iGEM 2024 — Team stanford

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# Wet Lab Notebook

English

Sorry, this browser does not support PDFs. Please view it at [here](https://static.igem.wiki/teams/5195/cloning-lab-notebook.pdf).

The Cloning Lab Notebook PDF outlines detailed protocols and experimental methods used in molecular cloning experiments. It includes step-by-step instructions on various techniques such as PCR, plasmid transformation, and DNA gel electrophoresis. This notebook is a comprehensive guide for lab members to follow and document experimental procedures.

# General Lab Notebook

English

Sorry, this browser does not support PDFs. Please view it at [here](https://static.igem.wiki/teams/5195/lab-notebook-stanford-igem-2024.pdf).

The General Lab Notebook PDF outlines detailed protocols and experimental methods used throughout our project. It also includes the people involved in the experiments as well as the dates.

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# Engineering

To test our hypothesis that shortened DUX4-DBD is capable of competitively inhibiting natural DUX4 binding to
DNA, the wet lab team made heavy use of the DBTL design cycle throughout our experimental efforts.

## DBTL cycles (wet lab)

### Cycle 1: Creating Initial Constructs (iDUX4-DBD, cDUX4-fl, and mScarlet DUX4 reporter)

**Design**
Prior to any wet lab experimentation, the first step of our project was to design the constructs we would use to
carry out our project. In our first round of cloning, we designed and created 3 plasmids: an inducible
mNG-P2A-DUX4-DBD plasmid, a constitutive DUX4-FL plasmid, and an mScarlet DUX4 reporter.

**imNG-P2A-DUX4-DBD**:
Upon obtaining a Tet-On doxycycline-inducible DUX4-fl plasmid (Addgene Plasmid #99281), we constructed a
DUX4-DBD plasmid by cloning out part of the DUX4-fl coding sequence and leaving only the first 217 amino acids
of DUX4. These 217 amino acids constitute the homeodomains and some of the C-terminal tail of the original
DUX4-fl protein, but lack the transcriptional activation domain. In order to be able to quantify the DUX4-DBD
protein in cells, we also inserted an mNeonGreen (mNG) sequence followed by a Peptide 2A (P2A) linker upstream
of the DUX4-DBD. The P2A linker causes ribosomal “skipping” during translation, and this effectively separates
the mNG and DUX4-DBD. In designing the coding sequence in this way, we enabled quantification of DUX4-DBD
without directly attaching another large protein to it.

**mTagBFP2-P2A DUX4-fl**:
We wanted to harness the inducible Tet-On system to more easily modulate the amount of DUX4-DBD produced, but
because we did not want to modulate the amount of DUX4-fl in a co-transfection, we also created a constitutive
version of the DUX4-fl from the original Tet-On inducible DUX4-fl plasmid we obtained from Addgene. We replaced
the original tight TRE promoter with an SV40 promoter and inserted an mTagBFP2 coding sequence followed by a P2A
linker downstream of the DUX4-fl coding sequence.

**mScarlet DUX4 reporter**:
We obtained a luciferase-based DUX4 reporter plasmid containing 6xDUX4 binding sites (pGL4-6X-DUX4-ffy) from the
Kyba Lab of the University of Minnesota Twin Cities. This construct produces firefly luciferase as its readout,
which requires an endpoint assay to quantify. In order to overcome this, we designed a reporter with mScarlet
fluorescent readout instead of luciferase, as this would allow us to skip the assay step, enable us to take
images of fluorescent readout at multiple time points throughout an experiment, and set up live cell fluorescent
imaging experiments.

#### Build

We first designed primers to cut out the fragments of interest for assembling our plasmids. Using PCR, we
amplified the fragments of the original DUX4-fl Addgene plasmid and the luciferase DUX4 reporter plasmid that we
obtained. After amplification, we ensured the purity and accuracy of the PCR fragments with gel purification,
quantification by Nanodrop, transformed into DH10B and DH5a cells, plating with antibiotic selection, and
finally, mini-prepping and sequencing (Plasmidsaurus).

For the imNG-P2A-DUX4-DBD plasmid, we first designed primers to amplify the entire plasmid except for the end of
the DUX4-fl coding sequence to exclude the transcriptional activation domain. Then, we amplified an mNG-P2A
fragment from an existing plasmid pK 381, provided to us by one of our mentors, Dr. Phillip Kyriakakis, from the
Stanford Coleman lab. The mNG-P2A fragment was then Gibson Assembled with the DUX4-DBD fragment to create our
imNG-P2A-DUX4-DBD plasmid.

To create the constitutive DUX4-fl plasmid, we amplified the original Addgene plasmid, but without the Tight TRE
promoter, and then Gibson assembled that fragment with a Geneblock containing an mTagBFP2-P2A sequence.

To create our DUX4 reporter construct, we amplified a backbone of the Kyba Lab reporter that excluded the
firefly luciferase fusion protein (fLuc Fusion) sequence. In parallel, we amplified an mScarlet sequence from
the plasmid pPK 381, which was provided to us by one of our mentors, Phil Kyriakakis, from the Stanford Coleman
Lab. After amplifying both fragments, we inserted the mScarlet sequence into the vector, pGL4-Amp(R)-6X-DUX4.
The resulting plasmid pGL4-DUX4-6X-UAS-mScarlet was subsequently utilized for nearly all other transfections as
our fluorophore reporter alternative.

The following schematic (from the 2023 Stanford iGEM team) describes our cloning methodology:

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-33-49-pm.png)

The below diagrams summarize our cloning efforts for our first round of cloning:

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-33-55-pm.png)

The key components of the final 3 plasmids we utilized for subsequent transfections are sketched below:

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-33-59-pm.png)

#### Test

We first needed to validate the functionality of our reporter constructs as it is critical to almost every
experiment we do from here. After that, we also used these initial constructs to test our hypothesis of
competitive inhibition.

To validate our reporter constructs, we transfected doxycycline-inducible DUX4-fl with DUX4 luciferase reporter
at different doxycycline concentrations. This was done in HEK293T cells, as they are an easily transfectable and
maintainable cell line.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-05-pm.png)

We transfected dox-inducible DUX4-fl with mScarlet DUX4 reporter to see if our new reporter construct reported
DUX4-fl activity accurately, and we also transfected imNG-P2A-DUX4-DBD by itself to check if it produces green
fluorescent protein. After observing that both our fluorescent reporter and DBD functioned as expected, we
conducted 4 repeat trials of transfection testing competition between DUX4 and DBD at different ratios.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-13-pm.png)

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-18-pm.png)

In these experiments, RFP served as the reporter readout, and GFP was the DBD fluorescence readout.

These experiments were all completed in HEK293T cells, which, though easy to work with, were not the most
representative of the environment our construct is targeting. Therefore, we repeated our experiments in C2C12
cells, which is a mouse muscle cell line. However, we noticed that our transfection efficiency became
significantly lower, leading us to optimize the Lipofectamine 3000 transfection protocol. After that, we were
able to conduct competitive inhibition experiments with C2C12 cells, yielding similar results to our HEK293T
experiments.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-22-pm.png)

To obtain higher quality data from our experiments, we used flow cytometry to view individual cells and their
fluorescence, which gave us a continuum of signals. The flow data supported our idea that as the DBD:FL ratio
increased, the corresponding reporter activity in those cells decreased.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-27-pm.png)

#### Learn

The reporter proved a critical part in verifying the competition between DUX4-FL and DUX4-DBD, offering a more
quantitative outlook. Both our luciferase reporter and mScarlet reporter functioned well, setting us up for
future experiments and validating our cloning and transfection techniques.

### Cycle 2: Creating DBD-KRAB

#### Design

After achieving successful DUX4 reporter knockdown with our DUX4-DBD construct, we explored fusing a repression
domain to the end of the DBD in order to further prevent the activation of any genes near the DUX4 binding
sites.

#### Build

Our KRAB repressor domain sequence was derived from AddGene plasmid pHAGE EF1α dCas9-KRAB, which was first
referenced as being an effective transcriptional repressor. We ordered the KRAB domain sequence as a gene block,
which we later Gibson Assembled with the backbone of the mNG-DBD 217 construct.

#### Test

Similar transfections to Cycle 1 have been done to test the competitive inhibition capabilities of our DBD-KRAB
construct. mNG-DBD-KRAB is transfected into HEK cells alongside full length and reporter fluorescence was
measured. DBD-KRAB consistently knocks down DUX4 fluorescent reporter activity at greater levels than DBD alone
(note the logarithmic scale).

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-32-pm.png)

DBD-KRAB appears to cause an exponential reporter knockdown, while DBD knockdown is linear. However, more
repeats and data points are needed to prove this hypothesis. (two repeat trials)

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-36-pm.png)

However, when the construct is tested in C2C12 cells, the inhibition trends are not as strong or apparent.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-40-pm.png)

#### Learn

From these transfections, we can conclude that KRAB can increase the inhibitive properties of DBD in HEK cells.
Though this still needs to be tested by more repeat trials and more intermediate data points. However, when it
comes to C2C12, KRAB seems to disrupt normal inhibition function. Since this was one trial, it could be human
error that led to this result. All together, more testing is needed to confirm KRAB functionality in muscle
adjacent systems, but the preliminary results in HEK cells are promising.

### Cycle 3: Creating NES Constructs (DBD-mScarlet-NES (designed but never built), NLS-DBD-mScarlet-NoLS-NES and for testing, DBD-GFP)

#### Design

In attempts to create a more “spreadable” DUX4-DBD that would enhance propagation of myoblasts and myofibers,
we planned to add an additional pair of NLS/NES to the plasmid. As a control, utilized as a way to visualize
migration differences between constructs, we made a constitutive DUX4-DBD fused directly to GFP. Finally, to
further assess the potency of a standalone NES in spreading DBD to distinct myonuclei, we planned to make a
DBD-mScarlet-NES construct; though we synthesized and ordered its parts, it was never cloned.

#### Build

We amplified the backbone of inducible mNG-DUX4-DBD, which excluded the fluorophore and DBD coding sequences.
For each backbone, we utilized different primers that would add overhangs and bind to their respective gene
fragments. The gene fragments, synthesized as gBlocks through IDT, consisted of NLS-DBD-mScarlet-NES, DBD-GFP,
and DBD-mScarlet-NES. During assembly, we inserted the fragments at a 3:1 insert to vector ratio, due to initial
difficulty growing isolated bacterial colonies with the fragments integrated into their sequence. Furthermore,
transformation via electroporation proved ineffective due to lack of bacterial growth on plates. Subsequently,
we transitioned to high-efficiency heat shock, eventually yielding our desired constructs.

#### Test

We designed and carried out an initial live-cell imaging experiment in which we co-transfected C2C12 cells with
DBD-GFP and NLS-DBD-mScarlet-NES, then lifted them and added them to a denser well plate of C2C12s and
differentiated them. We live-imaged the cells for 48 hours.

#### Learn

The live-cell imaging experiment was inconclusive because the fluorescent protein signal did not appear to move
much beyond individual cells. This is likely due to the imaging time frame being too short - imaging for 5-7
days would provide better visualization of C2C12 muscle cell fusion. We hope to carry out this experiment in the
future and observe results in more fully-formed myotubes.

### Cycle 4: reporter cell line and inducible full length cell line

#### Design

In order to achieve more accurate and precise results from our C2C12 transfections, we created a C2C12 cell
line stably expressing our mScarlet DUX4 reporter. With this cell line, all of the cells in a culture would
contain the reporter and therefore be a better experimental model than transfecting transiently, since not all
cells are transfected in that method. We also hoped to eventually use this cell line for live imaging of cell
fusion, as it would enable us to visualize reporter expression and DUX4 repression on the nuclear level.

#### Build

We transfected the DUX4 reporter into C2C12 cells, then added hygromycin as the DUX4 reporter plasmid contained
a hygromycin resistance gene, so this allowed us to select for the cells that got transfected and where the DUX4
reporter DNA was integrated into the cells’ genome. We continuously selected and cultured cells for 14 days,
until the control well with no DNA added died off while small colonies of the reporter-containing cells
remained.

#### Test

Next, we grew up the small remaining colonies and transfected them with DUX4-fl to test if the reporter could
successfully be activated. The below figure shows that the reporter was successfully activated, but its
expression level is not optimal. This may also be because of low Dux4-fl transfection efficiency.

![](https://static.igem.wiki/teams/5195/engineering/screenshot-2024-09-28-at-3-34-47-pm.png)

#### Learn

Transfecting the DUX4 reporter cell line with FL showed activation of DUX4 reporter, but we hope to further
increase its level of activation. We also created a DUX4 reporter + inducible DUX4-fl cell line, but have yet to
test its efficacy. In the future, we hope to generate stable cell lines through the use of lentiviral vectors.

### Cloning Overview

**Cloning Subsection**
Stanford iGEM 2024
To develop our cloning constructs, we followed a deliberate process of creating our plasmid vectors, connecting
fragments, and supplying exogenous DNA for bacterial cells.

For our entire cloning efforts, they consisted of resuspending primers and DNA fragments, then utilizing them to
PCR template backbones or add overhangs to linearized fragments. From there, we ran gel electrophoresis to
validate PCR products, imaged, and purified through gel extraction. The subsequent steps included Gibson
Assembly, electroporation, liquid culture, and mini-prep, yielding transfectable stocks of cloned DNA
constructs.

Through our initial tet-inducible DUX4-FL plasmid, AddGene pCW57.1-DUX4-CA, we yielded a multitude of
constructs. We made a constitutive BFP-P2A-DUX4-FL, in which an additional fluorophore mTAG-BFP2 fused to a P2A
linker was inserted into the backbone, preceding the DUX4-FL coding sequence. The fluorophore was attached in
attempts to view the binding of FL to the reporter.

From the aforementioned tet-inducible FL, we produced a doxycycline inducible DUX4-DBD, essentially removing the
C-terminal, including the transcription-activating domain. Building off of the inducible DUX4-DBD, we also added
an mNeonGreen protein attached to a P2A linker for fluorescent readout. The use of a P2A linker allowed us to
quantify the presence of our DUX4-DBD in a way that didn’t require the fluorescent protein to be directly fused
to it. In this manner, we were also able to visualize the competitive inhibition between both DUX4 isoforms.

We also designed a construct in which we only used the two homeodomains of the DBD, 157 amino acids. Initially,
our design included 217 amino acids to maintain most of the original DUX4-FL sequence, though we shortened its
length to test the effect of the extra C-terminal region.

Using the same DUX4-DBD backbone, we made three additional constructs, the first of those being Myospreader DBD.
In this case, we had attached an additional SV40 NLS on the N-terminus, an Alyref NES and HIV rev NoLS on the
C-terminus, and fused with a fluorescent mScarlet protein. We intended for the addition of an NLS/NES pair to
facilitate the propagation of DBD into more distant and scattered myonuclei.

As a control, we also made a constitutive DBD-GFP, where DBD was directly bound to GFP, in order to co-transfect
these and visualize migration differences. We also made a version of DBD with an attached repressor domain,
Krüppler-associated box (KRAB), to yield our DUX4-DBD-KRAB construct. We intended for the additional KRAB domain
to efficiently repress both DUX4-FL and its target genes in FSHD myocytes, targeting the promoter or exon1 of
DUX4.

We initially utilized a previously developed DUX4 reporter which featured 6 DUX4 binding sites of known DUX4
binding motifs upstream of a luciferase luminescent protein. The process proved somewhat inefficient, and we
decided to rely on fluorescent readouts instead. To obtain this construct, we replaced the luciferase sequence
with fluorescent mScarlet, yielding the plasmid 6x-DUX4-UAS-mScarlet.

And ultimately, utilizing the plasmid pAAV EF1A-eGFP-WPRE-hGHpA as a backbone, we cloned the plasmid
pAAV-EF1a-DBD-eGFP, which contains our desired cargo and is ready for transduction to create our AAV
therapeutic.

**References:**
[1] Poukalov, K. K., Valero, M. C., Muscato, D. R., Adams, L. M., Chun, H., Lee, Y. il, Andrade, N. S., Zeier,
Z., Sweeney, H. L., & Wang, E. T. (2023). Myospreader Improves Gene Editing in Skeletal Muscle by Myonuclear
Propagation. https://doi.org/10.1101/2023.11.06.565807
[2] Himeda, C. L., Jones, T. I., Virbasius, C.-M., Zhu, L. J., Green, M. R., & Jones, P. L. (2018).
Identification of epigenetic regulators of DUX4-FL for targeted therapy of facioscapulohumeral muscular
dystrophy. Molecular Therapy, 26(7), 1797–1807. https://doi.org/10.1016/j.ymthe.2018.04.019

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# Experiment Overview

English

Sorry, this browser does not support PDFs. Please view it at [here](https://static.igem.wiki/teams/5195/experiments.pdf).

This PDF contains the experiment overviews. It outlines the key protocols, steps, and methodologies followed during the project. The PDF describes in detail the experiments, providing step-by-step instructions for replicating the procedures and ensuring accurate results.

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Genetic Therapy Testing

DUX4-DBD Suppression in HEK293T Cells

Flow Cytometry Data

C2C12 Cell Line Analysis

Introduction of KRAB to DUX4-DBD

Future Plans

# Genetic Therapy Testing

## DUX4-DBD Suppression in HEK293T Cells

We tested multiple versions of our genetic therapy in both C2C12 and HEK293T cells. Our tests involved co-transfecting cells with a reporter plasmid, DUX4-fl plasmid, and DUX4-DBD plasmid for competitive inhibition of DUX4-fl.
The reporter produced fluorescent proteins at a rate correlated with DUX4-fl binding. As DUX4-DBD concentration increased, DUX4-fl binding and reporter activation decreased across trials.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-12-59-52-pm.png)

Figure 1: The mScarlet fluorescence from the DUX4 reporter decreases with increased ratios of DUX4-DBD to DUX4-fl when co-transfected, showing suppressed DUX4-fl binding to the DUX4 reporter’s binding sites.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-1-00-09-pm.png)

Figure 2: RFP decreases with decreased activation of the reporter by DUX4-fl while GFP increases with the amount of DUX4-DBD transfected.

## Flow Cytometry Data

We performed flow cytometry on HEK293T cells to quantify single-cell fluorescent protein expression. As DUX4-DBD concentrations rose, mScarlet reporter output decreased while GFP expression increased.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-1-00-21-pm.png)

Figure 3: B8-.5xfl
C1-.5xfl, .5xDBD
C6-.5xfl, 5x DBD,
C12, .5xfl, 12.5 DBD
Green name: 25x DBD:FL ratio
Orange name: 10x DBD:FL ratio
Red name: 1x DBD:FL ratio
Blue name: 0x DBD:FL ratio

## C2C12 Cell Line Analysis

A similar trend was observed in C2C12 cells, although transfection efficiency was lower. Fluorescence assays confirmed DUX4-DBD-mediated suppression in these cells, aligning with results from HEK293T assays.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-1-00-38-pm.png)

Figure 4: The mScarlet fluorescence decreases with decreased activation of the reporter by DUX4-fl.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-1-00-45-pm.png)

Figure 5: RFP decreases with decreased activation of the reporter by DUX4-fl while GFP increases with the amount of DUX4-DBD transfected.

## Introduction of KRAB to DUX4-DBD

We modified our DUX4-DBD plasmid by adding the KRAB repressor domain, which recruits chromatin-modifying proteins. In HEK293T cells, the DBD-KRAB construct further suppressed reporter activation compared to DUX4-DBD alone.

![](https://static.igem.wiki/teams/5195/results/screenshot-2024-09-28-at-1-00-55-pm.png)

Figure 6: The mScarlet fluorescence decreases with decreased activation of the reporter by DUX4-fl. The DUX4-DBD + Krab construct represses reporter activation via DUX4-fl at a higher rate than the DUX4-DBD construct.

## Future Plans

Post Wiki freeze, we aim to validate DUX4-DBD+KRAB in C2C12 cells with additional technical replicates. We also plan to test the construct in FSHD patient-derived cell lines and in vivo models using AAV gene therapy.

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