iGEM 2025 — Team utdallas

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of our team's progress throughout the season.{% endblock %}
{# Absolute base to avoid relative-path issues on iGEM renderer #}
{% set BASE = 'https://static.igem.wiki/teams/5774/wiki1/notebook/' %}
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# Lab Notebook

This serves as a chronological record of our team's progress throughout the season.

**Months**

---

* Wet Lab
* [April](#wet-april)
* [May](#wet-may)
* [June](#wet-june)
* [July](#wet-july)
* [August](#wet-august)
* [September](#wet-september)
* Dry Lab
* [Calendar](#dry-calendar)

Expand all
Collapse all

* Wet Lab
* Dry Lab

## April 2025

## April 1 – April 6

##### First run of plasmid designing

designIDPs

Determined 5 classes of IDPs and 2 species. Assigned UniProt sequence review and gBlock insert prep.

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27april-1.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27april-1.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27april-1.pdf%27%20%7D%7D).

## April 7 – April 13

##### Gene & primer design

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27april-7-april-13.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27april-7-april-13.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27april-7-april-13.pdf%27%20%7D%7D).

## April 14 – April 20

##### Codon optimization & ordering

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27april-14.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27april-14.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27april-14.pdf%27%20%7D%7D).

## May 2025

## May 27

##### Cloning kickoff

**PDF**

[Open](%7B%7B%20BASE%20~%20%27may-27.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27may-27.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27may-27.pdf%27%20%7D%7D).

## June 2025

## June 2 – June 8

##### Cloning continued

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27june-2.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27june-2.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27june-2.pdf%27%20%7D%7D).

## June 9 – June 15

##### Cloning + sequencing

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27june-9.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27june-9.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27june-9.pdf%27%20%7D%7D).

## June 16 – June 22

##### CHO line started

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27june-16-protocol.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27june-16-protocol.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27june-16-protocol.pdf%27%20%7D%7D).

## June 23 – June 29

##### Cell work & lysis

**Week PDF**

[Open](%7B%7B%20BASE%20~%20%27june-23.pdf%27%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20%27june-23.pdf%27%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20%27june-23.pdf%27%20%7D%7D).

## July 2025

{% set J = [
('July 1 – July 6','july-1.pdf'),
('July 7 – July 13','july-7.pdf'),
('July 14 – July 20','july-14.pdf'),
('July 21 – July 27','july-21.pdf'),
('July 28 – July 31','july-28.pdf')
] %}
{% for label, file in J %}

## {{ label }}

##### Week notes

**Week PDF**

[Open](%7B%7B%20BASE%20~%20file%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20file%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20file%20%7D%7D).

{% endfor %}

## August 2025

{% set A = [
('August 3 – August 10','aug-3.pdf'),
('August 11 – August 17','aug-11.pdf'),
('August 18 – August 24','aug1824.pdf')
] %}
{% for label, file in A %}

## {{ label }}

##### Week notes

**Week PDF**

[Open](%7B%7B%20BASE%20~%20file%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20file%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20file%20%7D%7D).

{% endfor %}

## September 2025

{% set S = [
('September 1 – September 7','sep-1.pdf'),
('September 8 – September 14','sep-8.pdf'),
('September 15 – September 21','sep-15.pdf'),
('September 22 – September 28','sep-22.pdf'),
('September 29 – September 30','sep-29.pdf')
] %}
{% for label, file in S %}

## {{ label }}

##### Week notes

**Week PDF**

[Open](%7B%7B%20BASE%20~%20file%20%7D%7D)
[Download](%7B%7B%20BASE%20~%20file%20%7D%7D)

PDF failed to load. [Open in a new tab](%7B%7B%20BASE%20~%20file%20%7D%7D).

{% endfor %}

2025

June 2025

‹
›

### Select a date

{% endblock %}

## 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](#pane-cloning)
* [Protein Expression](#pane-expression)
* [Protein Purification](#pane-purification)
* [Degradation Assays](#pane-assays)

## 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.

![](https://static.igem.wiki/teams/5774/wiki1/engineering/engineering-protein-expression-initial-protocol.avif)

### 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.

![](https://static.igem.wiki/teams/5774/wiki1/engineering/engineering-protein-expression-revised-protocol.avif)

### 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.

![](https://static.igem.wiki/teams/5774/wiki1/engineering/piggybac-sahs2-he-plasmid-map.avif)

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.

![](https://static.igem.wiki/teams/5774/wiki1/results/pb1.avif)

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).

![](https://static.igem.wiki/teams/5774/wiki1/results/pb2.avif)

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*

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![Gallery 8 Figure 1](https://static.igem.wiki/teams/5774/wiki1/engineering/cahs-he.avif)
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Figure 1.1: CAHS8 HE

Figure 1.2: CAHS8 RV

Figure 1.3: SAHS2 HE

Figure 1.4: SAHS2 RV

Figure 1.5: Dsup HE

Figure 1.6: Dsup RV

Figure 1.7: MAHS HE

Figure 1.8: MAHS RV

Figure 1.9: LEA HE

Figure 1.10: LEAM RV

### *1:1 mRNA to protein ratio*

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| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| CAHS8 HE | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 1: CAHS8 HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| CAHS8 RV | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 2: CAHS8 RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| SAHS2 HE | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 3: SAHS2 HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| SAHS2 RV | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 4: SAHS2 RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| Dsup HE | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 5: Dsup HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| Dsup RV | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 6: Dsup RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| MAHS HE | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 7: MAHS HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| MAHS RV | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 8: MAHS RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| LEA HE | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 9: LEA HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| LEAM RV | 1 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 10: LEAM RV

### *1:5 mRNA to protein Ratio*

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| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| CAHS8 HE | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 1: CAHS8 HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| CAHS8 RV | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 2: CAHS8 RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| SAHS2 HE | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 3: SAHS2 HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| SAHS2 RV | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 4: SAHS2 RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| Dsup HE | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 5: Dsup HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| Dsup RV | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 6: Dsup RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| MAHS HE | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 7: MAHS HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| MAHS RV | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 8: MAHS RV

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| LEA HE | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 9: LEA HE

| Molecule | Quantity |
| --- | --- |
| sfCherry3C mRNA | 1 |
| LEAM RV | 5 |
| K+ | 25 |
| Na+ | 25 |
| Cl- | 25 |

Figure 10: LEAM RV

### 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.

![](https://static.igem.wiki/teams/5774/wiki1/engineering/deg2.avif)

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) |

![](https://static.igem.wiki/teams/5774/wiki1/engineering/deg-mrna.avif)

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:

1. The optimal condition for observing a clear protective effect is an incubation of 9+ hours at 50°C.
2. 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.

{% extends "layout.html" %}
{% block page\_content %}

## Protocols

#

---

### Cloning Protocols

[PCR from G-blocks](https://static.igem.wiki/teams/5774/wiki1/protocols/pcr-from-g-blocks.pdf)
[Agarose Gel (Cast, Load, Image)](https://static.igem.wiki/teams/5774/wiki1/protocols/agarose-protocols.pdf)
[Gel Purification](https://static.igem.wiki/teams/5774/wiki1/protocols/gel-purification.pdf)
[Restriction Digestion](https://static.igem.wiki/teams/5774/wiki1/protocols/restriction-digestion.pdf)
[PCR Purification](https://static.igem.wiki/teams/5774/wiki1/protocols/pcr-purification.pdf)
[Ligation](https://static.igem.wiki/teams/5774/wiki1/protocols/ligation.pdf)
[Transformation](https://static.igem.wiki/teams/5774/wiki1/protocols/transformation.pdf)

[Bacterial Inoculation](https://static.igem.wiki/teams/5774/wiki1/protocols/bacteria-inoculation.pdf)
[Miniprep](https://static.igem.wiki/teams/5774/wiki1/protocols/miniprep.pdf)

### Expression Protocols

[Cell Culture (Passaging and Seeding)](https://static.igem.wiki/teams/5774/wiki1/protocols/passaging-and-seeding.pdf)
[Freezing and Thawing Cells](/https%3A//static.igem.wiki/teams/5774/wiki1/protocols/freezing-and-thawing.pdf)
[Transfection](https://static.igem.wiki/teams/5774/wiki1/protocols/transfection.pdf)
[Media Collection and Lysis](https://static.igem.wiki/teams/5774/wiki1/protocols/cell-lysis-and-media-collection.pdf)

### Purification Protocols

[Protein Purification](https://static.igem.wiki/teams/5774/wiki1/protocols/protein-purification.pdf)
[Western Blots](https://static.igem.wiki/teams/5774/wiki1/protocols/western-blot.pdf)

### mRNA Degradation Assay Protocols

[In Vitro Transcription](https://static.igem.wiki/teams/5774/wiki1/protocols/ivt.pdf)

[mRNA Stress Tests](https://static.igem.wiki/teams/5774/wiki1/protocols/stress-test.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

## Results

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](https://2025.igem.wiki/utdallas/contribution)> .

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.

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**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:

1. The protein was found almost exclusively in the cell lysate, indicating that while the proteins were expressed, they were not efficiently secreted.
2. 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.

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**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

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**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.

![](https://static.igem.wiki/teams/5774/wiki1/combined.avif)

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.

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**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.

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**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.

![](https://static.igem.wiki/teams/5774/wiki1/results/gels/fr.avif)

**Figure 5.1: Percentage mRNA intact at -80°C**

![](https://static.igem.wiki/teams/5774/wiki1/results/gels/done.avif)

**Figure 5.2: Percentage mRNA intact at 4°C**

![](https://static.igem.wiki/teams/5774/wiki1/results/gels/rt-new.avif)

**Figure 5.3: Percentage mRNA intact at Room Temperature(RT)**

![](https://static.igem.wiki/teams/5774/wiki1/results/gels/50-new.avif)

**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.

{% endblock %}