure 1. Initial SynLOCK Cassette Design
Initially, our team used the PSB1C5C plasmid backbone for assembly due to its size, high copy number, and availability in the 2024 iGEM Distribution kit, which was accessible to us and other teams.
Throughout our design stage, we used SnapGene software to simulate the integration of crRNA spacers into the cassette and the process of crRNA synthesis. This confirmed that in silico, molecules of the correct length and sequence could be obtained using our system, giving us confidence to proceed with the lab work.
We chose to use standard E. coli strains as the foundation for our design, specifically the NEB 5-alpha competent cells provided by our sponsors, New England Biolabs, and the commonly available Top10 competent cells from ThermoFisher.
We ordered the Cassette DNA, assembled it by hybridizing two DNA strands to create sticky ends, and successfully cloned it into the pSB1C5C plasmid backbone using cloning with BsaI enzyme.
Figure 2.Results of cloning the initial cassette design into the pSB1C5C plasmid backbone: Plates showing colonies post-transformation, with the negative control marked in the image.
Figure 3. The schematic representation of the initially assembled device.
We verified the sequence of our design using Sanger sequencing to ensure that the clones we selected contained the Cassette. The results confirmed that the Cassette was present and capable of accepting the crRNA spacers.
Figure 4. Sequencing data confirming the successful integration of the Cassette spine into the pSB1C5C plasmid backbone, with no detected mutations.
In this iteration, and after consulting with specialists, we learned the following:
Goal: To resolve the issues encountered in the first iteration by incorporating a different plasmid backbone and adding a reporter.
We designed an improved version of our Cassette with a reporter unit. We tested several reporter combinations assembled from basic parts available in the Distribution Kit. The reporter was strategically placed between the SapI sites to ensure it would be excised when a custom crRNA spacer was incorporated, allowing for easy visual screening of colonies.
Figure 5. Improved SynLOCK Cassette Design.
Additionally, we chose the pSB1C3 plasmid as the new backbone. This plasmid is a standard for storing BioBrick parts, meeting our criteria for being high-copy and accessible, and it supports cassette transfer using BioBrick prefix and suffix cutting sites.
We used SnapGene’s Golden Gate cloning simulator to predict our assembly process for the reporter. This involved simulating the construction of the reporter from basic parts. We then simulated the PCR reaction to amplify the reporter module with primers that introduced sticky ends, facilitating cloning into the backbone. Finally, we assessed whether the assembly would be successful by simulating the cloning of the product into a new backbone.
We introduced the reporter unit into the Cassette by amplifying it via PCR to obtain compatible sticky ends. Then we amplified the complete Cassette with the reporter and transferred it into the pSB1C3 plasmid backbone.
Figure 6. The schematic representation of the final assembled device.
Then we assembled the system by cloning in the custom crRNA spacers used in our PrymDetect test. We employed the Type IIS assembly standard to insert the crRNA spacers, ensuring that there were no scar sites in our final IVT template construct that could impact the length or sequence of the crRNA.
Figure 7. Schematic depiction of the improved system in action. On the right, a fragment of a Petri dish showing visible white and purple bacteria, indicating which colonies are ready for picking.
We conducted IVT on the plasmids according to a protocol included in the “Experiments” page.
Figure 8. RNA electrophoresis – IVT results. "SVR" indicates that the crRNA from that well was stored in the pSB1C5C backbone, while "USV" signifies that it was stored in the pSB1C3 backbone.
Conclusions: We successfully obtained crRNAs of the correct length, as indicated by the single band on the gel for most wells at around 64 bp. Some contamination is visible in certain wells, likely due to the presence of the plasmid IVT template.
We also tested the Cassette’s transferability between different backbones by excising it from the pSB1C3 backbone using the enzymes present in the BioBrick prefix and suffix. This step added to the system’s standardization.
Figure 9. Electrophoresis results showing the digestion of USV plasmid with the enzymes marked in the image.
Table 1. Expected band size the digestion of the USV plasmid.
| Enzyme/s used | Expected Band Size (bp) |
|---|---|
| EcoRI & SpeI | 2047 & 1031 |
Conclusion: The correct bands were obtained, demonstrating that the Cassette can now be transferred between other systems compatible with the BioBrick assembly standard.
In this iteration, we learned that:
Goals: Determining optimal lighting conditions for algae cultures containing Prymnesium parvum.
After setting up the initial algal cultures, we stored them in a growth chamber under constant light
conditions,
following the suggestion from our advisor – Dr Paweł Jedynak.
This environment proved suboptimal for Prymnesium parvum, as we observed a decline in cell numbers and reduced cell movement after about a month.
The lighting conditions that we utilize for our algae cultures should be optimized. It would be beneficial to contact a specialist who has expertise in Prymnesium parvum culture.
Seeking a solution to the problem of reduced algae growth, we consulted Dr Ewa Górecka from the Institute of Marine and Environmental Sciences, University of Szczecin, to learn about her algae culture conditions.
Dr. Górecka advised us to place some of our algal cultures in a growth chamber with a 14-hour light/10-hour dark cycle. We followed her recommendation and changed the lighting conditions of our cultures.
As a result, we observed improved algae growth after about a month, with an increase in cell numbers and enhanced movement.
A 14-hour light/10-hour dark cycle is more beneficial for algae growth compared to constant light conditions.
[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008 Feb;46(2):261-6. doi: 10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.
[2] Raport Kończący Prace Zespołu ds. Sytuacji w Odrze. Instytut Ochrony Środowiska – Państwowy Instytut Badawczy. Available at: https://ios.edu.pl/wp-content/uploads/2022/12/raport-konczacy-prace-zespolu-ds-sytuacji-w-odrze-2.pdf
PCR was performed to confirm the presence of the ITS2 sequence in DNA isolated from algal cultures. The ITS2 sequence is specific to Prymnesium parvum, allowing for its identification among other algal species. Primers targeting the ITS2 sequence were designed by Galluzzi et al. [1]. The goal of this iteration was to optimize conditions for this PCR reaction.
To determine the initial PCR conditions, we referred to the Final Report of the Team on the Situation in the Oder River [2], where Galluzzi primers were used in a standard PCR.
We conducted a PCR reaction on the Prymnesium parvum genome using Galluzzi primers and the reaction conditions sourced from the Final Report of the Team on the Situation in the Oder River [2]. Table 1 shows the implemented reaction conditions.
Table 1. Reaction conditions sourced from the Final Report of the Team on the Situation in the Oder River [2].
| Reaction step | Temperature [°C] | Time [s] |
|---|---|---|
| Pre-denaturation | 98 | 30 |
| Denaturationn | 98 | 10 |
| Annealing | 67 | 60 |
| Elongation | 72 | 5 |
| Final elongation | 72 | 300 |
Number of cycles: 40
Figure 1 presents the results of DNA electrophoresis of the PCR product.
Figure 1. Gel electrophoresis of the PCR product – results.
Legend:
“High-volume”, “2W1D”, “1BW11”, and “2W11” are names of DNA samples derived from different Prymnesium parvum cultures. For a more detailed description, please refer to the AlgaeLab documentation included in the “Notebooks.”
Not all PCR reactions yielded a specific product, even though identical DNA samples and preparation conditions were used for each reaction. This inconsistency suggests that the experiment cannot be reliably reproduced under the current conditions, indicating a need for optimization.
Nevertheless, in the case of sample 3 the PCR reaction did produce a specific product of the correct length, so we adopted these evaluated reaction conditions for subsequent PCR assays following each algal DNA isolation.
The PCR conditions were optimized for the Taq polymerase used, following the manufacturer's instructions (A&A Biotechnology). This polymerase was used due to its availability in the lab.
Table 2 presents the PCR conditions after optimization.
Table 2. PCR reaction conditions based on the manufacturer’s instruction (A&A Biotechnology).| Reaction step | Temperature [°C] | Time [s] |
|---|---|---|
| Pre-denaturation | 95 | 60 |
| Denaturationn | 95 | 15 |
| Annealing | 67 | 60 |
| Elongation | 72 | 15 |
| Final elongation | 72 | 300 |
Number of cycles: 30
We conducted a PCR with Galluzzi primers using the optimized reaction conditions. Simultaneously, we performed another PCR with the same reagents but under the previous conditions to compare the results.
Figure 2. presents the results of a DNA electrophoresis of the PCR product.
Figure 2. Gel electrophoresis of PCR product – results. Conditions A – PCR conditions before optimization. Conditions B – optimized PCR conditions.
Legend:
P, BOW 2, NOW 5, and Gdańsk 2 are names of DNA samples derived from different algal cultures. For a more detailed description, please refer to the AlgaeLab documentation included in the “Notebooks.”
PCR performed under Conditions A produced one product of the correct length, but this result was confounded by a product of the same size appearing in the negative control sample. In contrast, PCR under the optimized conditions yielded two specific products and did not show any visible product in the negative control sample.
The PCR under the optimized conditions was performed repeatedly, consistently yielding specific products.
The optimized PCR conditions are suitable for the reaction, as they consistently yielded a specific product of the correct length and the experiment demonstrated reproducibility.
[1] Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M. Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008 Feb;46(2):261-6. doi: 10.1111/j.1472-765X.2007.02294.x. Epub 2007 Dec 13. PMID: 18086191.
[2] Raport Kończący Prace Zespołu ds. Sytuacji w Odrze. Instytut Ochrony Środowiska – Państwowy Instytut Badawczy. Available at: https://ios.edu.pl/wp-content/uploads/2022/12/raport-konczacy-prace-zespolu-ds-sytuacji-w-odrze-2.pdf
We evaluated a rapid DNA isolation method that could potentially be performed in field conditions, simplifying the workflow required for the SHERLOCK assay. This method was originally described by Zou et al. [1]. The goal was to develop the most efficient DNA isolation procedure using an in-field method.
We implemented the in-field DNA isolation method for our algae cultures, following the original protocol described by Zou et al. [1]. This protocol has also been included in the “Experiments” page.
We performed DNA isolation followed by PCR according to the following steps:
Figure 1. DNA electrophoresis after the PCR – results. Electrophoresis performed after three separate PCR reactions. Results for the in-field isolation are labeled 11-19.
The PCR did not yield a specific product. The bands visible on the gel were determined to be primer dimers.
The evaluated in-field DNA isolation method proved ineffective, and improvements to the process are necessary. We hypothesize that the unsuccessful amplification may be due to a portion of the reaction mixture being absorbed by the dipstick and subsequently removed from the PCR tube.
The originally described in-field DNA isolation protocol was modified as follows:
We made a second attempt at DNA isolation using the in-field method, applying the modified protocol to improve the efficiency of DNA recovery for subsequent PCR amplification.
Figure 2. DNA electrophoresis after the PCR – results.
The subsequent PCR reaction still did not yield a specific product. The visible bands on the gel were identified as primer dimers, indicating that the DNA amplification was unsuccessful despite the modifications to the in-field isolation protocol.
Increasing the shaking time and number of dips with the isolation sticks did not improve the efficiency of DNA isolation. Thus, the method requires further optimization. Due to limited laboratory access, we were unable to continue working on the in-field DNA isolation process, and consequently, this goal was not achieved. However, the insights gained during this process provide a valuable foundation for future iGEM teams aiming to optimize in-field DNA isolation from Prymnesium parvum or other harmful species. We believe that using more than two ball bearings, as well as reducing the initial volume of the buffers used for the isolation are approaches worth testing in the further optimization process.
[1] Zou Y, Mason MG, Wang Y, Wee E, Turni C, Blackall PJ, et al. Nucleic acid purification from plants, animals, and microbes in under 30 seconds. PLoS Biol. 2017 Nov;15(11). doi: 10.1371/journal.pbio.2003916.
Goals: Design and 3D-print a functional device to excite fluorescence, then quantify the detected signal by analyzing the resulting image with a Python script.
We began designing a 3D-printed device aimed at detecting the green fluorescence signal generated by our SHERLOCK detection system. Our initial concept was inspired by the pe3Dish model (Figure 1A), which consists of a 3D-printed chamber with a network of channels formed by flexible tubes, allowing continuous perfusion of 3D cell cultures. These channels are designed to mimic the human vascular system. We adapted this approach for our project.
Figure 1B shows the preliminary model we developed, featuring a central chamber with several channels. Two channels function as inlets, separately delivering the SHERLOCK reagents and the DNA target, while a third channel acts as an outlet to remove waste liquid after the reaction.
Figure 1C illustrates the primary 3D-printed system, which was designed in TinkerCAD. Unlike the five inlets and outlets in the original pe3Dish model, our design incorporates one outlet for waste removal and three inlets for reagent delivery. This configuration allows us to separate the SHERLOCK reagents into two distinct channels. The exact dimensions of the design are detailed in Figure 1C. The STL file was processed using Prusa Slicer 2.7.1. This software was used in all following iterations.
Figure 1. Initial designs of the 3D-printed system. (A) Photo of the pe3Dish. (B) Scheme of the initial design of the 3D-printed device. (C ) Initial 3D-printed device designed in TinkerCAD.
The initial model of the device was successfully 3D-printed using a Prusa i3 MK3 printer with
Prusament PLA
Pristine White filament.
The same printer was used in all following iterations. The general print
settings were
based on the '0.10mm DETAIL' preset from the Prusa Slicer system. The specific modifications made to the default
system presets are as follows: Infill: 20%; Brim: enabled; Supports: none; Fill pattern: Rectilinear; Top fill
pattern: Rectilinear; Bottom fill pattern: Rectilinear; Perimeters: 20 mm/s; Small perimeters: 15 mm/s; External
perimeters: 15 mm/s; Top solid infill: 20 mm/s; Nozzle (first and other layers): 215 ⁰C; Bed (first and other
layers): 60 ⁰C; Filament type: PLA; Retraction length: 2 mm; Retraction speed: 40 mm/s.
The printing process was completed in 2 hours and 6 minutes.
The chamber was printed successfully, maintaining good structural integrity. However, the tube holders proved to be too thin, making them fragile and prone to breaking or detaching from the model. They were easily damaged by light pressure from a finger or by being dropped. The damaged tube holders are highlighted in Figure 2.
Figure 2. Photo of the 3D-printed initial 3D-printed device with damaged tube holders.
The most critical improvement needed for the model was to thicken the walls of the tube holders to enhance their durability. Additionally, the chamber required an external protective enclosure to secure and protect its components.
A new model with thicker walls for the tube holders was designed to improve durability. Additionally, drawing inspiration from the pe3Dish system, the model was adapted to fit into the bottom of a Petri dish, allowing the dish to function as an external protective enclosure. Figure 3 shows the updated model with, designed in TinkerCAD.
Figure 3. Improved 3D-printed device designed in TinkerCAD.
The new model was successfully printed using the Prusament PLA Pristine White filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. The specific modifications made to the default system presets were the same as for Iteration 1.
The printing process was completed in 2 hours and 26 minutes.
The chamber was printed successfully and fit well within the 90 mm Petri dish, allowing for
secure closure and
protection of the entire chamber. The tubes fit properly into the inlets and outlets of both the
chamber and the
tube holders, as shown in Figure 4.
The thicker tube holders proved to be sturdy and did
not
break. The
integrity of the channel system was tested using water and a 5 ml syringe, confirming that the design was
functional and robust.
Figure 4. Photos of the improved 3D-printed device. (A) Model in Petri dish without the tubes. (B) Model in Petri dish with the tubes
Although the design improvements were successful, we concluded that in future use, transferring reagents into the chamber with a syringe and fitted tubes would be challenging due to the small microliter volumes required for the SHERLOCK assay. The 3 mm diameter tubes necessitated a larger volume to fill the chamber, indicating the need for a different approach to the chamber design.
Additionally, it was noted that the PLA filament used to print the chamber is biodegradable, which could potentially affect biochemical reactions from environmental samples. This suggests that a different material or coating may be necessary to prevent interference with the assay.
We decided to abandon the use of tubes and redesign the chip. Our focus shifted to developing a smaller reaction chamber without tubes, drawing inspiration from the device described in the paper "Machine Learning-Driven and Smartphone-Based Fluorescence Detection for CRISPR Diagnostic of SARS-CoV-2" [1]. The new design features three separate compartments that can be easily attached and detached for convenient transportation. Additionally, we replaced the initial PLA filaments with PETG filaments, which are non-biodegradable but more suitable for preventing interference with biochemical reactions.
Each box was designated a specific function and in different colors of PETG filament, chosen to align with the colors in our team logo. The reaction chamber was placed in the black detection box, as this was where fluorescence was expected to be induced. A special compartment for the chamber was designed within this box (Figure 5 A).
The box containing the electronics necessary for generating blue light to induce green fluorescence was labelled the yellow electronics box. This box also included a separate compartment for the battery to power the LEDs and a hole for the LED switch (Figure 5B). Both the black and yellow boxes featured three holes for LEDs, designed to allow the LEDs to be inserted between the boxes within LED holders (Figures 5A and 5B).
The final box, intended for storing tools and reagents required for the experiment, was named the purple tool box. The initial box included designated spaces for a reaction chamber, glass cover slips and a 500 µL syringe (Figure 5 C).
The overall dimensions of the boxes were determined by the size of the phone intended to be placed on the device. The height of the boxes was minimized to accommodate the battery. Dedicated compartments within the boxes were designed to fit the dimensions of components prepared for the chip-based assay, including LED holders, battery, light switch, 500 µL syringe, and cover slips. Initially, the boxes were designed without lids to test the dimensions for the listed elements and to assess the printing quality of the filaments.
Figure 5 presents all the boxes, with exact dimensions, designed in TinkerCAD.
Figure 5. Models of the boxes designed in TinkerCAD
(A) The black detection box with a compartment for the chamber and holes for the LED holders.
(B) The yellow
electronics box with a separate space for a battery and holes for the light switch and LED holders.
(C) The
purple tool box with designated spaces for a reaction chamber, glass cover slips and a 500 µL syringe.
The models were printed with the following filaments:
The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system were the same as for Iteration 1, except for: Supports: everywhere (grid); Top fill pattern: Archimedean Chords; Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s; Gap fill: 20 mm/s; Nozzle (first and other layers): 250 ⁰C (black), 250 ⁰C (yellow), 240 ⁰C (purple); Bed (first and other layers): 70 ⁰C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing processes were completed in 4 hours and 35 minutes (black box), 7 hours and 6 minutes (yellow box) and in 8 hours and 8 minutes (purple box).
The boxes were printed, but the results were not optimal. The walls were too thin, resulting in areas that exhibited elasticity and transparency rather than stability and sturdiness (Figure 6A). Additionally, the supports inside the holes for the LED holders in the black and yellow boxes left filament residue, distorting the circular shapes of the holes (Figure 6B). The bottoms of each box did not print well; the layer height was uneven, leading to filament blobs, and stringing was particularly noticeable in the elements within the purple box. In some areas of the purple box, there were even holes in the bottom. Additionally, in the yellow box, the wall designed to create a separate space for the battery was too fragile due to its thickness and came off during the removal of the supports (Figure 6C).
The sizes of the boxes were adequate and did not require adjustments; however, increasing the height of the boxes would likely be beneficial.
Figure 6. Photos of the 3D-printed initial boxes.
(A) Yellow box displaying elastic and transparent walls.
(B) Holes in the
black and yellow
boxes showing residue
left after support removal.
(C) Poorly printed bottoms of the boxes.
The thickness of the walls should be increased to enhance the durability of the boxes. Additionally, after investigating the issues, we found that the problems with the printed boxes may have resulted from an incorrectly chosen nozzle temperature. To determine the optimal printing temperature for these filaments, it is essential to print calibration models, such as temperature towers. Increasing the wall thickness will further contribute to the overall durability of the boxes.
Since it was unclear which glass cover slip among the three ordered options (circular 9 mm, circular 15 mm, square 16 mm) would be the best fit for the lid of the reaction chamber, a box with dedicated holders for each slip was designed. This box was constructed to match the height of the boxes designed in Iteration 5, allowing us to evaluate which distance between the phone and the detection chamber would be optimal for capturing images. Figure 7 presents the model of the box, with exact dimensions, designed in TinkerCAD.
Figure 7. Model of the box with holders for different glass coverslips designed in TinkerCAD.
The new model was successfully printed with Prusament PLA Galaxy Silver filament. The general print settings were based on the '0.20mm QUALITY' preset from the Prusa Slicer system. Modifications to the default system presets were the same as for Iteration 1, except: Supports: on build plate only (grid); Top fill pattern: Octagram Spiral; Bottom fill pattern: Octagram Spiral; Nozzle (first and other layers): 220°C.
The printing process was completed in 3 hours and 13 minutes.
The box was printed successfully. Upon testing, the square coverslip appeared too large for the final chamber, while the 9 mm circular coverslip was too small. The medium-sized 15 mm circular coverslip emerged as the best option for the detection chamber (Figure 8). However, the box's height was insufficient, making it difficult to achieve optimal camera focus.
Figure 8. The gray box with holders for the coverslips in various sizes and shapes.
The circular 15 mm glass coverslip was selected for further improvements to the detection chamber.
It was decided to remodel the black box and increase its height from 29 mm to 41 mm to facilitate better camera focus and overall functionality.
Since the size of the glass coverslip was selected, the reaction chamber was designed in TinkerCAD specifically to accommodate it. The exact dimensions of the design are presented in the scheme shown in Figure 9.
Figure 9. Model of the reaction chamber designed in TinkerCAD.
The model was printed using the Prusa i3 MK3 printer with Transparent Devil Design PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets were the same as for Iteration 1, except: Supports: on build plate only (organic); Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s; Gap fill: 20 mm/s; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process was completed in 32 minutes.
The chamber was printed, but the results were unsuccessful. Significant stringing occurred, and the overall structure of the model was uneven. Additionally, the supports did not print correctly and remained inside the chamber, as shown in Figure 10.
Figure 10. The 3D-printed design of the initial reaction chamber.
Since the printing of the chamber was unsuccessful, it was decided that the filament needed to be printed at a more optimal temperature. Therefore, there was a need to print a temperature tower for this filament as well.
Temperature towers for optimizing the printing of PETG filaments were found online. The design selected from the website Printables.com (https://www.printables.com/model/20652-temp-tower-pla-petg-absasa-for-prusa-mini-mk3s-and/files) was chosen for optimizing the printing of all four previously mentioned PETG filaments. The file named ‘Temp_Tower_PETG.stl’ was downloaded and sliced using Prusa Slicer (Figure 11).
Figure 11. The temperature tower opened in Prusa slicer.
The models were printed with all four PETG filaments. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets were the same as for Iteration 1, except: Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s; Gap fill: 20 mm/s; Nozzle: 220°C – 260°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of each temperature tower was completed in 9 hours and 35 minutes.
Each of the printed temperature towers was carefully examined for stringing, even edges, detail quality, and the clarity of the circular holes and numbers to determine the optimal printing temperatures for each filament. The whole towers and their close-ups are shown in Figure 12. Generally, higher temperatures resulted in increased stringing, while lower temperatures produced clearer numbers and smoother circular holes, though the edges appeared more bent.
Figure 12. The 3D-printed temperature towers of the PETG filaments.
(A) Black temperature tower. (B) Close-up of the black temperature tower. (C) Yellow temperature tower. (D) Close-up of the yellow temperature tower. (E) Purple temperature tower. (F) Close-up of the purple temperature tower. (G) Transparent temperature tower. (H) Close-up of the transparent temperature tower.
Based on a careful evaluation, the following temperatures were chosen for further printing with each filament:
These temperatures were selected based on achieving minimal stringing, good circularity of the holes, and minimal bending of the edges while maintaining overall print quality.
The black box was redesigned with higher walls and a dedicated space for a cover lid, which will close the box using a sliding mechanism. The walls surrounding the chamber were also raised. Additionally, the holes for the LED holders were designed with organic supports this time. Figure 13 presents the model of the box, with exact dimensions, designed in TinkerCAD.
Figure 13. Model of the detection box designed in TinkerCAD.
The model was printed with black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except:
Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Perimeters: 15 mm/s; Small perimeters: 20 mm/s; External perimeters: 20 mm/s; Infill: 40 mm/s; Gap fill: 20 mm/s; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s.
The printing process of the model was completed in 11 hours and 48 minutes.
The box was printed successfully. The change in nozzle temperature significantly improved the quality of the design, and the results are satisfactory, as shown in Figure 14. Switching from grid supports to organic supports helped maintain the smooth, circular shape of the holes for the LED holders. The thickness of the walls was ideal, making the box sturdy, and the walls were no longer elastic. The spot for the lid also appeared well-constructed. However, one issue remained—the height of the box was still too low for taking clear photos.
Figure 14. Photo of the improved 3D-printed detection box.
The improvements made to the box were successful and will be incorporated into future iterations of the black box model. However, the height of the box needs to be increased by approximately 10 mm to address the issue with photo clarity.
The yellow box was redesigned with higher walls and a dedicated space for a cover lid, which will close using a sliding mechanism. The wall adjacent to the battery was also raised and thickened. Additionally, the hole for the light switch was redesigned to be circular, offering better protection, and the holes for the LED holders were designed with organic supports this time. Figure 15 presents the model of the box with exact dimensions, designed in TinkerCAD.
Figure 15. Model of the electronics box designed in TinkerCAD.
The model was printed with yellow PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 16 hours and 11 minutes.
The box was printed successfully. The change in nozzle temperature significantly improved the design quality, yielding satisfactory results, as shown in Figure 16. Switching from grid supports to organic supports helped maintain the smooth, circular shape of the holes for the LED holders. The wall thickness was ideal, making the box sturdy without elasticity, and the wall adjacent to the battery was well-constructed. Additionally, the circular spot for the light switch fits perfectly. However, one issue persisted—the height of the box was still too low, as the simultaneously printed black box also proved to be too low.
Figure 16. Photo of the improved 3D-printed electronics box.
The improvements made to the box were successful and will be incorporated into future iterations of the yellow box model. However, the height of the box needs to be increased by approximately 10 mm to match the height of the improved version of the black box.
The height of the black box was increased by 10 mm. Figure 17 presents the updated model of the box with exact dimensions, designed in TinkerCAD.
Figure 17. Model of the improved detection box designed in TinkerCAD.
The model was printed using black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 14 hours and 49 minutes.
The box was printed successfully, as shown in Figure 18. The increase in height yielded satisfactory results, providing the proper height for the detection box, making it suitable for further testing.
Figure 18. Photo of the improved 3D-printed detection box.
The improvement was successful, providing a properly designed detection box for further tests. The new height of the box can now be applied to the yellow and purple boxes, which will be printed next.
The height of the yellow box was increased by 10 mm. Figure 19 presents the updated model of
the box with exact
dimensions, designed in TinkerCAD.
Figure 19. Model of the improved electronics box designed in TinkerCAD.
The model was printed with yellow PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets were the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 19 hours and 18 minutes.
The electronics box was printed successfully, as shown in Figure 20. The increased height yielded satisfactory results, aligning perfectly with the height of the black box and providing the proper dimensions for further testing.
Figure 20. Photo of the improved 3D-printed electronics box.
The improvement was successful, providing a properly designed electronics box for further tests.
The properly printed boxes required well-designed lids that would function as a sliding mechanism. Figure 21 presents the yellow cover lid with exact dimensions, designed in TinkerCAD.
Figure 21. Model of the yellow cover lid designed in TinkerCAD.
The model was printed with yellow PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 225°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 3 hours and 36 minutes.
The lid was printed successfully as shown in Figure 22. It fit the yellow box well, and the sliding mechanism operated perfectly.
Figure 22. Photo of the yellow cover lid in the yellow box.
The lid was well designed and could be implemented in both the black and purple boxes.
Based on the design of the yellow lid, the black lid was created for the black detection box. It required an additional hole for the green filter and a magnifying lens. Figure 23 presents the lid with exact dimensions, designed in TinkerCAD.
Figure 23. Model of the black cover lid designed in TinkerCAD.
The model was printed with black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 3 hours and 55 minutes.
The lid was printed successfully. However, some of its dimensions were not precise enough, resulting in the filter protruding from the lid. This issue prevented the lid from being properly inserted into the designated space in the black box, as shown in Figure 24.
Figure 24. The photo of the black lid and the black box.
The lid had an appropriate size and did not require major improvements. However, the hole in the center needed to be widened slightly, and the protruding element in the center of the lid needed to be removed.
The black lid was redesigned to smoothly fit the green filter. Figure 25 presents the lid with exact dimensions, designed in TinkerCAD.
Figure 25. Model of the improved black lid designed in TinkerCAD.
The model was printed with black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 3 hours and 55 minutes.
The lid was printed successfully. This time, the dimensions were perfect, allowing it to fit the filter well. The lid was smoothly inserted into the black box using the sliding mechanism, as shown in Figure 26.
Figure 26. The improved black lid and the black box.
The black lid was printed successfully and did not require any further improvements.
The reaction chamber from Iteration 5 was printed again without any changes in the design. Figure 27 presents the model of the chamber, with exact dimensions, designed in TinkerCAD.
Figure 27. Model of the reaction chamber designed in TinkerCAD.
The model was printed with transparent PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 220°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 34 minutes.
The chamber was printed successfully, as presented in Figure 28A. It was tested in the black detection box to verify its visibility when the LEDs were turned on. The chamber was well illuminated, but a green signal was also observed within it, as shown in Figure 28B. To determine whether the filament emitted a green fluorescence signal, it was examined under a fluorescent microscope and excited with blue light. A fragment of the photo taken by the microscope is shown in Figure 28C, confirming that the filament did emit some green fluorescence. It also became apparent that the chamber did not hold water effectively and was leaking due to its thin bottom.
Figure 28. Reaction chamber printed with transparent PETG filament.
(A) Well-printed chamber. (B) Green signal produced by the chamber in the detection box. (C) Green fluorescence produced by the chamber, recorded by the fluorescence microscope.
Since the filament produced green fluorescence, it could not be used for creating the reaction chamber in the PrymChip. Therefore, the approach had to be revised.
It was decided to discontinue printing the reaction chamber. After exploring different options, it was determined to use a cap from a hair spray bottle as the reaction chamber, as the material did not produce a green fluorescence signal. The cap needed to be cut to a proper size, similar to that of the printed chamber as Figure 29 indicates.
Figure 29. The chosen hairspray bottle cap and the printed reaction chamber.
The cap was carefully cut to the proper height using scissors.
The cap had the proper height, and after placing it in the detection box, it was easy to capture a clear photo. It also did not leak and did not produce green fluorescence. The cut cap is shown in Figure 30 A. The reaction chamber made from the hair spray bottle was tested with fluorescein in the detection box. However, it was found that the green fluorescence of the fluorescein solution was uneven at the edges of the chamber, creating an intense green ring (Figure 30 B). Additionally, since the cap was inserted into the black box made in iteration 7, it did not fit well in the designated space for the reaction chamber.
Figure 30. The reaction chamber made out of the cut cap. (A) The cut cap and 3D-prited reaction chamber. (B) Green fluorescence of the fluorescein in the chamber with a visible intense green ring.
The cut cap was a good choice for the reaction chamber. However, a cover for the edges needed to be designed to even out the registered fluorescence signal. Additionally, the black box required adjustments to fit the chamber perfectly. In iteration 9, this issue was addressed, resulting in a larger space for the chamber in the box.
The cover for the reaction chamber was designed. Figure 31 presents the model of the cover, with exact dimensions, designed in TinkerCAD.
Figure 31. Model of the chamber cover designed in TinkerCAD.
The model was printed with black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process was completed in 10 minutes.
The cover was printed successfully. It fit the chamber well and the glass cover slip that was chosen to fit the reaction chamber fit well as Figure 32 A indicates. It was tested with the fluorescein solution and as Figure 32 B indicates it removed the need for further improvements of the chamber.
Figure 32. The 3D-printed cover for the chamber.
The printed lid cover was satisfactory and did not require further improvements. The issue with the intense green fluorescent ring was resolved.
The purple box was redesigned to feature an increased height that aligns with the improved black and yellow boxes. Additionally, a space was designated for the cover lid, incorporating a sliding mechanism. Figure 33 presents the model of the box with exact dimensions, designed in TinkerCAD.
Figure 33. Model of the tool box designed in TinkerCAD.
The model was printed using purple PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 230°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the model was completed in 24 hours and 12 minutes.
The purple box was printed successfully, matching the height and width of the black and yellow boxes, as indicated in Figure 34.
Figure 34. The three printed boxes aligned with each other.
The box was well designed and did not require any further improvements.
We concluded that the boxes may require additional symbols for easier recognition. Symbols indicating the use of each box were designed with varying heights to determine which would look best on the boxes. Figure 35 presents the plates with the chosen symbols, including their exact dimensions, designed in TinkerCAD.
Figure 35. Models of the plates with symbols designed in TinkerCAD.
The models were printed with black and yellow PETG filaments. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: none; Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C (black), 225°C (yellow); Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the concave model was completed in 1 hour and 32 minutes and the convex model in 49 minutes.
The printing process was successful, as shown in Figure 36. The symbols were evaluated to determine which version looked better: concave or convex. Subsequently, the convex symbols were assessed to identify which height appeared most suitable.
Figure 36. Convex and concave 3D-printed symbols.
The convex versions of the symbols were chosen for their better appearance, and a height of 1.5 mm was selected as the most suitable. These symbols can be incorporated into the designs of the cover lids in future steps.
The lids were redesigned to implement the convex 1.5 mm height symbols. The size of the lids was not changed. Figure 37 presents the models of the improved cover lids, with exact dimensions, designed in TinkerCAD.
Figure 37. Models of the cover lids with incorporated symbols designed in TinkerCAD.
The models were printed with black, yellow and purple PETG filaments. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: none (purple, yellow), on build plate only (black); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C (black), 225°C (yellow), 230°C (purple); Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process of the black model was completed in 3 hours and 58 minutes, yellow in 3 hours and 38 minutes, purple in 6 hours and 9 minutes.
The cover lids were printed successfully and fit the designated boxes, as shown in Figure 38. However, the smartphone placed on the connected boxes experienced stability issues due to the uneven height created by the protruding symbols.
Figure 38. The three boxes are connected to each other with cover lids that feature symbols.
Additional support was required on the opposite side of the boxes to stabilize the phone without removing the symbols, which are beneficial for colorblind individuals.
The supports for the smartphone were designed to stabilize the device when placed on the PrymChip. They are easily detachable and fit conveniently in the purple toolbox. Figure 39 presents the model of the support, with exact dimensions, designed in TinkerCAD.
Figure 39. Model of the support designed in TinkerCAD.
The model was printed with black PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are the same as for Iteration 1, except: Infill: 30%; Supports: on build plate only (organic); Top fill pattern: Monotonic Lines; Bottom fill pattern: Monotonic Lines; Nozzle (first and other layers): 240°C; Bed (first and other layers): 70°C; Filament type: PETG; Retraction length: 5.5 mm; Retraction speed: 50 mm/s. The printing process was completed in 1 hour.
The supports were printed successfully and fit the boxes, as shown in Figure 40. After placing the phone on the PrymChip with the supports, the level was even.
Figure 40. The supports attached to the PrymChip.
The design of the supports was effective and did not require further improvements.
[1] Samacoits A, Nimsamer P, Mayuramart O, Chantaravisoot N, Sitthi-amorn P, Nakhakes C, Luangkamchorn L, Tongcham P, Zahm U, Suphanpayak S, Padungwattanachoke N, Leelarthaphin N, Huayhongthong H, Pisitkun T, Payungporn S, Hannanta-anan P. Machine learning-driven and smartphone-based fluorescence detection for CRISPR diagnostic of SARS-CoV-2. ACS Omega. 2021;6(4):2727-2733. doi: 10.1021/acsomega.0c04929.
[2] Temp tower: PLA/PETG/ABS/ASA for Prusa Mini/MK3s and more. Available at: https://www.printables.com/model/20652-temp-tower-pla-petg-absasa-for-prusa-mini-mk3s-and/files.
