The aim of our project was to produce 10 synthetic tardigrade proteins and test each one’s ability to protect mRNA from temperature stress-induced degradation. The majority of the project focused on optimizing our cloning, protein expression, and protein purification workflows for downstream stress test applications. This page summarizes our results for each step of the project, including our successes, optimizations and remaining areas for improvement.
Cloning
Goal
To construct plasmids that, upon transfection, will express each of our 10 recombinant proteins.
Controls
Diagnostic Digest: A pCS2-NLS-GFP plasmid digested with the same restriction enzymes as the recombinant plasmids served as the negative control.
Results
We successfully constructed all 10 of our target expression plasmids. The process began with designing the coding sequence for each tardigrade gene as gBlocks (IDT), which we codon-optimized for expression in mammalian cells and fused with an N-terminal signal peptide for secretion and a 6xHis-tag for purification.
Each gBlock was amplified via PCR with primers designed to create an XhoI cut site, restriction digested with EcoRI and XhoI, and ligated into a pCS2 backbone digested with the same restriction enzymes to remove its previous insert, NLS-sfGFP. The resulting plasmids were transformed into E. coli and successful clones were screened through performing diagnostic restriction digests on miniprepped DNA, which confirmed the presence of an insert of the correct size (Fig. 1). As a final validation step, all successful constructs underwent whole-plasmid sequencing (Plasmidsaurus) to confirm the appropriate insert had been correctly inserted.
While most genes were cloned using this standard protocol, we had to troubleshoot two:
R. varieornatus Dsup: Required optimizing the PCR protocol and starting from a plasmid instead of a G-block
R. varieornatus MAHS: Required using a new vector backbone for ligation by digesting our previously-made recombinant plasmids
With these optimizations, both were also successfully cloned. Our recombinant plasmid sequences are deposited to the iGEM parts registry which can be found under our <Contributions Page> .
We are now in the process of cloning our most promising genes into the PiggyBac transposon system to create stable cell lines for future work.
To view all results, please hover and click the arrow.
Fig. 1.1: Diagnostic restriction digest of plasmids from 7 E. coli colonies to screen for a successful clone of H. exemplaris LEAM. Plasmids were digested with EcoRI and NotI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield two bands at 4 kb and 1 kb. The recombinant plasmid, pCS2-HE-LEAM, is expected to yield three bands around 4 kb, 850 bp, and 250 bp. Lanes 1, 2, 5: pCS2-NLS-sfGFP. Lanes 3, 4, 6: pCS2-HE-LEAM. Lane 7: pCS2-NLS-GFP with a high amount of undigested plasmid. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.2: Diagnostic restriction digest of plasmids from 5 E. coli colonies to screen for a successful clone of R. varieornatus LEAM. Plasmids were digested with EcoRI and XhoI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield two bands at 4 kb and 750 bp. The recombinant plasmid, pCS2-HE-LEAM, is expected to yield two bands at 4 kb and 1 kb. Lanes 1 and 4: pCS2-NLS-sfGFP. Lanes 2, 3, 5: pCS2-RV-LEAM. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.3: Diagnostic restriction digest of plasmids from 5 E. coli colonies to screen for a successful clone of R. varieornatus Dsup. Plasmids were digested with EcoRI and XhoI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield two bands at 4 kb and 750 bp. The recombinant plasmid, pCS2-RV-Dsup, is expected to yield two bands at 4 kb and 1.4 kb. Lanes 1, 2, 4: pCS2-NLS-sfGFP. Lanes 3, 5: pCS2-RV-Dsup. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.4: Diagnostic restriction digest of plasmids from 8 E. coli colonies to screen for a successful clone of H. exemplaris Dsup. Plasmids were digested with EcoRI and XhoI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield two bands at 4 kb and 750 bp. The recombinant plasmid, pCS2-HE-Dsup, is expected to yield two bands at 4 kb and 1.1 kb. Lanes 1, 3: pCS2-NLS-sfGFP. Lanes 2, 4, 6, 7, 8: pCS2-HE-Dsup. Lane 5: inconclusive. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.5: Diagnostic restriction digest of plasmids from 5 E. coli colonies to screen for a successful clone of R. varieornatus SAHS2. Plasmids were digested with EcoRI and PstI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield one band at 4 kb. The recombinant plasmid, pCS2-RV-SAHS2, is expected to yield two bands at 4 kb and 600 kb. Lanes 1, 5, 6: pCS2-NLS-sfGFP. Lanes 2, 3, 4, 7: pCS2-HE-SAHS2. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.6: Diagnostic restriction digest of plasmids from 5 E. coli colonies to screen for a successful clone of H. exemplaris SAHS2. Plasmids were digested with EcoRI and PstI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield one band at 4 kb. The recombinant plasmid, pCS2-HE-SAHS2, is expected to yield two bands at 4 kb and 575 kb. Lanes 1, 2, 3, 5: pCS2-HE-SAHS2. Lane 4: pCS2-NLS-sfGFP. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.7: Diagnostic restriction digest of plasmids from 5 E. coli colonies to screen for a successful clone of H. exemplaris CAHS8. Plasmids were digested with Xba1 and XhoI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield one band at 4 kb. The recombinant plasmid, pCS2-HE-CAHS8, is expected to yield two bands at 4 kb and 800 bp. Lane 1: pCS2-NLS-sfGFP. Lanes 2, 3, 4, 5: pCS2-HE-CAHS8. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.8: Diagnostic restriction digest of plasmids from E. coli colonies to screen for a successful clone of R. varieornatus CAHS8. Plasmids were digested with Xba1 and XhoI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield one band at 4 kb. The recombinant plasmid, pCS2-RV-DAHS8, is expected to yield two bands at 4 kb and 800 bp. Lanes 1, 3, 4, 5: pCS2-RV-CAHS. Lane 2: pCS2-NLS-sfGFP. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Fig. 1.9: Diagnostic restriction digest of plasmids from 6 E. coli colonies to screen for a successful clone of H. exemplaris MAHS. Plasmids were digested with EcoRI and NotI. The original plasmid, pCS2-NLS-sfGFP, is expected to yield two bands at 4 kb and 1 kb. The recombinant plasmid, pCS2-HE-MAHS, is expected to yield three bands at 4 kb, 1 kb and 250 bp. Lanes 1, 2, 3: undigested plasmid. Lanes 4, 5: pCS2-NLS-sfGFP. Lane 6: pCS2-HE-MAHS. Lane 7: pCS2-NLS-sfGFP positive control. Lane M: GeneRuler 1 kb DNA ladder (ThermoScientific).
Expression & Purification
Goal
To establish a reliable workflow to express, purify, and concentrate all 10 recombinant tardigrade proteins for use in downstream mRNA stress tests.
Controls
Positive Controls: We used a His-tagged purified protein 140 kDa in size to confirm our primary and secondary antibodies were operational and the blotting procedure was performed correctly. This positive control is dimeric and runs at half its typical size on a reducing blot, which allowed us to ensure the reducing blot procedure followed during optimization was also performed correctly.
Negative Control (Expression): We blotted mock-transfected cells’ conditioned media and cell lysate to account for background banding from native cellular proteins not removed during the purification process.
Negative Control (Purification): We purified conditioned media from mock-transfected cells to assess non-specific binding.
Results
We successfully expressed and purified 4 of our 10 target proteins: R. varieornatus CAHS, H. exemplaris CAHS, R. varieornatus SAHS, and H. exemplaris SAHS. Initially, each of the 10 constructed plasmids were transfected into CHO cells, and conditioned media and cell lysate were collected from each transfection. Initial Western blot analysis did not reveal any protein of interest expression, leading to an iterative optimization process.
Ultimately, we determined that HEK 293T cells consistently yielded higher quantities of proteins than the initial CHO LEC 2.8.3.1 system, that transfection needed to be scaled up to T75s to generate enough protein to visualize, that conditioned media and cell lysate needed to undergo nickel-NTA column purification and concentration in order to visualize protein of interest on the blot, and that cell lysate contained far more protein of interest than conditioned media. Fractions from each purification step — lysis, flow-through, wash and elution — were blotted to assess whether protein was lost during non-elution steps.
Western blot analysis revealed two important findings:
The protein was found almost exclusively in the cell lysate, indicating that while the proteins were expressed, they were not efficiently secreted.
The protein bands appeared at a slightly higher molecular weight than predicted, suggesting they underwent post-translational modifications, such as glycosylation.
Our team is currently optimizing purification procedures for the 6 proteins whose expression has not yet been confirmed. We suspect the high isoelectric point of the remaining 6 proteins, which are outside the buffering range of the Tris-HCl used in our purification protocol, is causing ineffective matrix binding and protein loss during purification. Experimentation with different buffers and purification systems will continue in order to minimize production costs and maximize yield for each protein.
After confirming expression of a given protein of interest, we performed additional expression and purification experiments to gather enough to run multiple stress trials. Each additional purification was blotted to confirm protein presence. From these blots, we estimated that 1% of the total protein recovered during elution was protein of interest for all 4 successfully expressed proteins.
After purification, the recovered protein was concentrated, quantified via BCA assay, and mixed with experimental aliquots containing fluorescent protein mRNA and buffer salts, and stress trials were performed.
To view all results, please hover and click the arrow.
Figure 2.1: Negative Control(NC) CHO 3.2.8.1 Blot ,Western blot of non-transfected CHO LEC 3.2.8.1 cells serving as a negative control. A prominent band around 18 kDa suggests the presence of native cellular proteins of a similar size to one of the proteins of interest, recombinant H. exemplaris SAHS protein. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M: molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.2: Negative Control(NC) HEK293T Blot , Western blot of a non-transfected HEK 293T t-75 serving as a negative control. A prominent band around 18 kDa suggests the presence of native cellular proteins of a similar size to one of the proteins of interest, recombinant H. exemplaris SAHS protein. Lane 1: positive control; lane 2: conditioned media; lane 3: cell lysate; lane 4: flow-through fraction #1 from purification; lane 5: flow-through fraction #2 from purification; lane 6: elution fraction #1 from purification; lane 7: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.3: CAHS HE CHO 3.2.8.1 Blot, Western blot showing H. exemplaris CAHS protein expression in a CHO cell line around 26 kDa. The band around 33 kDa is possibly a glycosylated version of the protein. Lane 1: positive control; lane 2: elution fraction #1 from purification; lane 3: elution fraction #2 from purification; lane 4: flow-through fraction #1 from purification; lane 5: flow-through fraction #2 from purification; lane 6: desalting waste; lane 7: concentrated conditioned media; lane 8: negative control conditioned media.
M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.4: CAHS HE HEK293T Blot, Western blot showing H. exemplaris CAHS protein expression in a HEK 293T cell line at 26 kDa, with banding between 25-35 kDa corresponding to post-translationally modified or aggregated versions of the protein of interest. Lane 1: positive control; lane 2: conditioned media; lane 3: cell lysate; lane 4: flow-through fraction #1 from purification; lane 5: flow-through fraction #2 from purification; lane 6: elution fraction #1 from purification; lane 7: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.5: CAHS RV CHO 3.2.8.1 Blot, Western blot showing R. varieornatus CAHS protein expression in a CHO LEC 3.2.8.1 cell line with banding around 26 kDa. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Fig 2.6:CAHS RV HEK293T Blot, Western blot showing R. varieornatus
CAHS protein expression in a HEK293T cell line with banding around 26 kDa.
Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.7:SAHS HE HEK293T Blot, Western blot showing inconclusive H. exemplaris SAHS protein expression in a HEK 293 cell line with an expected band size of 18 kDa. The HEK cell negative control blot also had prominent banding around 18 kDa. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.8: SAHS RV HEK293T Blot, Western blot showing inconclusive R. varieornatus SAHS protein expression in a HEK 293 cell line with an expected band size of 18 kDa. The HEK293T cell negative control blot also had prominent banding around 18 kDa. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification.
M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.9: MAHS HE HEK293T Blot, Western blot showing no expression of H. exemplaris MAHS protein in a HEK 293 cell line. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.10: Dsup HE HEK293T Blot, Western blot showing no expression of H. exemplaris Dsup protein in a HEK 293 cell line. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification.
M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific)
Figure 2.11: Dsup RV HEK293T Blot,
Western blot showing no expression of R. varieornatus Dsup protein in a HEK 293 cell line. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification.
M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.12: LEAM HE HEK293T Blot, Western blot showing no expression of H. exemplaris LEAM protein in a HEK 293 cell line. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification.
M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Figure 2.13: LEAM RV HEK293T Blot, Western blot showing no expression of R. varieornatus LEAM protein in a HEK 293 cell line. Lane 1: conditioned media; lane 2: cell lysate; lane 3: flow-through fraction #1 from purification; lane 4: flow-through fraction #2 from purification; lane 5: elution fraction #1 from purification; lane 6: elution fraction #2 from purification. M - molecular weight ladder in kDa, PageRuler Plus Prestained Protein Ladder, 26620 (Thermo Scientific).
Protection of mRNA by Purified Protein
Goal
To determine whether combining fluorescent protein mRNA with tardigrade proteins prevents or reduces temperature stress-induced mRNA degradation.
Results
We heat-stressed vaccine-buffered sfGFP mRNA in combination with R. varieornatus and H. exemplaris CAHS8 and SAHS2 proteins at -80°C, 4°C, 25°C, and 50°C.
Stress Test Aliquot Recipe
100ng sfGFP mRNA
2uL Vaccine Buffer
20U RNase Inhibitor
8.69ug protein
RNase inhibitor was added to each aliquot to prevent mRNA degradation caused by contaminating RNases, which had significantly degraded the mRNA in our initial round of stress testing.
Controls
Negative control: sfGFP mRNA was combined with purified protein from mock-transfected HEK293T cells and underwent each temperature stress condition.
Positive control: sfGFP mRNA alone in nuclease-free water underwent each temperature stress condition.
Initial degradation assays suggested that holding each temperature condition for 1 hour was insufficient time to see differences in degradation between experimental groups. We ran a degradation assay of mRNA in water and mRNA in imitation vaccine buffer, where each temperature condition was repeated at various time lengths to determine the optimal heat shock length.
The sfGFP mRNA has an expected band size of 600 bp and this was verified before starting each trial.
To view all results, please hover and click the arrow.
Intact mRNA Analysis for 1 hour Stress Test
Figure 1.1: Trial 1 for RT and 4C Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1-4 consists of the sfGFP mRNA mixture with proteins CAHS RV, CAHS HE, SAHS RV, and the negative control respectively, incubated at 4°C for 1 hour. Lane 5 is the positive control with only sfGFP mRNA and no protein. Lane 6 consists of the GeneRuler 1Kb+ DNA ladder. Lane 7-10 consists of the sfGFP mRNA mixture with proteins CAHS RV, SAHS HE, SAHS RV, and the negative control respectively, incubated at room temperature for 1 hour. Lane 6 is the positive control with no protein, incubated at room temperature for 1 hour.
Figure 1.2: Trial 2 for RT and 4C Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1 consists of the GeneRuler 1Kb+ DNA ladder. Lane 2 is the room temperature (positive) control that consists of sfGFP mRNA and no protein. Lanes 3-6 are the sfGFP mRNA mixture with the proteins CAHS RV, SAHS HE, SAHS RV, and negative control respectively, incubated at room temperature for 1 hour. Lane 8 is the positive control (no protein) incubated at 4°C for 1 hour. Lanes 9-12 are the sfGFP mRNA mixture with the proteins CAHS RV, SAHS HE, SAHS RV, and negative control respectively, incubated at 4°C for 1 hour.
Figure 1.3: Trial 3 for 4C Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1 consists of the GeneRuler 1Kb+ DNA ladder. Lane 2 is loaded with the sfGFP mRNA positive control with no added protein. Lanes 3-7 are the sfGFP mRNA solution with proteins CAHS HE, CAHS RV, SAHS HE, SAHS RV, and the negative control respectively, incubated at 4°C for 1 hour.
Figure 1.4: Trial 3 for Room Temperature(RT) Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1 consists of the room temperature negative control. Lanes 2-4 consist of sfGFP mRNA combined with the proteins SAHS RV, SAHS HE, CAHS HE respectively, incubated at room temperature for 1 hour. Lane 5 is the positive control with no protein, incubated at room temperature for 1 hour. Lane 6 is the GeneRuler 1Kb+ DNA ladder.
Figure 1.5: CAHS proteins (CAHS RV from Trial 3, CAHS HE from Trial 2) Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1 consists of the room temperature positive control with no protein. Lanes 2-4 consists of the sfGFP mRNA mixture combined with the proteins CAHS HE, CAHS RV, and the negative control respectively, incubated at room temperature for 1 hour. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lane 6 is the positive control with no protein incubated at 4°C. Lanes 7-9 consists of the sfGFP mRNA mixture combined with the proteins CAHS HE, CAHS RV, and the negative control respectively, incubated at 4°C for 1 hour.
Figure 1.6: Trial 3 for CAHS HE: Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in two temperature conditions for 1 hour. Lane 1 consists of the positive control with no protein, incubated at 4°C for 1 hour. Lane 2 is the sfGFP mRNA solution with protein CAHS HE. Lane 3 is the sfGFP mRNA mixture with the negative control. Lane 4 is the GeneRuler 1Kb+ DNA ladder. Lane 5 is the positive control incubated at room temperature for 1 hour. Lane 6 is the sfGFP mRNA mixture with protein CAHS HE that has been incubated at room temperature for 1 hour. Lane 7 is the sfGFP mRNA mixture with the negative control, incubated at room temperature for 1 hour.
Image aquisition and quantification:
mRNA degradation was assayed via agarose gel electrophoresis and intact mRNA was quantified using Fiji Software from the agarose gels.
To perform the quantifications, random background areas of the gel image were selected and measured for area and integrated density to obtain an average background density. Then, uniform areas were selected at the expected size for the band of interest across the gel and measured for area and integrated density. This was repeated with the smear area of the whole lane, and then areas of the bands and smears were used to calculate the background density in the selected area. This background correction value was subtracted from both the band and the smear measured integrated densities to obtain the true band and smear densities. Once the true values were obtained, the percentage of mRNA intact was calculated by dividing the true band density by the true smear density. To effectively compare the percentage of intact mRNA across trials, the positive control group was normalized to 100% and the calculated densities were normalized to the positive control group (no protein) in each gel.
To view all results, please hover and click the arrow.
Fig. 2: Percentage of intact mRNA after 1 hour incubation at room temperature and 4°C.
Statistical Analysis:
To analyze the differences in mRNA degradation for each protein, a statistical comparison of the mRNA integrity was conducted relative to the positive control. This was done using GraphPad Prism through a paired t-test between the mRNA percentages of each protein and the positive control. The paired t-test generated p-values for each comparison of a protein to the positive control, and the p-values were used to determine the significance of the difference between the intact mRNA of the positive control and the intact mRNA with the protein experimental condition.
Figure 3.1: Statistical Analysis for Intact mRNA after 1 hr at Room Temperature.
Figure 3.2: Statistical Analysis for Intact mRNA after 1 hr at 4°C.
Significance:
% mRNA Intact vs Control at 4°C
Protein
p-value
Significance
CAHS HE
0.1128
Not Significant
CAHS RV
0.0127
Significant
SAHS HE
0.0942
Not Significant
SAHS RV
0.0797
Not Significant
NC
0.3950
Not Significant
% mRNA Intact vs Control at Room Temperature
Protein
p-value
Significance
CAHS HE
0.1264
Not Significant
CAHS RV
0.1356
Not Significant
SAHS HE
0.0207
Significant
SAHS RV
0.0320
Significant
NC
0.2867
Not Significant
Discussion: At both room temperature and 4°C, the positive control (no protein) mRNA sample and the Negative Control mRNA sample demonstrate the highest amounts of mRNA intact overall after one hour. In contrast, mRNA samples containing one of the added proteins show a noticeable decrease in the percentage of intact mRNA after one hour. This loss of intact mRNA potentially implies RNase contamination-induced degradation or other mRNA-degrading catalytic properties across proteins. For both SAHS2 proteins, the statistical analysis shows that there is significant degradation at room temperature, which may be due to increased RNase activity in warmer temperatures. For the CAHS8 proteins, the degradation at room temperature is lower than the degradation at 4°C. This may be related to temperature-specific properties of CAHS8 as an intrinsically disordered protein or the optimal working temperature of the RNase inhibitor.
The results indicate that the mRNA itself is stable under the experimental conditions and that a longer heat shock duration is needed to quantifiably degrade the mRNA. In other words, the mRNA is not experiencing degradation for the proteins to protect against in the first place, rendering the results inconclusive.
Comparison of Duration of Incubation
Further optimizations of the stress test protocol were performed, including degradation assays of mRNA in water and mRNA in mock vaccine buffer(VB) where each temperature condition was repeated at various time lengths to determine an optimal heat shock length. The most pronounced differences in mRNA degradation occurred at and after 9 hours of heat shock.
Figure 4.1: 1 hour mRNA stress test. Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in various temperature conditions for 1 hour. Lanes 1-2 show sfGFP mRNA that has been incubated at -80°C for 1 hour. Lane 1 contains only sfGFP mRNA diluted in water, lane 2 consists of the sfGFP mRNA diluted in water alongside our mock vaccine buffer (VB). Lanes 3-4 show mRNA that has been incubated at 4°C for 1 hour. Lane 3 is the sfGFP mRNA, lane 4 is the sfGFP mRNA with the VB. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lanes 6-7 is the sfGFP mRNA that has been incubated at room temperature for 1 hour. Lane 6 is the sfGFP mRNA dilution, lane 7 is the sfGFP mRNA, water, and VB. Lanes 8-9 is the mRNA that has been incubated at 50°C for 1 hour. Lane 8 is the sfGFP mRNA, lane 9 is the sfGFP mRNA with VB.
Figure 4.2: 2 hour mRNA stress test. Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in various temperature conditions for 2 hours. Lanes 1-2 show sfGFP mRNA that has been incubated at -80°C for 2 hours. Lane 1 is sfGFP mRNA, lane 2 consists of the sfGFP mRNA alongside our mock vaccine buffer (VB). Lanes 3-4 show mRNA that has been incubated at 4°C for 2 hours. lane 3 is the bare sfGFP mRNA, lane 4 is the sfGFP mRNA with VB. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lanes 6-7 is the sfGFP mRNA that has been incubated at room temperature for 2 hours. Lane 6 is the bare sfGFP mRNA, lane 7 is the sfGFP mRNA with VB. Lanes 8-9 is the mRNA that has been incubated at 50°C for 2 hours. Lane 8 is the bare sfGFP mRNA, lane 9 is the sfGFP mRNA with VB.
Figure 4.3: 5 hour mRNA stress test. Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in various temperature conditions for 5 hours. Lanes 1-2 show sfGFP mRNA that has been incubated at -80°C for 5 hours. Lane 1 is sfGFP mRNA diluted in water, lane 2 consists of the sfGFP mRNA and water solution with added mock vaccine buffer (VB). Lanes 3-4 show mRNA that has been incubated at 4°C for 5 hours. lane 3 is the bare sfGFP mRNA, lane 4 is the sfGFP mRNA with VB. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lanes 6-7 is the sfGFP mRNA that has been incubated at room temperature for 5 hours. Lane 6 is the bare sfGFP mRNA, lane 7 is the sfGFP mRNA with VB. Lanes 8-9 is the mRNA that has been incubated at 50°C for 5 hours. Lane 8 is the bare sfGFP mRNA, lane 9 is the sfGFP mRNA with VB.
Figure 4.4: 9 hour mRNA stress test. Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in various temperature conditions for 9 hours. Lanes 1-2 show sfGFP mRNA that has been incubated at -80°C for 9 hours. Lane 1 is sfGFP mRNA diluted in water, lane 2 consists of the sfGFP mRNA and water solution with added mock vaccine buffer (VB). Lanes 3-4 show mRNA that has been incubated at 4°C for 9 hours. lane 3 is the bare sfGFP mRNA, lane 4 is the sfGFP mRNA with VB. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lanes 6-7 is the sfGFP mRNA that has been incubated at room temperature for 9 hours. Lane 6 is the bare sfGFP mRNA, lane 7 is the sfGFP mRNA with VB. Lane 8 is the bare sfGFP mRNA, lane 9 is the sfGFP mRNA with VB.
Figure 4.5: 12 hour mRNA stress test. Agarose gel electrophoresis (1% agarose gel) showing mRNA degradation after being incubated in various temperature conditions for 12 hours. Lanes 1-2 show sfGFP mRNA that has been incubated at -80°C for 12 hours. Lane 1 is bare sfGFP mRNA diluted in water, lane 2 consists of the sfGFP mRNA in water alongside our mock vaccine buffer (VB). Lanes 3-4 show mRNA that has been incubated at 4°C for 12 hours. lane 3 is the sfGFP mRNA in water, lane 4 is the sfGFP mRNA with VB. Lane 5 is the GeneRuler 1Kb+ DNA ladder. Lanes 6-7 is the sfGFP mRNA that has been incubated at room temperature for 12 hours. Lane 6 is the bare sfGFP mRNA in water, lane 7 is the sfGFP mRNA with VB. Lanes 8-9 is the mRNA that has been incubated at 50°C for 12 hours. Lane 8 is the bare sfGFP mRNA diluted in water, lane 9 is the diluted sfGFP mRNA with VB.
Figure 5.1: Percentage mRNA intact at -80°C
Figure 5.2: Percentage mRNA intact at 4°C
Figure 5.3: Percentage mRNA intact at Room Temperature(RT)
Figure 5.4: Percentage mRNA intact at 50°C
Discussion: The electrophoresis gels were used to quantify the percentage of mRNA intact on Fiji, as done in previous experiments. The obtained percentages of intact mRNA were normalized to the positive control (-80°C mRNA without the mock vaccine buffer) and then used to construct graphs comparing the percentage of intact mRNA over increasing incubation time periods. The mRNA samples incubated at room temperature had the highest intact percentages overall compared to the samples at -80°C. During longer incubation periods of nine and twelve hours, the 50°C mRNA samples start to appear visibly degraded on the gels and are supported by the graph. The addition of the mock vaccine buffer potentially has a stabilizing effect on the mRNA, particularly at higher temperatures seen through the increased percentages at room temperature and 50°C. Overall, since we did not see visible degradation temperatures other than 50°C, experiments with longer incubation time periods will be performed to determine when mRNA visibly degrades at lower temperatures.
The remaining 6 proteins will be used in stress tests upon purification and expression confirmation.
