iGEM 2025 — Team westlake


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{% block title %}Engineering{% endblock %}
{% block lead %}"I have not failed. I've just found 10,000 ways that won't work." - Thomas Edison{% endblock %}
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**Content**

* [Introduction](#intro)
* [Design](#design)
* [Build](#build)
* [Test](#test)
* [Learn](#learn)

## Engineering Success

---

As Thomas Edison once said, "I have not failed. I've just found 10,000 ways that won't work." And this year,
the Westlake iGEM team has found 10,000 ways to not incorporate a CRISPRa system in vitro. However, true to the
spirit of Edison's quote, we're not just here to present why and how we failed, but also what we learned.

### D is for Design

---

Design. The first part of the iGEM Engineering Success Cycle. A phase where everything looks perfect on paper,
until you try to make it real. This was where our journey began. Our goal was simple enough: build a construct
that could be delivered into cells in vitro and turn on antiviral protection when needed. Easy, right?

Inspired by existing gene therapy delivery systems, and advice from our Westlake professors, we developed a
CRISPRa/TetON fusion system. By combining the two, we could fine-tune gene expression that promised quick
activation when needed and silence when not, while avoiding the risk of leaky expression.

The system comprised three key components: dCas9-VP64 (the transcriptional activator), sgRNAs (our
gene-targeting guides), and rtTA (the switch for the TetON promoter system). Simple ingredients. Complicated
recipe. Together, they made up nearly 1700 amino acids, not even counting the three 100 nt sgRNAs. Delivering
all that into a single cell would be no easy feat.

This was the first of many hurdles to come, but eventually, we decided to bite the bullet and made the decision
to split the system: three separate parts, one for each major component. While it wasn’t perfect, this kind of
setup meant more flexibility and control, letting us test individual and combined sgRNAs and explore how
different ratios affected activation. The downside? Fewer cells would get the full trio, gene expression might
vary more, and the cells would be put under serious metabolic strain. Regardless, every design starts as a
compromise. What mattered was that we now had a clear direction for the next step of our journey: the Build
Phase.

### B is for Build

---

Of all the steps in our project, the Build Phase seemed like it would be the easiest, just basic
bioengineering. Each component had its purpose, and all we had to do was assemble them according to the
underlying mechanism. The TetON system operates through Doxycycline (Dox), which activates the rtTA protein.
Once active, rtTA binds to a promoter known as TRE3G, enabling transcription of any downstream gene. This setup
meant two important things: first, rtTA had to be expressed before Dox was added, and second, at least one of
our other constructs needed to be under the control of a TRE3G promoter for inducibility.

As such, we built our system as follows. First, a plasmid containing rtTA, driven by an EF-1α promoter to
ensure consistent expression and easy delivery. Then, a second plasmid containing dCas9-VP64, where we swapped
the CMV promoter for TRE3G, making it Dox-inducible. And finally, the sgRNAs, delivered not as plasmids but as
2'-O-methylated ssRNA, to reduce metabolic burden and simplify delivery.

For this, we turned to lipid nanoparticles (LNPs), and after combing through literature, we settled on fairly
simple composition, just DOTAP, a cationic lipid that complexes with nucleic acids, and cholesterol, to
stabilize the particle. Previous studies had shown this mixture to be efficient for both mRNA and pDNA delivery,
making it an ideal choice for our hybrid system.

With the plan in place, the workflow was straightforward, and for a brief, shining moment, it seemed like
smooth sailing. We ordered the vectors, made the edits, and did simple cloning and isolation of the samples.
What happened next, dear reader, was the problem.

![dCas9 vector map](https://static.igem.wiki/teams/6014/icon/vector-dcas9.avif)

Image of our dCas9-VP64 + TRE3G vector.

### T is for Test

---

To see if our LNPs actually worked, we started simple: an EYFP plasmid as a reporter for gene delivery
efficiency. This was our first challenge. The paper we’d modeled our LNPs on reported robust gene expression
even after 48 hours, but in our hands? Barely a flicker after 24 hours. Three separate transfections later, and
our EYFP signal remained stubbornly minimal. It was a crushing contradiction to the goal of our system. Still,
we held out hope: perhaps the problem was specific to EYFP, and our real construct would behave differently.

Armed with our understanding of hydrogel, LNP, and pDNA ratios, we moved on to test the actual system. We
transfected cells and applied Dox induction across three concentrations and four 12-hour intervals, aiming to
build a comprehensive map of how timing and dosage affected expression. The results? Uniform disappointment.
dCas9 expression remained almost completely absent across all conditions. Western blotting confirmed that,
despite a clear GAPDH reference signal in some blots, dCas9 was barely detectable. The experiment, as far as
protein induction went, had failed. As a consequence, we were unable to even begin work with sgRNA testing,
limiting our ability to effectively characterize the system in its full potential.

![Western blot results for dCas9 expression](https://static.igem.wiki/teams/6014/icon/result/3.avif)

Figure 1: The image above shows the four different plates transfected at
various Dox timings. Each plate followed the same layout A—L only varying in Dox timing. A—D were all at a Dox
concentration of 200 ng/mL, E—H at 500 ng/mL, and I—L at 1000 ng/mL. Columns A, E, and I possessed a 0.5:1
ratio of plasmid, B, F, and J had a 1:1 ratio, C, G, and K had a 1.5:1 ratio, and D, H, and L had a 0:1 ratio.
The membrane was split into two halves for each protein (GAPDH at ~35 kDa and dCas9-VP64 at ~170 kDa).
Resultant chemiluminescent blots are shown above.

![Flow Cytometry Report of Specimen 1](https://static.igem.wiki/teams/6014/icon/result/2.avif)

### L is for Learn

---

So, what did we learn from all of this? Well, first and foremost, the major takeaway from this protocol is that
our system is extremely bulky and hard to deliver. Although this was expected, we severely underestimated the
extent to which is would inhibit our protein expression. Secondly, we learned that something in our system needs
to change, most likely with Dox addition. Although it will usually take around 24 hours for expression to fully
kick in, we should still be seeing some minimal expression at the 12-hour mark, but despite a clearly defined
GAPDH band, we lacked any dCas9 expression. As such, we need to run some more thorough experiments in order to
characterize our protein expression system, testing rtTA levels and dCas9 levels in a more refined way to
examine the root cause of our issue.

Thirdly, and finally, we considered that with the difficulties of adding even just two plasmids, perhaps it
would be better to create a barebones plasmid, synthesized almost fully from scratch, allowing us to pack the
rtTA and dCas9 together. While this does go against our initial decision to split the system, we theorize that
it may be necessary for us in order to achieve effective gene expression. However, the biggest take away is that
our work lays the foundation for other groups to look into a fusion system like this, and further evaluate if it
has the potential to become a genuine therapeutic.

**Link to Part Registry Collection:** <https://registry.igem.org/collections/df8d0b8e-5baa-4573-986d-c8e995ada034>

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{% block title %}Results{% endblock %}
{% block lead %}This page documents the results of our key experiments, including transfection efficiency validation and
protein expression analysis.{% endblock %}
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**Content**

* [EYFP Flow Cytometry Results](#flow-cytometry)
* [Western Blot Results](#western-blot)

## EYFP Flow Cytometry Results

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![Flow Cytometry Report of Specimen 1](https://static.igem.wiki/teams/6014/icon/result/2.avif)

![LNP Volume vs EYFP Delivery Efficiency](https://static.igem.wiki/teams/6014/icon/result/1.avif)

##### Data Summary

| Sample # | LNP (μL) | % FITC-H | Total Count |
| --- | --- | --- | --- |
| 1 | 0 | 0.19 | 11295 |
| 2 | 5 | 0.69 | 11315 |
| 3 | 10 | 6.33 | 10732 |
| 4 | 20 | 14.76 | 8075 |
| 5 | 30 | 34.34 | 7195 |
| 6 | 50 | 32.20 | 8769 |

##### Analysis

The data above shows the results of our Flow Cytometry Data from transfections with EYFP plasmid in order to
test the efficiency of our Hydrogel-LNP Matrix. Total concentration of Hydrogel at a constant 0.2% per the
manufacturer instruction, and LNP volume was varied according to experimental plan. In total, we tested a set of
six volumes ranging from 0 μL to 50 μL. After incubation, cells were then lifted and suspended in PBS before
measuring the number of cells that showed a measure of fluorescence intensity. According to the data, increasing
the volume of LNP solution used directly correlated to an increase in the transfection efficiency, with our
highest candidate being 30 μL at 34.34% (which we used in all subsequent transfections). The efficiency then
seemed to decrease or plateau, although we were unsure which. Interestingly, the total viable cell count taken
by the machine varied (12000 total events per sample), decreasing until the 30 μL mark before increasing again.
This suggests that the LNPs may induce some form of strain on the cell when delivering the gene, and thus,
further experiments should be conducted to determine whether or not the LNPs have any measurable cytotoxic
effect.

## Western Blot Results

---

![Western Blot results for dCas9 expression](https://static.igem.wiki/teams/6014/icon/result/3.avif)

Figure 1: The image above shows the four different plates transfected at
various Dox timings. Each plate followed the same layout A—L only varying in Dox timing. A—D were all at a Dox
concentration of 200 ng/mL, E—H at 500 ng/mL, and I—L at 1000 ng/mL. Columns A, E, and I possessed a 0.5:1
ratio of plasmid, B, F, and J had a 1:1 ratio, C, G, and K had a 1.5:1 ratio, and D, H, and L had a 0:1 ratio.
The membrane was split into two halves for each protein (GAPDH at ~35 kDa and dCas9-VP64 at ~170 kDa).
Resultant chemiluminescent blots are shown above.

##### Analysis

The WB above shows the four different plates transfected at various Dox timings. Each plate following the same
layout as follows: A—D were all at a Dox concentration of 200 ng/mL, E—H at 500 ng/mL, and I—L at 1000 ng/mL.
Columns A, E, and I possessed a ratio of 0.5 : 1 (for rtTA to dCas9), Columns B, F, and J had a 1 : 1 ratio,
Columns C, G, and K had a 1.5 : 1 ratio, and lastly Columns D, H, and L had a 0 : 1 ratio (Cntrl). The membrane
was split in half for each protein (GAPDH at ~35 kDa and dCas9-VP64 at ~ 170kDa). The above is after
chemiluminescent HRP activation soln. was added to the blots, and imaged under UV light.

As shown in the data, the weak GAPDH signals completely invalidate the experimental soundness, necessitating a
second round of work to be done. Although faint signals can be detected at a high mass, we determined that the
signals present were around 200 kDa, much too large to be the dCas9-VP64 complex. This was further supported by
the presence of protein in columns D/H/L, which lacked the rtTA plasmid (and should therefore be unable to
induce dCas9 translation). Ultimately, the data is poor and inconclusive, and needs further revision (as of
October 8th, 2025) to draw any sort of conclusions about the nature of the pTRE3G-dCas9-VP64-3xFLAG (NTD)
plasmid.

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