Engineering
Project TaRNAdigrada has been based in iGEM’s iterative engineering framework from the first whiteboard sketches through each troubleshooting session to the long-term planning necessary to continue future research, across each challenge and each success. Every step of the project has undergone multiple iterations of the Design-Build-Test-Learn cycle to ensure process optimization and ensure our team never lost sight of our goal.
Cloning: DBTL
Design Phase: Choosing the Right Vector
We aimed to create recombinant plasmids containing tardigrade protein genes for protein production in mammalian cell lines.
We selected a variety of tardigrade-specific genes by surveying existing literature and the UniProt protein sequence database. We made our selections while aiming to cover five major classes of significantly-disordered tardigrade proteins known to contribute to macromolecular and/or whole-cell stress tolerance in vitro and in vivo. They include the three tardigrade-specific disordered protein (TDP) classes—Cytosolic Abundant Heat-Soluble (CAHS), Secretory Abundant Heat-Soluble (SAHS), and Mitochondrial Abundant Heat-Soluble (MAHS) proteins—as well as Late Embryogenesis Abundant (LEA) proteins and Damage suppressor (Dsup) proteins.
We chose to look at two different species of tardigrade: Hypsibius exemplaris(HE) and Ramazzottius varieornatus(RV). The proteins and their respective accession numbers for Uniprot:
- HE CAHS8 (P0CU50)
- RV CAHS8 (A0A1D1UQN2)
- HE Dsup (A0A1W0XB17)
- RV Dsup (P0DOW4)
- HE LEAM (P0CU49)
- RV LEAM (A0A0E4AVP3)
- HE MAHS (A0A1W0X402)
- RV MAHS (LC002822)
- HE SAHS2 (P0CU40)
- RV SAHS2 (J7MAN2)
Five proteins were selected from Ramazottius varieornatus (RV) and five proteins were selected from Hypsibus exemplaris (HE), both model species in tardigrade research, to compare the extent to which proteins of the same class across different species varied in their stress-protection capabilities.
All 10 genes were purchased as G-blocks after codon optimization for mammalian system expression, 6x-His tag insertion, CMV promoter, a Kozak sequence for efficient translation initiation, and secretory signal pepetide. Mammalian expression was chosen to ensure the expressed proteins’ post-translational modifications would closely match those of native tardigrade proteins and to circumvent inclusion body generation in an E. coli expression system, as experienced by previous iGEM teams working with tardigrades.
The 6x-His tag was chosen for a simple and low-cost purification process not requiring tag cleavage steps, and the secretory sequence was chosen to simplify purification by only requiring the harvesting and purification of cell conditioned media.
Each gene would be inserted into a pCS2 backbone integrated with sfGFP.
Build Phase: PCR and Assembly
Cloning was performed via PCR amplification of each gene, restriction digestion of the G-block and cut sites in the pCS2 backbone, ligation into a recombinant plasmid, transformation into chemically competent E. coli and confirmation via diagnostic digestion and sequencing.
For the R. varieornatus Dsup gene, PCR amplification of the G-block failed despite multiple attempts, and for multiple genes, few or no correct transformants were observed despite multiple cloning rounds, necessitating refinements to the cloning process.
Test Phase: Colony PCR and Sequencing
Colony sequencing for multiple cloning rounds returned only the original pCS2-sfGFP plasmid and no recombinant plasmid, suggesting incomplete digestion of the plasmid backbone interfering with the cloning process. In the case of the R. varieornatus Dsup gene, sequencing frequently revealed partial or incorrect ligation of the gene into the pCS2 backbone, suggesting the large G-block (# base pairs in length) was ill-suited to the traditional cloning technique.
Learn Phase: Optimization
Our team made the following optimizations to our cloning protocols:
- Touchdown PCR to successfully amplify the R. varieornatus Dsup G-block
- Restriction digestion of successful recombinant plasmids to generate vectors for insertion of other genes, such as R. varieornatus MAHS
- Religation to generate fewer transformants containing the recombinant plasmid instead of many transformants containing pCS2
- Our initial ligation protocol called for 14 μL of insert and 3 μL of backbone vector. After measuring concentrations of restriction-digested inserts and vectors following PCR purification, we determined that adjusting volumes to ensure an insert-to/vector ratio between 6:1 and 20:1 in ng yields a higher quantity of successful ligations.
These modifications resulted in the successful generation of all 10 recombinant plasmids.
Alternative plasmid designs were explored after the generation of recombinant plasmids as part of troubleshooting downstream inefficiencies. To solve the problem of repeated expensive and time-consuming transfections to generate sufficient amounts of each protein, we explored the PiggyBac transposon system as a tool to integrate each tardigrade protein gene into a mammalian genome, requiring only one successful transfection per protein. After noticing nonspecific binding of our His-specific primary antibodies during Western blotting, we also designed primers to perform site-directed mutagenesis (SDM) to add FLAG tags onto our genes in order to more accurately visualize protein of interest expression via Western blot.
The construction of both genome-integrating recombinant plasmids and FLAG-tagged recombinant plasmids is currently in the “Build” stage, with R. varieornatus and H. exemplaris SAHS gene inserts currently being cloned into the PiggyBac vector and a FLAG tag being added onto the H. exemplaris Dsup gene.
Protein Expression: DBTL
DBTL Cycle #1: Initial Expression & Scaling
Design Phase: Expression System
For our expression system, we selected Chinese Hamster Ovary (CHO) cells to produce all ten tardigrade proteins. This decision was based on two primary factors. First, as noted by previous iGEM teams, expressing these specific tardigrade proteins in E. coli often results in their aggregation into inclusion bodies. This suggests that the prokaryotic environment lacks the necessary machinery to correctly process these unique proteins. Second, both tardigrades and CHO cells are eukaryotic. They share more compatible cellular machinery for protein folding and post-translational modifications compared to prokaryotic systems like E. coli. Therefore, using a mammalian CHO cell line increases the likelihood of producing soluble, correctly folded, and functional tardigrade proteins that more closely resemble their native state. Well plates seeded with CHO cells were transfected with a recombinant plasmid, allowed to generate protein for three days, and collected and assayed the conditioned media for protein expression via Western blot. Nickel column purification would then be employed to purify the generated His-tagged proteins after expression confirmation.
Build Phase: Expressing the Protein
We transfected the CHO cells at 30-45% confluency and changed the media to serum-free media(Gibco EXPI-293) 6 hours post-transfection for ideal protein expression. Using media without penicillin-streptomycin (pen-strep) could theoretically increase protein yield. The rationale is that without the antibiotic, cells do not expend energy (ATP) to combat it, freeing up those resources for protein synthesis.
We started with 6-well plates and then moved on T75 cell culture flasks as we scaled up to see protein expression.
Test Phase: SDS-PAGE Analysis
We ran the collected conditioned media on an SDS-PAGE gel, followed by Coomassie staining, to verify the presence and size of our target protein. No protein of interest was detected via Western blot.
Learn Phase: Optimization
Multiple experimental modifications were investigated to optimize the protein expression and purification process. The first major redesign involved scaling up the amount of total protein collected — conditioned media was collected from multiple wells in a six-well plate instead of just from one well, and eventually from daily harvests of transfected T75 flasks over the course of three days. The collected conditioned media were also further concentrated prior to blotting.
After scaling up failed to demonstrate protein on a Western blot, cell lysate was collected and blotted along with the conditioned media for all future transfections in the event the tardigrade proteins were not being secreted.
DBTL Cycle #2: Western Blot Optimization
Design Phase: Western Blot
To improve the detection of our target proteins and confirm their expression, we designed a series of optimization experiments for our Western blotting protocol. Key variables included the blocking agent (BSA vs. non-fat milk powder), blocking agent concentration (5% vs. 10%), primary antibody dilution (1:500, 1:1000, and 1:2000), and the HRP substrate (Enhanced Chemiluminescence [ECL] vs. TMB).
Additionally, we planned to use RT-qPCR to quantify gene expression for all 10 synthetic tardigrade proteins in transfected CHO cells to verify expression at the transcript level.
Build Phase: Conduct the Assays
We prepared blots testing each variable: BSA vs. milk, different concentrations of the selected blocking agent, and various primary antibody dilutions. For detection, we implemented the ECL substrate protocol.
Test Phase: SDS-PAGE Analysis and RT-PCR Data
We analyzed the results from the optimized Western blots and the RT-qPCR. All blots showed bands for postive control.
Learn Phase: Upstream Issue not the Western Blot Protocol
The test results showed that none of the optimization procedures significantly improved blot quality. While the positive control protein was consistently detected, our target proteins of interest remained undetectable. This suggests the issue may not be with the Western blot protocol itself but rather with upstream processes like insufficient protein expression or stability. The results from the RT-qPCR will be used to determine if the lack of protein detection is due to a failure in transcription or a post-transcriptional issue.
DBTL Cycle #3: Alternative Cell System
Design Phase: Exploring an Alternative Expression System
A HEK293T cell line was established alongside the CHO cell line as an alternative expression system, and collected condition media was put through the complete nickel column purification process, and the purified fractions across the process was run on the Western blot along with the cell lysate and positive control.
Build Phase: HEK293T Cell Line
We established a HEK293T cell line and scaled up cell growth in a T75 flask. Cells were transfected and media was changed 6-8 hours post-transfection, after which conditioned media was collected once daily over the course of 3 days and the cells were lysed on the third day.
We also performed nickel-NTA column purification on the collected conditioned media prior to blotting in an effort to concentrate our protein of interest and lessen nonspecific banding on our expression confirmation blot, a major change from our initial plan to purify after confirming protein expression. The collected conditioned media underwent our purification protocol, and the flow-throughs, purified elutions, conditioned media before purification, and cell lysate were assayed via Western blot for expression confirmation.
Test Phase: SDS-PAGE Analysis
We analyzed the results from the blots. Protein collected from the cell lysate of HEK cells seeded in a T75, when assayed via Western blot, demonstrated the presence of H. exemplaris and R. varieornatus CAHS and SAHS proteins.
Learn Phase: Isoelectric Point Mismatch
The successful detection of CAHS and SAHS proteins confirms that the HEK cell line is a viable expression system. We now suspect the failure to detect the remaining LEA, Dsup, and MAHS proteins is due to a biochemical incompatibility in our purification step. These specific proteins have a high isoelectric point (pI), which falls outside the effective buffering range of the Tris-HCl buffer used during nickel column purification.
DBTL Cycle #4: Engineering a More Efficient Production System
Design Phase: A Stable Expression Strategy Using the PiggyBac System
To address the need for constant, costly re-transfection to generate enough protein for our experimental trials — a problem only exacerbated if these proteins were to be manufactured at scale for vaccine applications — we designed an alternative expression method using the PiggyBac transposon system. PiggyBac presents an alternate to transient transfection by integrating each synthetic gene into host cells’ genomes and forcing constitutive expression using two co-transfected plasmids, one containing a transposon and one coding for a transposase. The transposon vector contains a multiple-cloning site for gene insertion inside an FP locus flanked by inverted terminal repeat sequences, which the transposase enzyme recognizes and excises for integration at genomic TTAA sites. Any genes inserted into the multiple-cloning site are controlled by a CMV promoter. We aimed to insert each of our four successfully-expressed genes — R. varieornatus and H. exemplaris CAHS8 and SAHS2 — into PiggyBac transposon vectors by following the cloning protocol we used to construct our 10 pCS2-backbone plasmids.
Fig. 1: PiggyBac-HE-SAHS2 plasmid map showing antibiotic resistances, promoters of interest and the multiple-cloning site wherein the H. exemplaris SAHS2 gene is inserted.
Build Phase: Assembling the Plasmid via Restriction Ligation
The PiggyBac vector was digested with EcoRI and SwaI to produce a sticky and blunt end, respectively, within the MCS. Our constructed plasmids pCS2-RV-SAHS2 and pCS2-HE-SAHS2 were digested with EcoRI and PvuII to produce a compatible sticky and blunt cut on either side of the gene of interest. Initially, all digestions were done using CutSmart buffer (NEB), but then double digestion with CutSmart and NEBuffer r3.1 was performed upon discovering that the EcoRI and SwaI enzymes require different buffers for optimal activity. Both the PiggyBac and SAHS2 insert digestion products were run on an agarose gel to isolate the fragments of interest, gel-purified, and ligated together using T4 ligase (NEB). The ligation product was transformed into chemically competent E. coli, which were then inoculated and miniprepped to isolate the resulting plasmid.
Test Phase: Agarose Gel Analysis
Whole-plasmid sequencing revealed that the miniprepped PiggyBac vector contained no insert. To determine whether the digestion was successful, we compared the undigested PiggyBac vector, the EcoRI-digested PiggyBac vector, the SwaI-digested PiggyBac vector, and the PiggyBac vector sequentially digested by EcoRI and SwaI. The gel displayed clear size differences between each digestion.
Fig. 1: Restriction digestion gel of PiggyBac plasmid (lane 1), pCS2-RV-SAHS2 (lane 2), and pCS2-HE-SAHS2 (lane 3). PiggyBac was digested with EcoRI and SwaI. Both SAHS2 plasmids were digested with EcoRI and PvuII. The very faint band in lane 1 suggests incomplete digestion due to performing a single digestion instead of double digestion. M: GeneRuler 1kb Plus DNA ladder (ThermoScientific).
Fig. 2: Sequential digestion gel of PiggyBac plasmid showing successful restriction digestion. Lane 1: undigested PiggyBac plasmid. Lane 2: PiggyBac digested with EcoRI. Lane 3: PiggyBac digested with SwaI. Lane 4: PiggyBac digested sequentially with EcoRI and SwaI. M: GeneRuler 1kb Plus DNA ladder (ThermoScientific).
Learn Phase: Identifying Ligation as the Point of Failure
With confirmation that PiggyBac restriction digestion was not the issue, we turned to troubleshooting the ligation process as the next most probable cause of cloning failure.
DBTL Cycle #5: Optimizing the Ligation Protocol for PiggyBac
Design Phase: Revised Ligation Protocol
Keeping the restriction digestion steps the same, we replaced T4 ligase with QuickLigase (NEB) and followed its corresponding ligation protocol.
Build Phase: Executing the Revised Ligation Protocol
PiggyBac vector was double digested with EcoRI and SwaI, and the constructed SAHS2 plasmids were each digested with EcoRI and PvuII. Each digestion was run on an agarose gel, excised, gel-purified, and ligated using QuickLigase.Unlike T4 ligase, QuickLigase only needs to incubate for 5 minutes at room temperature and does not need to be heat-inactivated prior to transformation into competent cells.
Test Phase: Confirmation of Cloning Success
Whole-plasmid sequencing revealed that the PiggyBac vector contained no insert.
Learn Phase: Investigating a Revised Ligation Strategy and Alternative Assembly Methods
Further optimizations to our ligation procedure are being investigated, including overnight QuickLigase ligation at both room temperature on the benchtop and at 25℃ in a thermocycler. Overtime sequential digestion for both the SAHS2 plasmids and the PiggyBac insert is also being explored for potentially providing more total cut ends for ligase enzymes to act on, as well as using SfoI in place of PvuII for the SAHS2 digestions. Our team is also developing a PiggyBac Gibson assembly procedure as an alternative to traditional digestion and ligation, currently in the primer design stage. The SAHS2 inserts and the PiggyBac vector will be blunt-cut, modified with overlapping DNA sequences via PCR, digested to produce complementary sticky ends, and ligated together to generate a recombinant plasmid with a SAHS2 insert in the multiple-cloning site.
Protein Purification: DBTL
Design Phase: Nickel-NTA affinity chromatography
We purified the proteins using the His-Tag through Nickel-NTA affinity chromatography and optimized the wash buffer to contain 20 mM imidazole to remove non-specific binding proteins. To purify our high pI proteins while preventing them from precipitating, we used a Tris-HCl buffer at a basic pH of 9.0.
Build Phase: Running the Column
We purified the protein from our conditioned media using a Nickel-NTA column. After the initial protein binding, fractions were collected during the subsequent washing and elution steps. The purified protein was desalted. We learned that the Tris-HCl pH 9.0 buffer is chemically incompatible with the nickel-NTA resin, which was confirmed by a distinct color change in the nickel upon adding the pH 9.0 Tris-HCl.
Test Phase: SDS-PAGE Analysis
We analyzed the collected fractions using Coomassie-stained SDS-PAGE gels and Western blotting with an anti-His antibody. The results confirmed that our target protein was successfully captured by the nickel resin and was strongly seen in elution 1 after desalting. However, the Coomassie gel revealed that several other contaminating native cell proteins were also present in the final sample. The Western blot also showed some non-specific bands in addition to our strong target band.
Learn Phase: Improving Purity
While the nickel column works for capturing our protein of interest, a single affinity step is not sufficient to achieve high purity. The contaminants that remain are likely native proteins that have some affinity for the nickel resin. We would consider an adiitional purification step like ion-exchange or size-exclusion chromatography to separate the remaining contaminants based on charge or size. We could also consider FLAG-tagging which enables a more selective column chromatography than Nickel resin.
Degradation Assays: DBTL
DBTL Cycle #1: In Vivo mRNA Protection Assay
Design Phase: In Vivo Transfection and Fluorometry
Our initial design involved stressing mRNA encoding for a fluorescent protein, after which the stressed mRNA would be transfected into a mammalian cell line seeded in well plates and fluorometric analysis would quantify the relative amounts of non-degraded, successfully translated mRNA in each well. The mRNA stress tests involved static-temperature heat shock at 4C, 25C, 35C, 45C, 55C, 65C and 75C for 72 hours, multiple rounds of freeze-thawing, and UV irradiation delivered by a gel imager for 30 minutes. The stressed mRNA would be co-transfected with non-stressed mRNA encoding another fluoroprotein to adjust for different transfection efficiencies between wells.
Each experimental aliquot was designed to contain 1 ug mRNA, an equimolar amount of tardigrade protein, and a buffer solution designed to mimic the buffer found in the Pfizer vaccine.
Build Phase: mRNA IVT and Cell Transfection
We generated sf-Cherry3C and sfGFP mRNA through in vitro translation, with mRNA acting as the experimental aliquot to be stressed and sfGFP acting as the non-stressed counterpart to compare transfection efficiencies. To establish baseline fluorescence readings for both fluoroprotein mRNAs, equal amounts of sfCherry3C and sfGFP mRNA were transfected into CHO cells seeded in well plates and assayed with fluorometry.
Test Phase: Agarose Gel Analysis and Computational Modeling
Aliquots of the sfCherry3C mRNA produced from IVT was run through a preliminary stress trial, held at multiple different temperatures for 72 hours, and assayed via gel. The temperatures higher than 4°C showed significant to total degradation with no visible differentiation among the highest temperature trials, suggesting the 72-hour period was too long for the heat shock to last. Gel electrophoresis assays of stressed and unstressed sfCherry3C yielded significant differences in band size between assays, prompting sfGFP mRNA be explored as the stressed fluoroprotein mRNA instead should it provide a more consistent band size readout.
Transfected sfGFP mRNA and transfected sfCherry3C mRNA did not produce any detectable fluorescence during our attempt to establish a baseline for degradation assays in vivo.
The cost of mRNA transfection reagents — especially when added to the transfection reagents needed to maintain regular transfection of recombinant plasmids across 10 different protein-expressing cell lines — prompted us to look for a lower-cost alternative to assay mRNA degradation that would require less fine-tuning.
Additionally, molecular interactions between sfCherry3C mRNA and each of the 10 tardigrade proteins in a buffered system were modeled using AlphaFold 3 and suggested that a 1:10 mRNA to protein molar ratio resulted in more intermolecular interactions than the initial 1:1 ratio. This suggested a redesign of the experimental aliquots could maximize the chances of observing stress protection from the proteins. AlphaFold modeling also suggested that multiple protein classes such as CAHS and LEA are structurally similar and perform similar molecular interactions with a given mRNA, suggesting the results of one protein’s degradation assay will likely resemble the other’s.
To view all models, please hover and click the arrow.
Protein
1:1 mRNA to protein ratio
1:5 mRNA to protein Ratio
Learn Phase: Pivot to an In Vitro Approach
We redesigned our assays entirely in vitro instead of in vivo to eliminate costs, as well as refining the stress conditions the fluoroprotein mRNA would experience. Since vaccines are most liable to temperature fluctuations during transport, we eliminated UV radiation as a stressor, and reduced the static temperature shocks to 4C, 25C and 50C for one-hour exposures in addition to the -80C negative control to eliminate the possibility of total degradation or compounding effects of RNase activity.
Degradation was now primarily measured via gel assay, with the differences in degradation quantified using the PRISM biological image analysis tool. The amount of mRNA present in each experimental aliquot was lowered to 100 ng at a 1:10 molar ratio with tardigrade proteins to fit with our most successful computational models.
DBTL Cycle #2: Initial mRNA Degradation Assay
Design Phase: mRNA assay
To evaluate the ability of four synthetic tardigrade proteins (CAHS HE, CAHS RV, SAHS HE, SAHS RV) to protect mRNA, we designed a temperature stress experiment. Formulations of mRNA mixed with mock vaccine buffer and each protein were to be subjected to a range of temperatures (-80°C, 4°C, Room Temperature, and 50°C). The integrity of the mRNA post-incubation would be assessed by agarose gel electrophoresis to determine the protective capacity of each protein.
Build Phase: Sample Formulation and Stress Test
Samples were formulated by mixing 100 ng of mRNA with 8.69 µg of protein in a vaccine buffer, with the total volume brought to 20 µL with nuclease-free water. The components were incubated together for one hour under the different conditions. We also had control experiments which included a sample of the stock mRNA with water and mRNA mixed only with the vaccine buffer, to isolate any effects seen by the addition of the protein.
Test Phase: Agarose Gel Electrophoresis Analysis
We observed severe and widespread mRNA degradation in all samples containing protein, irrespective of the temperature condition. Even the -80°C sample, which should have preserved the mRNA, showed complete degradation. In contrast, control gels confirmed the integrity of the initial mRNA stock. We know that the vaccine buffer is not the primary cause of degradation as we tested a control with just the buffer and the mRNA which still showed a band. To establish whether doubling the amounts of mRNA and protein allows for better band visualization on the gel, we ran an additional round of stress tests and found similarly severe degradation across all protein-containing samples.
Fig. 1: Degradation assay showing different 1-hour heat shocks’ effects on mRNA intactness and the effects of combining each of 4 purified proteins with mRNA prior to any stress testing. The addition of proteins caused mRNA degradation before any stress was applied. “2x” denotes that the quantity in grams of mRNA and protein were both doubled compared to previous degradation assays in an effort to visualize stronger bands.
| Lane | Sample Description |
|---|---|
| 1 | mRNA control (nuclease-free water) |
| 2 | -80℃ mRNA |
| 3 | 4℃ mRNA |
| 4 | RT mRNA |
| 5 | 50℃ mRNA |
| M | GeneRuler 1kb Plus DNA ladder (ThermoScientific) |
| 6 | H. exemplaris CAHS8 + mRNA (2x) |
| 7 | R. varieornatus CAHS8 + mRNA (2x) |
| 8 | H. exemplaris SAHS2 + mRNA (2x) |
| 9 | R. varieornatus SAHS2 + mRNA (2x) |
| 10 | Negative control protein + mRNA (2x) |
| 11 | mRNA control (nuclease-free water) (2x) |
Fig. 2: Degradation assay showing the effects of combining each of 4 purified proteins with mRNA prior to any stress testing, suggesting the protein addition is responsible for the observed degradation.
| Lane | Sample Description |
|---|---|
| M | GeneRuler 1kb Plus DNA ladder (ThermoScientific) |
| 1 | H. exemplaris CAHS8 + mRNA (2x) |
| 2 | R. varieornatus CAHS8 + mRNA (2x) |
| 3 | H. exemplaris SAHS2 + mRNA (2x) |
| 4 | R. varieornatus SAHS2 + mRNA (2x) |
| 5 | mRNA control (nuclease-free water) |
| 6 | mRNA control (mock vaccine buffer) |
| 7 | 50℃ mRNA |
Learn Phase: RNase Contamination Identified
The test results strongly indicated that the failure was not due to the experimental stress conditions but rather to RNase contamination within the purified protein preparations. The protective effects of the tardigrade proteins can not be evaluated until the RNases are removed.
DBTL Cycle #3: Incorporating an RNase Inhibitor
Design Phase: Protocol Optimization
To counteract the RNase contamination identified in Cycle 2, we redesigned the protocol to include a commercial RNase inhibitor in the formulation. Additionally, we observed some degradation due to a 65°C sample pre-heating step which was done to denature RNA secondary structure. The heating step was removed and kept to determine its impact on the inhibitor's efficacy.
Build Phase: Sample Formulation and Stress Test
Each 20 µL sample was formulated with 8.69 µg of protein, 100 ng of mRNA (added as 0.906 µL of a 1104.3 ng/µL stock), 2 µL of vaccine buffer, and 0.5 µL of RNase Inhibitor (providing a final concentration of 20 Units per reaction).Samples were prepared for analysis both with and without the 65°C pre-heating step before loading the gel.
Test Phase: Agarose Gel Electrophoresis Analysis
Samples prepared without the 65°C heating step showed complete recovery of intact mRNA across all protein conditions, indicating the inhibitor successfully neutralized the contaminating RNase activity. Conversely, samples subjected to the 65°C heating step showed complete mRNA degradation, confirming that the inhibitor denatures and loses function at elevated temperatures.
Learn Phase: RNase Inhibitor Works and Pre-heating Step to be Removed
The addition of an RNase inhibitor is an effective and necessary modification to the protocol, successfully resolving the contamination issue. The data also clearly show that the 65°C sample pre-heating step is detrimental and must be omitted prior to gel loading from the procedure.
DBTL Cycle #4: mRNA Stress Test Duration
Design Phase: Establishing a Time-Based Assay
To identify the ideal conditions for observing mRNA degradation, we designed a comprehensive matrix experiment. The plan was to test a broad range of incubation times (1, 2, 5, 9, and 12 hours) across multiple temperatures representing both storage and high-stress conditions. We wanted to establish a baseline by testing only naked mRNA and mRNA within our formulation buffer.
Build and Test Phase: Execution of Stress Test
We prepared the baseline samples and incubated them at the specified temperatures. Aliquots from each condition were collected at every time point and run on an agarose gel to visualize degradation.
Test Phase: Agarose Gel Electrophoresis Analysis
The results were very clear: the most dramatic and measurable differences in degradation occurred at the 9-hour mark and beyond. Specifically, at 50°C, we observed a perfect assay window: the naked mRNA was completely degraded (no band), while the mRNA in our formulation buffer remained significantly protected (strong band).
Learn Phase: Establishing the Standard mRNA Degradation Assay Duration
From this experiment, we learned two critical things:
- The optimal condition for observing a clear protective effect is an incubation of 9+ hours at 50°C.
- Our formulation buffer alone has a significant protective effect on mRNA, establishing a baseline that our proteins must outperform.
This directs our final experimental design: we will now run the full trials with our tardigrade proteins under these optimized conditions (9+ hours at 50°C) to definitively measure their protective capacity above and beyond the buffer alone.