iGEM 2024 — Team dtu-denmark

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# Notebook

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Our notebook contains chronological notes of what our team has been doing in the lab along with who was responsible for each task. It is uploaded as a PDF below.

Your browser does not support PDFs. Please download the PDF to view it: [Download PDF](https://static.igem.wiki/teams/5107/document/wiki-colors.pdf).

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# Engineering Success

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We successfully developed an **in-cell biosensor** based on multiple different human receptors (Cycle
1). Additionally, we constructed a proof of concept for the **cell-free biosensor called EndoSense**
(Cycle 2) and attempted to **produce the receptors** ourselves (Cycle 3). We overcame many challenges
and uncertainties by adhering to three engineering cycles.

# Engineering Success

With EndoSense, we wanted to develop a broad range cell-free biosensor for measuring the influence of
endocrine-disrupting compounds (EDCs) on human hormone receptors. The initial idea was a system based on
human endocrine receptors functioning as allosteric
transcription factors (aTFs). Such aTFs should bind to a receptor-binding element (RE) on a DNA strand,
thereby regulating the transcription of the DNA placed downstream. Upon binding of EDCs, the affinity of the
receptor for the RE is altered, resulting in a change in transcription. By using a part with a measurable
output, this change in transcription can be used to determine the presence or absence of EDCs in your
sample.

We started off the project with a couple of engineering challenges, summarized here:

* The human endocrine receptors: How do we obtain functional and stable eukaryotic receptors?
* The interaction between receptor and DNA: How do we ensure high enough affinity of the receptor for the
  receptor response elements?
* The measurable read-out: How do we get a fast and reliable signal?

We decided to start addressing the engineering challenges in this order and went through multiple cycles to
repeatedly improve upon our design. Some questions were partly answered by our stakeholders on our [Integrated Human Practice](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) page, while other
questions were analyzed through the engineering cycles.

![eng-cylce-overview](https://static.igem.wiki/teams/5107/eng-cycles1.webp)

Cycle
1: In-cell assay
Cycle 2: Cell-Free
Biosensor
Cycle 3: Receptor
Production

* [Engineering success](#Engineering-success)
* [Cycle 1: In-cell Assay](#cycle1)
* [Cycle 2:
  Cell-free Biosensor](#cycle2)
* [Cycle 3:
  Receptor Production](#cycle3)

* [Design](#design1)
* [Build](#build1)
* [Test](#test1)
* [Learn](#learn1)

# Cycle 1: In-cell system

While the final aim of the project was to develop a cell-free biosensor, multiple stakeholders
recommended that we start by trying to create an in-cell system as described in our Integrated Human
Practice page. This was done to test some important aspects of the system. Additionally, another aim of
this engineering cycle was to create a **system for multiple different receptors**. This is
necessary to broaden the range of detectable compounds.

## Design

### Design - selection of receptors

The first step was to identify interesting and functional receptors along with the EDCs that can be
sensed by the receptors from a literature search. This step proved very difficult and required us to
read many articles relating EDCs to receptors. This sparked the idea for the software tool we have
developed: An NLP tool that processes articles for you and creates a database of EDCs and their target
receptors. This is explained in depth on our [Software
page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D).

We set the following requirements for these receptors:

* They should function as transcription factors directly.
* We need to be able to acquire a natural hormone for the receptor to test the system on.
* They should have multiple known EDCs related to them.

Using the NLP tool, we landed on working with **six receptors** for this initial system;
ERα, ERβ, GR, AR, MR, and PR. Advantageously, these six receptors can be covered by only two different
operator sites (HRE and ERE) and five different natural hormones. The system will be used in
*Saccharomyces cerevisiae* (yeast) strain CENPK113-3C, as the receptors have previously been
produced successfully in yeast [[1](%E2%80%9D#ref1”)]. This is a *TRP* deficient strain to allow
selection for successful transformants.

In eukaryotic cells, these nuclear receptors are anchored to chaperones in the cytoplasm. Upon ligand
binding, they dimerize, dissociate from chaperones, and translocate to the nucleus where they initiate
transcription (facilitated by co-activators) [[2](%E2%80%9D#ref2), [3](%E2%80%9D#ref3)].

### Design - interaction of receptor with DNA

This brought us to the second engineering challenge, concerning the interaction between the receptor
and DNA. Literature searches showed us that the receptor response elements do function as singular
inverted repeats, but that tandem repeats have been proved to have a much higher effect [[1](%E2%80%9D#ref1)]. Previously, in-cell systems have successfully used **five tandem repeats of
the REs** for different human hormonal receptors [[1](%E2%80%9D#ref1)]. Therefore, the final
tandem operator site sequences, denoted ERE5 and HRE5, are shown below.

ERE5

TTGGATCCC**AGGTCAGAGTGACCT**GC**AGGTCAGAGTGACCTGCAGGTCAGAGTGACCT**GGTCGACC**AGGTCAGAGTGACCT**GC**AGGTCAGAGTGACCT**GCTTTTTCCCGGGTTT

HRE5

TGGATCCGTCT**GGTACAGGGTGTTCT**TTTTGTCT**GGTACAGGGTGTTCT**TTTTGTCT**GGTACAGGGTGTTCT**TTTTGTCGACGTCT**GGTACAGGGTGTTCT**TTTTGTCT**GGTACAGGGTGTTCT**TTTTCCCGGGT

Receptor response elements ERE5 and
HRE5. The consensus operator site sequences are shown in bold green.

The six receptors, their natural hormones and response elements are outlined below in [Table 1](#table1):

Table 1: Overview of receptors along with corresponding response elements
and natural hormones [[2](#ref2)]. Finally the plasmid used for the in-cell assay is noted.

| **Human receptor** | **Response Element (operator site)** | **Natural hormone** | **Plasmid name (in-cell system)** |
| --- | --- | --- | --- |
| Estrogen Receptor α (ERα) | ERE | 17β-Estradiol | pRR-ERalpha-5Z |
| Estrogen Receptor β (ERβ) | ERE | 17β-Estradiol | pRR-ERbeta-5Z |
| Glucocorticoid Receptor (GR) | HRE | Dexamethasone | pRR-GR-5Z |
| Androgen Receptor (AR) | HRE | Testosterone | pRR-AR-5Z |
| Mineralocorticoid Receptor (MR) | HRE | Aldosterone | pRR-MR-5Z |
| Progesterone Receptor (PR) | HRE | Progesterone | pRR-PR-5Z |

### Design - generating a measurable read-out

To complete the design of the first engineering cycle, we had to consider the measurable read-out that
could be used for the in-cell system. We landed on a well-characterized beta-galactosidase enzyme as a
reporter for the system, inspired by the protocol by Edwards, T. M. et al. [[4](#ref4)].
Using ONPG as a
substrate, activity of the β-galactosidase enzyme can be assessed by measuring absorbance at 405 nm.

## Build

This system is simply a proof of concept to test the functionality of the receptors and their response
elements, and therefore a simple plasmid-based system can be used.
The plasmids utilized were purchased as complete plasmids from Addgene, originally deposited by Miller
et al. [[1](#ref1)]. The plasmids were later sequenced by an external company
using nanopore. This
allowed us to save time we could otherwise have spent cloning.

The plasmids are low-copy CEN/ARS plasmids containing a β-lactamase and a *TRP1* gene for
selection of successful transformants in  *E. coli* and yeast, respectively. Additionally, they
carry one of the hormonal receptors listed in [Table 1](#table1) transcribed from a GAL1,10 or GPD promoter [[1](#ref1)].
Finally, they contain a LacZ gene transcribed from a minimal cytochrome C (CYC) promoter. The receptor
response element is located upstream of the CYC promoter. The full plasmid map is shown in  [Figure 1](#fig1)
below.

![In-cell plasmid](https://static.igem.wiki/teams/5107/eng-incellplasmid1.webp)

Figure 1: Plasmid map of the pRR plasmids utilized in the in-cell system.
Contains a LacZ gene encoding β-galactosidase downstream a minimal CYC promoter in which a corresponding
receptor response element (RE\*: blue) is incorporated. Additionally, this plasmid carries a hormonal
receptor (HR\*: purple). Finally it carries a TRP1 gene (green) catalyzing tryptophan synthesis, an AMP
gene (green) encoding β-lactamase, and CelE1 Ori and CEN/ARS for replication in E. coli and yeast,
respectively. Not to scale.

The *S. cerevisiae* strain CENPK113-3C was transformed with the plasmids using a heat-shock method
(according to this protocol), which disrupts the cell membranes using LiAc. After the addition of the
plasmids, the yeast was grown on a YNB medium without tryptophan to select for successful transformation
carrying the TRP1 gene.

The design of the plasmid-based in-cell system is shown in [Figure 2](#fig2) below.

![In-cell system](https://static.igem.wiki/teams/5107/eng-incellsystem.webp)

Figure 2: Overview of the plasmid-based in-cell system to detect EDCs. A
hormonal receptor (HR) binds the receptor response element (RE) and activates the transcription of a
β-galactosidase reporter gene (LacZ) upon binding an EDC. A) shows the system when no EDC is present,
while B) shows the system under the presence of an EDC. Not to scale.

## Test

To assess whether the system worked, a protocol was designed inspired by the protocol by Edwards, T. M.
et al. (2018) [[3]](#ref3). As shown in [Figure 2](#fig2), the β-galactosidase
enzyme was used as a
reporter of EDCs, and ONPG was used as a substrate, which upon cleavage will absorb at 405 nm. For
comparisons between samples, the protocol was based on the principle of measuring the OD610 and OD405 to
be able to calculate values independent of the cell density. The complete protocol can be found in our
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

For data handling, the OD values were compared with appropriate media controls (without cells) and
vehicle controls (without EDC). The final LacZ value was calculated as the relationship between the
OD405 and the OD610 as shown below:

![LacZ calculation](https://static.igem.wiki/teams/5107/eng-lacz.webp)

Here, t is the incubation time of the enzymatic reaction

The initial tests consisted of creating **dose response curves using the natural
hormones**
for the receptors (shown in [Table 1](#table1)). The LacZ values were converted to a
percentage of the maximum value
for easy comparison between the curves. Unfortunately, we were not able to obtain progesterone to test
the
PR, and when testing the MR we saw unrealistically high standard deviations and no significant response.
The four functional dose-response curves are shown in [Figure 3](#fig3) below.

![Dose response curves](https://static.igem.wiki/teams/5107/eng-incellresults1.webp)

Figure 3: Dose-response curves for the in-cell assay. Percent maximal
response is shown for easy comparison. The concentration of the added hormone is shown - not the
concentration in the reaction. All measurements were done in triplicates and standard deviations are
represented by error bars. The sample positive controls are shown from left to right; ERb, ERa, AR, GR.

Finally, the system was tested on four water samples. We tested two different types of bottled water,
tap water, and a lake sample from a local lake. The data is shown in [Table 2](#table2) below. As a positive
control, the natural hormones (diluted in 50% ethanol) were added to the tap water sample to show that
we can replicate the data from the dose-response curves. These positive controls are shown in [Figure 3](#fig3) and fit the dose-response curves nicely.

Table 2: Overview of percent maximal response
for the in-cell assay on water samples. All measurements were done in triplicates and standard
deviations are represented by error bars. Colors correspond to the receptors shown in [Figure 3](#fig3).

|  | Bottled water #1 | Bottled water #2 | Tap water | Lake water |
| --- | --- | --- | --- | --- |
| ERa | 1.3% ± 5.9% | 5.3% ± 3.7% | 5.3% ± 3.6% | -0.8% ± 3.0% |
| ERb | 2.4% ± 16.6% | 1.4% ± 6.0% | 5.3% ± 4.9% | -1.8% ± 1.8% |
| GR | -13% ± 50.9% | 9.4% ± 11.3% | 10.5% ± 10.8% | -2.2% ± 31.5% |
| AR | 15.7% ± 53.4% | 28.9% ± 24.7% | 28.3% ± 24.0% | 33.7% ± 53.0% |

None of the measurements displayed in [Table 2](#table2) are significantly different from the lowest value on the
corresponding dose-response curve in [Figure 3](#fig3). Therefore we can conclude that
**none of these
water samples contain EDCs binding the tested receptors** above our measurement threshold.

If we had some results that showed a significant difference, the procedure would be to calculate sample
hormone equivalents (EHQs) by fitting the dose-response curves to a logistic model and using the model
to interpolate EHQs [[1](#ref1)].

## Learn

These experiments lead us to conclude three important things. First of all, the system functions,
providing an intermediate proof of concept of the in-cell system. Importantly, four of the
**yeast-produced receptors functioned**, and the design with tandem response elements (HRE5
and ERE5) functioned as expected.

Secondly, as seen in [Figure 3](#fig3), the biosensor sensitivity differs a lot between
receptors. This is however not too important, as the sensitivity for the cell-free assay should be
different.

Another conclusion was that while working with β-galactosidase as a reporter is reliable, it is also a
slow and tedious process involving a lot of pipetting. Therefore, in the cell-free assay, another
reporter method will be utilized.

Finally, while working with β-galactosidase as a reporter is reliable, it is also a slow and tedious
process. Therefore, in the cell-free assay, another reporter method will be utilized.

* [Engineering success](#Engineering-success)
* [Cycle 1: In-cell Assay](#cycle1)
* [Cycle 2:
  Cell-free Biosensor](#cycle2)
* [Cycle 3:
  Receptor Production](#cycle3)

* [Design](#design2)
* [Build](#build2)
* [Test & Learn I](#test-learn1)
* [Test & Learn II](#test-learn2)
* [Conclusions & Future Perspectives](#conclusions)

# Cycle 2 - Cell-free Biosensor: EndoSense

Having proved that the yeast-produced receptors are functional, we moved outside the cells to develop
**the cell-free biosensor: EndoSense**. We took the learned aspects from the in-cell system
and used
this knowledge to create a few different designs for the cell-free biosensor.

We have divided this engineering cycle in the three parts below.

### Design & Build: EndoSense

We designed an ON/OFF cell-free biosensor able to detect EDCs: EndoSense.
Different DNA template versions were engineered by varying the responsive elements, both in sequence
and
in number. Assumptions and uncertainties are also addressed.

### Test & Learn I: Cell-free Transcription Optimization

The biosensor's fluorescence output from the broccoli aptamer was tested and optimized without
receptor
or ligand by adjusting reaction parameters (buffer, DNA concentration, wavelengths, etc.).

### Test & Learn II: EndoSense Proof of Concept

The EndoSense biosensor was tested with the androgen receptor to find the optimal receptor
concentration. A proof of concept was demonstrated.

## Design

As mentioned, we faced many uncertainties in the design of the cell-free biosensor. We got a lot of
answers from our stakeholders, but other uncertainties still persisted - the most relevant being that
the human receptors, to our knowledge, were not fully tested in a cell-free environment. We therefore
had the challenge of reducing the complexity of the environment of the receptors, designing a cell-free
system around them.

For the structure of the biosensor, we took inspiration from the **ROSALIND cell-free
biosensor** [[5](#ref5)], modifying their design to match our goals. We kept the
general idea of having a Transcription Factor (TF) altering the activity of a RNA polymerase, and the
output signal as a consequence. We tailored the ROSALIND concept by selecting specific custom
transcription factors (TFs) as receptors and designing unique operator sequences to serve as responsive
elements. The exact parts are described on our [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
page.

### Selection of Receptors

## Selection of Receptors

Based on the findings from Cycle 1, where no results were obtained for the Mineralocorticoid
and
Progesterone receptors, we excluded them from this second cycle of testing. We only proceeded
with
Estrogen Receptor α (ERα), Estrogen Receptor β (ERβ), Glucocorticoid Receptor (GR) and Androgen
Receptor
(AR). It was decided to focus particularly on the androgen receptor, as we learned from Svend
Lindeberg
that the effect of EDCs on male health has been better characterized as it can be easily
measured
by
testing sperm quality (see [Human
Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)).

One significant challenge of working in a cell-free environment is the uncertainty surrounding
the
dynamics of receptor-DNA interactions. Given the intricate mechanisms involved with receptors in
human
cells (as previously described in Cycle 1) we opted to simplify the system by excluding
chaperones
and
co-activators. However, this decision necessitated the assumption that nuclear receptors
bind
to
DNA exclusively in the presence of a ligand (Assumption n°1).

### Receptor Responsive Elements

## Receptor Responsive Elements

We designed DNA templates using two response elements: ERE and HRE, designed to interact with
the
above-listed receptors (see [Table 1](#table1) for details).

Since we didn’t know the dynamic of the receptor-DNA interaction, we decided to test two
variants
for
each receptor element (so 4 variants in total). The DNA devices denoted
**T7-HRE5-dB-T**
and **T7-ERE5-dB-T** carry the same five-tandem repeat of response elements as in
the
in-cell assay (see in Cycle 1).

Additionally, we designed another version with only a single response element and only a single
repeat.
For its minimalistic approach, we denoted these parts **HREminimal** and **EREminimal**. The sequence for these parts can be seen below:

EREminimal

**CCAGGTCAGAGTGACCTG**

HREminimal

**AGAACANNNTGTTCT**

The devices incorporating these parts were named **T7-HREmin-dB-T** and
**T7-EREmin-dB-T** for the HREminimal and EREminimal respectively.

### Readable output

## Readable output

As for the output, we decided to go for a less tedious reporter than the one previously used.
For
our
cell-free system, the simplest and fastest way to have an output is to use an aptamer, and we
opted for
the fluorescent broccoli aptamer, which, when bound to the fluorophore DFHBI-1T, will produce
green
fluorescence upon excitation.

We implemented two different designs for the broccoli aptamer:

1. The single broccoli aptamer (abbreviated “**sB**”) surrounded by two tRNA
   scaffolds to
   increase its stability (inspired from BBa\_K3380101) in combination with the HREminimal and
   EREminimal
   responsive elements, in the parts T7-HREmin-**sB**-T and
   T7-EREmin-**sB**-T.
2. The three-way junction dimeric Broccoli (3WJdB, BBa\_K3777000, abbreviated
   “**dB**”) was
   used in the for the T7-HER5-**dB**-T and T7-ERE5-**dB**-T templates

The reason for this choice is that we discovered a synthesis limitation during a preliminary
check on
our sponsor IDT's website (details in the build section below).

### Selection of RNA Polymerase

## Selection of RNA Polymerase

Around those listed components, we choose the other parts to build a cell-free DNA-based
biosensor. The
essential parts still needed are an RNA polymerase with a promoter, NTPs and a proper buffer.

In our decision, we got inspiration from the ROSALIND biosensor [[8](#ref8)]. We
used the T7 RNA
polymerase
(T7 RNAP) from the T7 bacteriophage, as it is commercially available and well-described.
Additionally,
it doesn’t require any co-factor for the transcription process [[6](#ref6)],
simplifying the
interactions in
the systems, and also in regards to modeling (see [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)).
This choice, however, gives rise to uncertainty, as the interaction with the human receptor is
not
known
yet. Due to the structural difference from the human RNA polymerase [[7](#ref7)],
we assumed
no
significant interaction except for sterical inhibition (assumption n°1)  (see table
below:
Uncertainty n°1).

After we had determined the polymerase for the cell-free system, we then decided to use a
standard T7
promoter (link to part here) and a standard T7 terminator (link to part here - proven to
increase
the
efficiency of the transcription [[5](#ref5)]).

### Design - Working Mechanism of the Biosensor

![mechanism of biosensor 1](https://static.igem.wiki/teams/5107/eng-cellfreesystem-p1.webp)

When no EDC is present, the receptor won't bind the DNA, thus the T7 RNAP is
free to transcribe the Broccoli aptamer. The aptamer binds to DFHBI-1T and enables fluorescence,
by absorbing light at 472 nm and emitting it at 507 nm.

![mechanism of biosensor 2](https://static.igem.wiki/teams/5107/eng-cellfreesystem-p2.webp)

When an EDC is present, it binds the hormone receptor and induces a
conformational change that will allow it to bind the receptor response element. Once the receptor
is bound to the DNA, it will act as a repressor, suppressing the transcription.

Figure 4: Mechanism of the cell-free system. Output is shown when no EDC is present and when EDCs are
present. Not to scale.

Design - Detailed considerations & discussion

## ON/OFF vs OFF/ON biosensor design

The interaction between our receptor with T7 RNAP is essential for induction of transcription
upon ligand binding. Since this is very challenging to engineer in a cell-free environment with
our current system (as mentioned before), we opted in this engineering cycle for an
**ON/OFF-type biosensor** to start with. In such a biosensor, the default state is
ON, and only in the presence of the EDC this state is changed by binding (and thereby hindering
transcription) of the receptor to the DNA, leading to a lower signal.

As explained on our Integrated Human Practice page, we discussed the possible flaws of this
design in depth with our stakeholders, and especially Dr. Gerd M. Seibold, Associate Professor at
DTU, provided valuable insights into this.

Ideally, an **OFF/ON-type biosensor** would be more robust, as the signal will
represent both a functional biosensor and the presence of the analyte. Conversely, in our
ON/OFF-type biosensor, seeing a signal difference can either mean that there is an EDC present, or
that the biosensor is malfunctioning, like in the presence of a transcription inhibitor.

### Challenges with the OFF/ON system

Going with the more robust OFF/ON-type biosensor would require a different DNA template, but also
would require using the human RNA polymerase. This would allow the receptor to recruit the RNA
polymerase to the promoter, which in turn could be used to design an OFF/ON-type biosensor. There
are two main reasons why we didn’t choose to use this system:

* The interaction between the human RNA polymerase and the hormone is mediated by a series of
  cofactors. These cofactors are not yet well established and we would therefore **increase
  the complexity and introduce additional uncertainties**. Additionally, with the
  increased complexity and number of interactions, the modeling procedure will also be more
  difficult.
* As we are dealing with a limited budget, carrying out the full engineering cycle with more
  expensive reagents wouldn't be feasible for us. This would **increase the cost** of
  the testing procedure.

## Build

### DNA Templates: Synthesized and Assembled

In the interest of saving time, we decided to combine the minimal responsive elements with the single
Broccoli aptamer, as it could be *de novo* synthesized and used. On the other hand, we coupled in
our design the responsive elements with multiple binding sites (HRE5 and ERE5) with the more efficient
3WJdB double Broccoli aptamer (dB), as these templates needed to be manually assembled.

From the parts listed above (see [Figure 4](#fig4)), we created and assembled four different DNA templates
to be
used in our EndoSense biosensor.

T7-HRE5-dB-T & T7-ERE5-dB-T

The DNA templates denoted **T7-HRE5-dB-T** and **T7-ERE5-dB-T**,
carrying the same five-tandem repeat of response elements as in the in-cell assay, along with
the more efficient double Broccoli aptamer (3WJdB), could not be ordered due to the presence of
multiple repetitive sequences in both the response element region and the 3WJdB. As a result,
these templates had to be manually assembled. Their structure is represented in the [Figure 5](#fig5)
below:

![](https://static.igem.wiki/teams/5107/eng-parts-re5.webp)

Figure 5: Overview of the HRE5 and ERE5 DNA fragments used for the
cell-free
system. T7 promoter, response elements, aptamer parts and terminators are shown. Not to scale.

We used USER cloning to assemble the T7-HRE5-dB-T and T7-ERE5-dB-T. For the design of the
assembly we used the [AMUSER tool](https://services.healthtech.dtu.dk/services/AMUSER-1.0/). The USER assembly consists of 2 steps - PCR and the USER
reaction.

## PCR

First, we ran PCR reactions using primers from the AMUSER tool for the following parts:

* pUC19-T7-3WJdB-T (Addgene)
* HRE5 (pRR-PR-5Z template)
* ERE5 (pRR-ERalpha-5Z template)

Each of the primers used to amplify a given response element part (HRE5, ERE5) was equipped with
a specific USER overhang, complementary to the overhangs produced in plasmid backbone
(pUC19-T7-3WJdB-T(Addgene)). For the PCRs, a *Phusion U Hot Start polymerase* that tolerates
uracil bases was used. As a result, we obtained PCR products ready for USER cloning procedure.

## USER cloning

USER cloning is a uracil-based excision technique that utilizes USER (Uracil-Specific Excision
Reagent) enzyme to create specific 3’-overhangs on a DNA template. PCR products with
double-strand USER overhangs (each containing uracil base) are subdued to the activity of USER
and DpnI enzymes, resulting in an assembly of complementary overhangs and ligation of the
templates.

In our case, each of the responsive elements (HRE5, ERE5) was cloned into a backbone plasmid
pUC19-T7-3WJdB-T (Addgene). As a result, we produced two plasmids:

* pUC19-T7HRE5-3WJdB-T (pUC19\_HRE5)
* pUC19-T7\_ERE5-3WJdB-T (pUC19\_ERE5)

Each plasmid was transformed into and amplified in *E. coli* strain DH5-α. The final
ROSALIND templates (T7-HRE5-dB-T and T7-ERE5-dB-T) were obtained by PCR of the target sequences containing only
the DNA parts necessary for Cell-Free Transcription System protocol out of the novel pUC19
plasmids.

## Validation

To check the effectiveness of the assembly, we performed gel electrophoresis using HindIII and
SspI restriction enzymes on the templates. The procedure produced bands of expected length for
pUC19\_HRE5, which sequence we later confirmed by sequencing the plasmid with Nanopore
technology.

In the end, we were unable to confirm the proper assembly of pUC19\_ERE5 plasmid.

T7-HERmin-sB-T & T7-EREmin-sB-T

The ready-to-be-synthesized DNA templates named **T7-HREminimal-sB-T** and
**T7-EREminimal-sB-T**, carrying only one single binding site and the single
Broccoli aptamer flanked by two scaffold, are shown in the image below ([Figure
6](#fig6)):

![](https://static.igem.wiki/teams/5107/eng-parts-re.webp)

Figure 6: Overview of the T7-HREminimal-sB-T and T7-EREminimal-sB-T DNA
fragments used for the cell-free system. T7 promoter, response elements, aptamer parts and
terminators are shown. Not to scale.

These DNA templates were ordered as G-blocks from IDT and were amplified with PCR.

T7 RNA polymerase & Receptors: Purchased

The T7 RNA polymerase was purchased from New England Biolabs (catalog #: M0251L), and the only
receptor purchased, the human androgen receptor, was purchased from OriGene (CAT#: TP761906).

The human androgen receptor was expressed in *E. coli*, exhibiting a lower molecular
weight (72.5 kDa) compared to the version expressed in human cells (110 kDa). This difference is
likely due to the absence of post-translational modifications (PTMs), which may play a role in
influencing receptor function. Since we lacked the resources to further analyze this disparity,
we proceeded under the rationale that the lower molecular weight would not impair receptor
functionality.

## Reference template: T7-3WJdB-T

Additionally, we acquired the plasmid pUC19-T7-3WJdB-T from Addgene, which contains the T7
promoter, double Broccoli aptamer, and T7 terminator. We amplified this core region (T7-3WJdB-T)
using the same primers, as it has a compatible sequence. This plasmid was used as a control
because it lacks any response elements for the receptor to bind.

Finally, we developed a model of the system with the different DNA templates with data found in
literature, providing valuable insights before conducting the initial experiments. For further
details, please refer to the Modeling page.

## Test & Learn

To test the created parts, we performed two iterations. Firstly, we tested and optimized the
fluorescence output without the presence of any receptor or ligand (Test & Learn I), to ensure that the
design at its basic level works properly.

Secondly, we proceeded by testing the biosensor on its whole with the receptor and the ligands (Test &
Learn II).

For the testing process, we followed the protocols from our [Experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

### Test & Learn I: Cell-free Transcription Optimization

The aim of this first iteration of testing is to ensure that all the build parts are behaving properly
in the absence of both receptor and ligand, as well as optimize the output signal.

In our testing, we also used as a reference the T7-3WJdB-T template, without any responsive element, to
use it as a benchmark in fluorescence readings.

X

### Adjusting Wavelength and Plate reader setting

## Wavelength and Plate reader setting

**Rationale:** As the signal for the first experiments was erratic and sometimes
incoherent, we tried to improve the reading settings.

**Result:** Higher fluorescence output was yielded by:

* Using the wavelength couple 488/530 nm.
* Reading from the top (instead of from the bottom).

During the experiments so far, we encountered issues with the plate reader that we used.
Non-zero values and high peaks were present also in empty or water-filled (blank) wells, and
reaction
wells had very erratic measurements, as they were below the detection limit of the machine. Once
the
settings have been changed, the readings were much better.

We recorded the measurements using 2 different couples of wavelengths: 477/507nm (as
recommended from the ROSALIND protocol [[8](#ref8)] and 488/530 nm (also used to
measure the
fluorescence of the
fluorescein for reference). The latter wavelength couple yielded higher signal:

![Wavelength Comparison](https://static.igem.wiki/teams/5107/engineering/wavelength-comparison.webp)

Figure 7: Wavelength comparison for different DNA templates. Fluorescein
sodium salt was used as a reference.

The broccoli aptamer had clearly higher output on using the 488/530 nm couple of
excitation/emission.

### Buffer test

## Buffer test

* **Rationale:** Optimize the reaction to increase the signal
* **Result:** The commercial In Vitro Transcription (IVT) buffer was better than
  the custom-made one.

Initially, for the first transcription test, we used a custom In Vitro Transcription (IVT)
buffer recommended from the ROSALIND protocol (where we took the inspiration for the cell-free
system).
However, we didn’t get any fluorescence emission by using that custom buffer.

When using the commercial buffer, we could see a much higher output signal. The custom buffer
clearly is not ideal for the cell-free transcription, while the commercial buffer seems to work
much better. A possible explanation for this, is the ionic concentration, which is much higher
in
the custom buffer (especially NaCl). Either the indicated concentrations were wrong (we in fact
acknowledge a mistake in the ROSALIND protocol, as the indicated concentration of the NaCl ion
was
too high) or we made a mistake in the process of preparing it.

### DNA concentration: 10 nM

## DNA concentration

* **Rationale:** Increase the sensitivity of the biosensor, enlarge the limits of
  detection, and reduce the cost of testing.
* **Result:**  A concentration of 10 nM of DNA was sufficient to yield a
  substantial
  signal and the best one to perform the next experiments, also according to the [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) analysis.

Reducing the amount of DNA enhances both sensitivity and the limit of detection, which are
critical factors for our stakeholders. This reduction will ultimately lower the detection
threshold, allowing even trace amounts of EDCs in the tested water to produce measurable
inhibition. Additionally, it enables us to minimize the use of the receptor, the most expensive
component of the biosensor system.

The cell-free transcription was carried out with different DNA concentrations, to assess the
lowest one that would lead to an optimal detection:

![DNA concentration tests engineering](https://static.igem.wiki/teams/5107/engineering/dna-comparison-narrow.webp)

Figure 8: Test for different DNA concentrations in order to have a readable
and measurable signal. Measurements were taken after 1 hour for 20 minutes. Error bars represent
standard deviation of triplicates.

We didn’t test for lower DNA concentration, as, for the first iteration, it was not recommended
by the initial modeling analysis with the data from literature (see [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)).

### Effects of Interferents & Ligands in the assay

## Effects of Interferents & Ligands in the assay

* **Rationale:** Scout and test the effects of inhibitors and interferents on the
  signal output.
* **Result:**  Some compounds can negatively affect the aptamer production:
  + **Testosterone** (dissolved both in water and ethanol at 4µM
    concentration). We took into account this inhibitory effect when analyzing the data with
    the receptor present
    (a more detailed explanation is available in [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)).
  + Sarkosyl (above 0.25%) [[9](#ref9)];
  + Ethanol (above 2.5%) [[10](#ref10)].

We tested the hormone dissolved in both ethanol and water to assess the influence of each
solvent. Additionally, we examined the effect of sarkosyl, a surfactant included in the buffer
of the
purchased receptor.

To standardize and make comparable the values from different experiments, we included in each
measurement a serial dilution of Fluorescein Sodium Salt (FSS), from the distribution kit, and
normalized
the values as Micromolar Equivalent Fluorescein (MEF).

These results are summarized in the [Figure 9](#fig9) below:

![Inhibitors engineering](https://static.igem.wiki/teams/5107/engineering/inhibitors-effect.webp)

Figure 9: Effects of inhibitors and interference on the fluorescence output.
The T7-HREmin-sB-T DNA template was used where DNA is indicated. The intensity of the signal is
expressed as Micromolar Equivalent Fluorescein (MEF), more specifically Fluorescein Sodium Salt
(FSS). Error
bars indicate standard deviation of triplicates of measurements taken after 1h incubation with
the T7
polymerase.

As the hormone itself has an influence on the fluorescence, we took that into account for the
next tests. In particular, for testosterone dissolved in ethanol (used in future experiments),
**the
signal was reduced to ~65%** when compared to the transcription alone (from a [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) analysis).

Moving on to sarkosyl, seeing the results prompted us to change the receptor buffer before
using it for the reactions.

## Learnings from Iteration I: Key Points

1. The commercial RNAPol Reaction Buffer was employed to optimize cell-free transcription.
2. The optimal wavelength for enhanced signal measurement was found to be 488/530 nm. Additionally,
   measuring from the top, rather than the recommended from the bottom (as recommended by the ROSALIND
   protocol [[8](#ref8)]), resulted in significantly higher signals, improving the detection
   limit.
3. The lowest and best DNA concentration tested that produced a significant signal was 10 nM of the
   DNA template.
4. The hormone itself inhibits transcription, a factor that must be considered when comparing
   fluorescence in the presence and absence of the ligand, especially in the next testing phase, when
   the receptor is also used in the assay.

## Test & Learn II: EndoSense Proof of Concept

For the second iteration of testing, we focused solely on the androgen receptor, as limiting the scope
simplified the process (as we learned in the Cycle 1) and additional receptors were unavailable due to
failed production and cancelled orders. Only the T7-HREmin-sB-T and T7-HRE5-dB-T, compatible with the
androgen receptor, were tested using testosterone (the natural ligand of the receptor).

The main aim of this iteration is to **validate the effectiveness of the biosensor in detecting
EDCs**.

### Optimal Receptor Concentration & Validity of Assumptions

## Optimal Receptor Concentration & Validity of Assumptions

**Rationale:** Determine the optimal receptor concentration for the best detection
and
investigate the mechanism of the designed biosensor, evaluating the assumptions
previously made. As the receptor is our limiting resource, we prioritized this test, as being
able
to use less receptor would mean for us performing more testing.

**Results:**

**Receptor concentration:** The optimal concentration determined for the androgen
receptor is in the range of 0.75-1 µM. To determine the optimal receptor concentration for the
best
detection, we measured and compared the fluorescence output at various receptor concentrations,
analyzing the signal both in the presence and absence of the ligand.

After a first test with concentrations ranging from 0.025 µM to 2 µM, the most promising
results
were around 1 µM, so we decided to confine deeper testing around that concentration (see figure
12
below).

In fact, for receptor concentrations above 1.25 µM, there is a complete and irreversible
inhibition
of transcription, probably due to very stable binding of the receptor to the DNA. On the other
hand,
for concentrations below 0.5 µM of the receptor, there was no difference between the presence
and
absence of the ligand (data not shown).

In particular, the difference between the presence and absence of the ligand is the
evident
when the concentration of the receptor is 1 µM (see [Figure 10](#fig10) below),
reason which is why we decided to
further the analysis on this concentration.

![Receptor concentration](https://static.igem.wiki/teams/5107/engineering/receptor-conc-comparison-narrow.webp)

Figure 10: Comparison of different receptor concentrations after 1h
incubation
with the T7 polymerase. R - androgen receptor, H - testosterone hormone (4µM in all reactions
where
indicated). The T7-HREmin-sB-T DNA template (at a concentration of 10 nM) was used where DNA is
indicated. The signal has been adjusted considering the negative effect of the hormone on the
fluorescence. Error bars, when present, represent standard deviation of triplicates (duplicates
only
for receptor concentration 0.75 µM).

For all receptor concentrations here tested, there is a noticeable difference between the
absence
and presence of the ligand, and it is more relevant the higher the receptor concentration
(although it needs to be below 1.25 µM). For this reason, we chose to proceed with the 1 µM
concentration.

More data is required for the 0.5µM concentration, as it was only performed with a single
replicate.

### Validity of our assumptions

Firstly, upon adding the receptor, we observed a significant decrease in the signal,
invalidating
our initial 2nd assumption about the receptor-DNA interaction. It is clear that the
receptor binds to the DNA and inhibits the signal even in the absence of the ligand.

However, our first assumption regarding the receptor's interaction with RNA
polymerase
was **confirmed**. Even in the presence of the ligand, the signal is lower compared
to when the receptor is absent, validating this part of our hypothesis.

The validity of our assumptions can be summarized as follows:

* Absence of Ligand → No inhibition: Invalidated. The receptor binds to the DNA and
  inhibits the signal even in the absence of the ligand.
* The Receptor is not able to recruit the T7 RNAP: Confirmed. The signal is lower in
  the presence of the ligand, validating this part of our hypothesis.

| Uncertainties | Assumptions | What we learned |
| --- | --- | --- |
| Receptor & T7 RNAP: Which interaction takes place between these two entities? | The Receptor is not able to recruit the T7 RNAP | Due to the different conformation, the T7 RNAP will not be recruited by the nuclear receptor as it happens for the eukaryotic RNAP (also because the co-activator proteins are absent). Correct. |
| Receptor: Which conformation binds the DNA? | Absence of Ligand → No inhibition | The receptor will be able to bind the DNA only after the dimerization induced by the binding to the ligand, as that is what happens in the human cells. Wrong: The receptor can bind to the DNA even when the ligand is not present. No dimerization is needed for the binding. |

### Revised Working Mechanism: OFF/ON Type

In light of these results, we reconsidered the working mechanism of the biosensor: It behaves
as
an **OFF/ON type, instead of ON/OFF**. Which is in fact the opposite of what we
were expecting: An increase rather than a decrease in the signal. When
the
receptor alone is added, it binds to the DNA template, inhibiting the transcriptional activity
of
the T7 RNAP.

On the other hand, when also the ligand is present, the affinity of the receptor for the DNA
seems to be lower,
and, as a consequence, the activity of the T7 RNAP is partially restored, resulting in an
increase
of the signal. This suggests that the receptor is not able to bind DNA when the ligand is
present,
similarly to what happens in case of the Lac repressor when lactose is added. This behavior is
most likely due to a change of affinity for the DNA when ligand is bound to the receptor. This
working mechanism is shown in [Figure 11](#fig11) below.

![Figure 11: Actual working mechanism of the cell-free biosensor](https://static.igem.wiki/teams/5107/results/actual-working-mechanism.webp)

Figure 11: OFF/ON behavior of the EndoSense biosensor. When the water sample
does not contain EDCs, the fluorescence signal is inhibited. When EDCs are present, this will
result in the transcription of the broccoli aptamer and ultimately a fluorescence signal.

The identification of an OFF/ON working mechanism significantly **enhances the robustness
of the EndoSense biosensor**. Unlike the ON/OFF biosensor, which we discussed during
our
meeting with DTU Associate Professor Gerd, this design avoids the flaws inherent to the ON/OFF
system. In fact, when the signal is increased, this also validates that the assay is working
properly, and that there is definitely an EDC in the water sample.

#### Proof of Concept & Predicted Limit of Detection (LOD)

**Rationale:** Even though the working mechanism of the biosensor is opposite than
designed, we can still see a difference between the presence and absence of the ligand, which we
want to further investigate.

**Results:** The biosensor is able detect with statistical significance the presence
of the ligand.

As the difference between the presence and absence of the ligand is more evident from the
receptor concentration of 1 µM, we examined it more deeply.

As represented in [Figure 12](#fig12) below, the significance (expressed as the p-value of a
t-test between
the triplicates in each time point) is below the 0.05 threshold for all measurements, and below
0.01 after 45 minutes the addition of the T7 RNAP.

![Figure 12: Statistical significance of ligand detection](https://static.igem.wiki/teams/5107/engineering/proof-of-concept-motherfucker3.webp)

Figure 12: Difference in signal between presence and absence of the
testosterone hormone (indicated as *H*, at 4 µM) with the androgen receptor (*R*, 1 µM), and
the DNA template (at a concentration of 10 nM).
For each time point, a statistical t-test was performed between the two shown measurements. The
p-value for each test is shown as stars symbols above the top point.

### Test with EDCs

## Test with EDCs

**Rationale:** After having demonstrated that the EndoSense biosensor can detect
the
natural ligand of the receptor (testosterone), we wanted to test it with some proven EDCs, which
we got thanks to our **collaboration with the 2024 [2024 UCopenhagen iGEM team](https://2024.igem.wiki/ucopenhagen/). In particular, we tested with PCB
(polychlorinated biphenyls) and BAC (Benzanthracene).**

**Results:** Unfortunately, we noticed that when the receptor was added, there is
no
decrease in the signal, regardless of the hormone presence, as can be seen in the [Figure 13](#fig13)
below:

![EDC Test Results](https://static.igem.wiki/teams/5107/engineering/final-ecd-test1.webp)

Figure 13: Measured signal for two different DNA templates (DNA1:
T7-HRE5-dB-T
grouped on the left; and DNA2: T7-HREmin-sB-T grouped on the right), addition of androgen
receptor
(*R*) and testosterone (*H*).
For the T7-HREmin-sB-T two different EDCs have been tested: Polychlorinated biphenyls (PCB) and
benzanthracene (BAC).

From this observation, we concluded that **the receptor in this experiment was denatured
and not functional**, likely due to the buffer exchange process and/or repeated
freeze-thaw cycles. Thus, the results regarding the detection are not to be considered in this
experiment.

Despite this, the data clearly indicate a **negative effect of the tested EDCs on the
transcription process**. With the receptor's functionality ruled out, it is evident
that
the EDCs directly influenced the fluorescence output.

This finding suggests that, for future experiments, we should normalize the signal using
samples
with the EDCs, following the same approach applied with the hormone in earlier tests.

### Learnings from Iteration II: Key Points

1. The optimal range of concentration for the androgen receptor for the EndoSense cell-free biosensor
   was predicted to be around 0.75~1 µM, but below 1.25 µM, as an irreversible inhibition is detected above
   that.
2. The **working mechanism** of the receptor was of the **OFF/ON type**, with improved robustness.
3. We demonstrated a **proof of concept** for the EndoSense biosensor, by detecting testosterone with the Androgen Receptor.
4. We tested the biosensor with EDCs, but we were only able to assess the negative influence of those
   on
   the fluorescence output.

## Conclusions & Future Perspectives

From the experiments and results obtained so far, we can confirm that the **EndoSense biosensor
can
effectively detect analytes**. This has been clearly shown through our **proof of
concept**, where we used the T7-HREmin-sB-T DNA template to detect testosterone using the
androgen receptor.

Our biosensor detects EDCs in less than two hours compared to the current detecting methods, which take
several days to obtain results. The discovery of an OFF/ON-type biosensor mechanism enhances the robustness of the system. This unexpected finding strengthens the biosensor
reliability
due to its clear signal in the presence of EDCs, where the negative influence of the ligand can be
normalized when testing it in the absence of the receptor. Indeed, the signal increase not only
indicates
the presence of the analyte but also is a guarantee that the reaction was completed.

From the modeling analysis of this and the previous data, we were able to predict the **Limits Of
Detection
(LOD)** of biosensors: The upper LOD is 8.39 µM and the lower 0.1 µM (as shown in [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)). The obtained
lower value of LOD is still too high for a practical detection; however, one should underline that only
a
preliminary prediction has been done here and several factors have not been investigated in depth yet.
For
the real LODs more data would be needed, starting by testing the use of different ligand concentrations.
This would also allow to determine the dynamic range and the linearity of the biosensor, which are crucial parameter for an optimal detection.

Most notably, there are **numerous opportunities for improvement**. The receptors'
concentration has not been optimized yet, as only a few arbitrary concentrations were used. The results
are
probably more promising when the receptor concentration is between 0.75 and 1.1 µM, which, from our
analysis in the [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D), most likely contains the
optimal receptor concentration. Furthermore, the
optimization of DNA concentration, currently set around the thoroughly well-understood 10 nM, can be
refined
further, not only above but also below this value.

Also, other nuclear receptors can be screened, since our templates are compatible, allowing a more
platform-based screening for EDCs. This system will enable the testing of a wide number of EDCs and
their
action on different receptors.

By continuing to address current limitations, such as receptor stability and interaction dynamics,
**EndoSense has the potential to become a highly valuable tool for efficient environmental
monitoring of EDCs**. With future developments, this technology could not only reduce detection time but
also
provide a more accessible and cost-effective alternative to traditional testing methods.

* [Engineering
  success](#Engineering-success)
* [Cycle 1: In-cell Assay](#cycle1)
* [Cycle
  2:
  Cell-free Biosensor](#cycle2)
* [Cycle
  3:
  Receptor Production](#cycle3)

* [Design](#design3)
* [Build](#build3)
* [Test](#test3)
* [Learn](#learn3)

# Cycle 3 - Production of receptors

In Cycle 2 we showcased that our biosensor using the human androgen receptor can detect testosterone.
Although this is a favorable outcome, we used a factory-produced human androgen receptor for these tests.
To make the testing of our biosensor more affordable, we aimed to produce human hormone receptors
in-house
in Cycle 3. In the first iteration of this cycle, the goal was to produce the human androgen receptor in
yeast

## Design

In the design phase, we identified what properties we wanted for the production process and the
produced receptor itself:

* The receptor must be able to be purified
* The produced receptor should be exported outside of the cell to make the purification process
  easier
* The produced human androgen receptor (AR) should have the same properties as the androgen
  receptor
  from the human body
* The production should preferably use the same *TRP* deficient  *S. cerevisiae* strain as
  we used in Cycle 1 as we have experience with it.

To make the receptor purifiable we decided to use a 6xHIS tag at the N-terminal of the receptor. To
make the yeast excrete the protein we included an alfa-tag at the N-terminal of the HIS tag.

To make the yeast excrete the protein, we went with the alfa-tag at the N-terminal of the HIS tag.

We assumed the following:

* The AR produced in *S. cerevisiae* mimics the properties of the androgen receptor from the
  human
  body.
* The HIS-tag doesn’t affect the properties of the receptor.

The Alfa-6xHIS-AR sequence was retrieved using the online tool [vectorbuilder.com](https://www.vectorbuilder.com).

We decided to use services from Genscript for synthesizing the construct, as they offer the pESC-TRP
backbone, which is compatible with the S. cerevisiae trp1 strain we want to use. This backbone has a
GAL1,10 promoter which we decided to use for inducing expression of our construct. Thus, we could use
the strains tryptophan auxotrophy for selecting transformed colonies (which would be prototroph).
Outsourcing the synthesis and assembly of this part would also save us very valuable lab time.

We used the codon optimization tool from Genscript to optimize the sequence for expression in *S.
cerevisiae* and DNA synthesis. We also used it to make the sequence legal for the iGEM part
repository standards.

[Figure 14](#fig14) below shows a schematic of the final design of the plasmid, denoted
pGS-AR:

![pGS-AR](https://static.igem.wiki/teams/5107/eng-cycle3plasmid.png)

Figure 14: Plasmid map of the pGSAR plasmid acquired from Genscript. Carries a CelE1
Ori (red) and 2U Ori (red) for replication in E. coli and yeast, respectively. Additionally, it
carries
a TRP1 gene (green) catalyzing tryptophan synthesis, an AMP gene (green) encoding β-lactamase. Finally
it carries the human androgen receptor (AR: yellow) transcribed from a pGAL1,10 promoter. Upstream of
the AR is an alfa tag and a 6xHIS tag. Not to scale.

## Build

After receiving the plasmid from Genscript, we transformed the plasmid in *E. coli*. Then, after
making a glycerol stock of the transformed *E. coli* and purifying the plasmid from an overnight
culture of the *E. coli* using miniprep, we used 10uL of the purified plasmid for *S.
cerevisiae* transformation. All the protocols are described in our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

After failing to produce colonies from the yeast transformation, we finally got 2 colonies by using
1uL
of the stock from Genscript using our yeast transformation protocol.

## Test

After getting 2 colonies from the yeast transformation, we made 5 mL overnight cultures in yeast
nitrogen base (YNB) media with 2% glucose. The next day we inoculated 250 mL YNB + 2% galactose media
with the 5 mL overnight culture for one of the colonies.

After incubating the flask in a shaker at 180 rpm 30C for 2 days, we span down the culture to
separate
the cells from the broth. We performed SDS-PAGE and a Western blot to analyze the proteins found in
the
culture as described on our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.
The resulting gels and blots are shown in [Figure 15](#fig15) below.

![Cycle 3 Result Gels](https://static.igem.wiki/teams/5107/eng-cycle3resultgels.webp)

Figure 15: Analysis of culture containing the pGSAR plasmid. A: SDS page gel. Lane 1
contains a Precision Plus standard, while lane 2, 3, and 4 contains culture, crude protein and pellet
samples mixed with a Laemmli Loading buffer and DTT respectively.

## Learn

From the SDS-PAGE and the Western blot, we concluded that we failed to produce any AR - both in the
cells (pellet) and out of the cells (culture/crude protein). This lead us to propose the following two
explanations:

* The induction and expression of the GAL1 promoter failed.
* The colony was a false positive and does not contain our construct.

As we had a lot of trouble performing a successful transformation, we believe that it is much more
likely that the second option is true. To confirm this, a colony PCR could be performed on the
colonies,
thus determining if the colonies contain our construct or are false positives. However, due to time
limitations, we couldn’t perform this analysis, thus we left this cycle as is after this first failed
iteration.

Cycle 1: In-cell Assay
Cycle 2: Cell-Free
Biosensor
Cycle 3: Receptor
Production

---

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Note: Figures were generated using BioRender

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# Experiments

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This page contains all the protocols we used in our wetlab work. For each of them we’ve
added a small description, list of reagents and equipment, and a detailed procedure. Click each field to read the
corresponding protocol.

Buffers, media and solutions

### Lac Z Buffer

For the beta-galactosidase assay utilized in the in-cell assay [[8]](#ref8).

#### *Reagents*

* 2.13 g Na2HPO4 (dibasic)
* 1.20 g NaH2PO4 or 1.38 g NaH2PO4•H2O (monobasic)
* 23.8 mg MgCl2 (corresponds to 9.99 mL of a 25 mM MgCl2 solution)
* 186.4 mg KCl
* 0.5 g N-lauroylsarcosine sodium salt
* 100 mg ONPG
* Deionized water to a final volume of 250 mL

#### *Consumables*

* Weighing boats

#### *Equipment*

* 1 L flask
* Decimal scale

#### *Procedure*

1. Mix everything to a final volume of 250 mL.
2. Store the buffer in 20 mL aliquots at -20°C (sufficient for one 96 well plate in the in-cell
   assay assay).

### Glucose/Galactose Media

Minimal defined yeast media without tryptophan. Allows for selection of successful transformants
using the TRP1 marker.

#### *Reagents*

* 1.675 g Yeast Nitrogen Base without amino acids (YNB) (Should be 0.67%)
* 25 ml 20% glucose & galactose
* 0.48 g Yeast Synthetic Drop-out Medium Supplements without Tryptophan (Should be 0.192%)
* 225 ml distilled water
* 5 g Bacto agar

#### *Consumables*

* Weighing boats

#### *Equipment*

* Autoclave
* 500 ml bluecap bottle
* Scale

#### *Procedure*

1. Add YNB, Drop-out Medium Supplements to a 500 ml bluecap bottle.
2. Add 450 ml water and mix with a stirring bar until dissolved.
3. Add 5 g Bacto agar.
4. Autoclave.
5. Add 25 ml 20% glucose/galactose to each bottle (25 ml).

### LB amp agar

Ensures a 100ug/mL ampicillin conc. applicable with E. coli with amp resistance gene

#### *Reagents*

* 10 g LB broth with agar (miller)
* 250 ml ddH2O
* 100 mg/ml ampicillin solution

#### *Consumables*

* Weighing boats

#### *Equipment*

* 500 ml bluecap bottles

#### *Procedure*

1. Add 10 g LB broth with agar (miller) to a 500 mL bluecap bottle.
2. Add 250 ml water and mix with a stirring bar while heating until dissolved.
3. Autoclave.
4. Add 250 µL 100 mg/mL amp stock solution.
5. *NOTE:* Ensure that the media is cooled to around 50°C before adding ampicillin. Higher
   temperatures degrade it.

### LB Amp Broth Preparation

This protocol ensures a 100 µg/mL ampicillin concentration, applicable for use with E. coli strains
carrying the ampicillin resistance gene.

#### *Materials:*

* **Reagents:**

+ 5 g LB broth
+ 250 mL ddH2O
+ 100 mg/mL ampicillin solution

* **Consumables:**

+ Weighing boats

* **Equipment:**

+ 500 mL bluecap bottles

#### *Procedure:*

1. Add 5 g of LB broth to a 500 mL bluecap bottle.
2. Add 250 mL ddH2O and mix with a stirring bar while heating until completely
   dissolved.
3. Autoclave the solution to sterilize.
4. Once cooled to approximately 50°C, add 250 µL of 100 mg/mL ampicillin stock solution.
5. **Note:** Ensure the media is cooled before adding ampicillin, as higher
   temperatures degrade the antibiotic.

### Hormone Dilution Standards

Protocol for diluting the desired hormone standards for both the assays, In-cell and Cell-free.
**CAUTION:** These hormones are harmful if inhaled, swallowed, or absorbed through the
skin. Use appropriate personal protection (gloves, fume hood, dust mask) and avoid exposure during
pregnancy and lactation. Dispose as hazardous waste and do not release into the environment.

#### *Materials*

* Reagents:

+ Hormone powders
+ Ethanol
+ 50% ethanol

* Consumables:

+ Eppendorf tubes
+ Parafilm

* Equipment:

+ Scale
+ 1000 μl pipette
+ 100 μl pipette
+ 10 μl pipette

#### *Procedure*

##### Dilution of Estradiol to Working Conditions

1. Mix 1 mg estradiol powder with 1 ml ethanol and let it dissolve. This yields a 3.67 mM temporary
   stock #1.
2. Dilute 10 μl of stock #1 in 990 μl ethanol. This yields a 36.71 μM temporary stock #2.
3. Dilute 100 μl of stock #2 in 900 μl 50% ethanol. This yields a 3.6713 μM temporary stock #3.
4. Dilute 619.7 μl of stock #3 in 380.3 μl 50% ethanol. This yields the 2.275 μM working stock for
   the testrun (E1).
5. Dilute 100 μl of stock #3 in 900 μl 50% ethanol. This yields a 367.13 nM temporary stock #4.
6. Dilute 619.7 μl of stock #4 in 380.3 μl 50% ethanol. This yields the 227.5 nM working stock for
   the ERα receptor (E2).
7. Dilute 26.56 μl of stock #4 in 973.44 μl 50% ethanol. This yields the 9.75 nM working stock for
   the ERβ receptor (E3).
8. Seal tubes with parafilm and store at -20°C or -70°C.

##### Dilution of Testosterone to Working Conditions

1. Mix 22.6 mg testosterone powder with 1 ml ethanol and let it dissolve. This yields a 78.4 mM
   temporary stock #1.
2. Dilute 10 μl of stock #1 in 990 μl 50% ethanol. This yields a 784 μM temporary stock #2.
3. Dilute 128 μl of stock #2 in 872 μl 50% ethanol. This yields the 100 μM working stock for the
   AR.
4. Seal tubes with parafilm and store at -20°C or -70°C.

##### Dilution of Aldosterone to Working Conditions

1. Mix 5 mg aldosterone powder with 1 ml ethanol and let it dissolve. This yields a 13.9 mM
   temporary stock #1.
2. Dilute 72.3 μl of stock #1 in 927.7 μl 50% ethanol. This yields the 1 mM working stock for the
   MR.
3. Seal tubes with parafilm and store at -20°C or -70°C.

##### Dilution of Dexamethasone to Working Conditions

1. Mix 18.5 mg dexamethasone powder with 1 ml ethanol and let it dissolve. This yields a 47.1 mM
   temporary stock #1.
2. Dilute 21.3 μl of stock #1 in 978.7 μl 50% ethanol. This yields the 1 mM working stock for the
   GR.
3. Seal tubes with parafilm and store at -20°C or -70°C.

Transformations

### Plasmid Transformation of *E. coli*

Introduction: Transformation protocol for DH5alpha or BL21 E. coli cells that were prepared using the
Mix&Go *E. coli* Transformation Kit developed by [[2]](#ref2)

#### *Materials*

Reagents:

+ 100 μL x (N+2) LB (use SOC for better results)
+ MQ water
+ N+2 competent E. coli tubes (N+1 if no positive control)
+ N amount of cloning products
+ Negative control (NC) from cloning (can use water instead)
+ Positive control (PC) (optional but suggested)

Consumables:

+ N+2 LB antibiotic plates (N+1 if no positive control). Can multiply this amount if plating
  different amounts (step 10).
+ Pipettes
+ 200 μL tips
+ 10 μL tips

#### *Procedure*

##### Prepare

1. Take competent cell tubes and thaw them on ice.
2. If competent cells are in larger volumes, split them into tubes of 50 μL each.
3. Label each tube, remembering NC (Negative Control) and PC (Positive Control).
4. Take plates and prewarm them in the incubator.

##### Transform

1. While cells are still on ice, add 2 μL of cloning product or controls. Mix gently with a
   pipette. **Note:** Quantify the DNA! This protocol is optimal with ~100 ng/μL of
   DNA, so a total of 200~250 ng of DNA.
2. The optimal protocol states that no more than 5% of volume (of DNA) should be added to the
   competent cells. So, 5% of 50 μL = 2.5 μL.
3. Incubate on ice for 5 minutes.
4. Add 100 μL of SOC medium to each tube.
5. Incubate at 37°C for 1 hour. This can be done in an incubator or shaker (preferably sideways for
   better mixing; tape the tubes so they don't fall out).
6. Label the plates while waiting.

##### Plate

1. Plate the appropriate amount of media. It could be 50 μL, 100 μL, or 200 μL. You can plate
   different volumes on different plates for each tube.
2. Incubate plates at 37°C overnight. If no results appear, incubate longer. **Note:**
   Beware of satellite colonies if using ampicillin and incubating for a long period.

### Yeast Transformation

This protocol is used to make competent yeast cells and then transform them with the desired plasmid

#### *Reagents*

* 1 ml Frozen yeast glycerol stock
* 150 ml YPD media
* ca. 30 ml Sterile H2O
* 1.5 ml 0.1M lithium acetate (LiAc)
* 0.5 ml 1M lithium acetate (LiAc)
* 2.4 ml PEG 3350 (50% w/v)
* 0.5 ml Pre-denatured 2.0mg/ml single-stranded carrier DNA (salmon sperm derived). To prepare,
  boil 1 ml of the ss DNA for 5 min and quickly chill it on ice. Aliquote it and store at -20°C.

#### *Consumables*

* Spectrophotometer cuvettes
* Sterile 50 ml falcon tube
* Sterile 1.5 ul eppendorf tubes

#### *Equipment*

* Incubator set at 30°C and 200 rpm
* Spectrophotometer set at 660 nm
* Centrifuge for 50 ml falcon tubes set at 3000g
* Centrifuge for 1.5 ml eppendorf tubes set at 6000-8000rpm
* Water bath / heating block set at 42°C
* Pipettes
* Vortex
* Culture flask

#### *Procedure*

##### Day 1

Inoculate 100 ml YPD with 1 ml of a glycerol yeast stock. Grow O/N at 30°C with 200 rpm.

##### Day 2

After 12-16h determine the titer of the yeast culture using a spectrophotometer: Pipette 10 μl of
cells into 1.0 ml of water in a spectrophotometer cuvette, mix thoroughly by pipetting and measure
the OD at 660 nm.

1. Determine the titer in the cell culture (remember the dilution factor). A suspension containing
   1\*106 cells ml-1 will give an OD660 of 0.1.
2. Add 2.5\*108 cells to 50 ml of pre-warmed YPD in a pre-warmed culture flask: this will
   give a final solution of 5\*106 cells ml-1.
3. Incubate the flask in the shaking incubator at 30°C with 200 rpm until the cell titer is at
   least 2\*107 cells ml-1. This should take about 4 hours. Follow the OD
   closely toward the end.
4. Transfer the culture to a sterile 50 ml falcon tube and centrifuge at 3000g for 5 minutes.
5. Pour off the medium, resuspend the cells in 25 ml of sterile H₂O, and centrifuge again.
6. Pour off the H₂O, resuspend the cells in 1.0 ml of 0.1M lithium acetate (LiAc), and transfer the
   suspension to a sterile 1.5 ml microfuge tube.
7. Centrifuge the cells at top speed for 5 seconds and remove the supernatant with a pipette.
8. Resuspend the cells in 0.1M LiAc to a final volume of 500 μl (by adding around 400 μl of 0.1M
   LiAc). Aliquot 50 μl per transformation.
9. Spin down the cell suspension and remove the supernatant.
10. Add the following transformation mix to each of the eppendorf tubes in the specified order:
    * 240 μl PEG 3350 (50% w/v)
    * 36 μl 1.0M LiAc
    * 10 μl pre-denatured single-stranded carrier DNA (5.0 mg/ml) (thaw on ice)
    * 65 μl total: H₂O + plasmid DNA (0.1-10 μg)*Note:* The order is important! The PEG, which shields the cells from the detrimental
    effects of the high concentration of LiAc, should go in first.

    *Note 2:* You can also make a premix of all the ingredients without the plasmid DNA.
    Add this to the cells first, followed by the DNA. This mix can be stored at -80°C before adding
    the DNA for future use.
11. Mix by pipetting up and down slowly until the cell pellet is completely mixed (about 1 minute).
12. Incubate for 30 minutes at 30°C.
13. Heat shock for 30 minutes in a water bath at 42°C.
14. Centrifuge the tubes at 6000-8000 rpm for 15 seconds and remove the transformation mix with a
    pipette.
15. Add 0.2-1.0 ml of sterile H₂O to each tube and resuspend the pellet by pipetting up and down
    gently.

#### *Plating Procedure:*

1. If using a dominant marker for transformation, add 1 ml YPD to each tube, resuspend the pellet
   by pipetting up and down gently, and incubate for 1-2 hours at 30°C.
2. If performing a plasmid transformation, plate both a small amount (10 μl + 100 μl H₂O) and a
   larger amount (200 μl) of the transformation mix onto selective plates.
3. For integration transformations, plate the entire amount of the transformation mix onto
   selective plates.
4. Incubate the plates at 30°C for 3 days.

### Preparation of Glycerol Stocks

Preparation of glycerol stocks for long-term storage at -80°C, suitable for both
*E. coli* and yeast strains. The primary difference is the choice of media and incubation
temperature.

#### *Materials*

##### Reagents

* 50% glycerol (Add 500 mL glycerol to a 1L glass bottle. Fill up to 1L with distilled water.
  Autoclave)
* Broth for the O/N culture (e.g., YPD)

#### *Consumables*

* Inoculation needles
* 1000 μL pipette tips
* Freezing vials

#### *Equipment*

* Incubator (set to 30°C and 200 rpm for yeast, or 37°C and 200 rpm for *E. coli*)
* 1000 μL pipette
* Glass test tube

#### *Procedure*

##### Day 1

1. Inoculate 5 mL of broth (e.g., YPD for yeast, or LB for *E. coli*) with the strain in a
   glass test tube.
2. Incubate overnight in an incubator (set to 30°C and 200 rpm for yeast, or 37°C and 200 rpm for
   *E. coli*).

##### Day 2

1. Label cryotubes and record corresponding information in Benchling (name of culture, name, and
   date).
2. Pre-fill the cryotube with 750 µL of 50% glycerol solution.
3. Add 750 µL of the overnight culture to the cryotubes.
4. Invert the tubes several times to ensure proper mixing.
5. Store the tubes in the -80°C freezer.

DNA amplification, purification and cloning

### PCR with Phusion U Hot Start Polymerase

Protocol adapted from [[3]](#ref3). We use it to amplify the G-blocks attached with
primers compatible for the USER reaction in the following steps.

#### *Reagents*

* 10 mM Primers
* 10 mM dNTPs mix
* PCR grade water
* Template DNA
* Phusion U DNA Polymerase
* 5X Phusion HF Buffer

#### *Consumables*

* desired number of PCR tubes
* Pipettes
* 20 µL or 10 µL tips
* 200 µL tips

#### *Procedure*

##### Preparation

1. Label PCR tubes, including negative and positive controls. Thaw the reagents. Only take the
   Phusion U Polymerase out right before use.
2. Calculate the template concentration (using gel or Nanodrop) to determine the amount of template
   to add.
3. Prepare the Master Mix without the template/primers if these differ between reactions.
4. Remember to dilute primers to 10 µM - they are 100 µM when resuspended from the supplier.

##### Master Mix Composition

| Component | 50 µL Reaction | Final Concentration in Reaction |
| --- | --- | --- |
| H2O | 32.5 µL |  |
| 5X HF Buffer | 10 µL | 1x |
| 10 mM dNTPs | 1 µL | 200 µM (each) |
| Primer FWD (10 µM) | 2.5 µL | 0.5 µM |
| Primer RV (10 µM) | 2.5 µL | 0.5 µM |
| Phusion U Polymerase | 0.5 µL | 0.02 U/µL |
| Template | 1 µL | 1 pg - 10 ng DNA per 50 µL |

##### Reaction Setup

Add the DNA template/primers to each tube. For the negative control tube, add water (or nothing)
instead of the DNA template.

##### Thermocycler Program

| Step | Temperature (°C) | Time | Repeats |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 2 min |  |
| Denaturation | 98 | 15 sec | 25-30x |
| Annealing | Tm - 5 | 20 sec |  |
| Elongation | 72 | kb x 15-30 sec |  |
| Final elongation | 72 | 5 min |  |
| Hold | 4 or 12 | infinite |  |

### Massive PCR for Cell-Free Assay Template

We use the AQ97 High Fidelity DNA Polymerase 2x Master Mix
to amplify massive amounts of the templates for our cell-free biosensor [[4]](#ref4).

#### *Process*

Split the 1000 µL into 2 PCR strips of 8 wells each, resulting in 16 wells, with 62.5 µL in each
well.

##### Reaction Preparation

| Component | Volume (µL) | Final Conc. | Initial Conc. |
| --- | --- | --- | --- |
| H2O | up to 1000 |  |  |
| 2X Master Mix Buffer | 500 | 1x |  |
| Primer FWD (10 µM) | 30 | 0.3 µM | 10 µM |
| Primer RV (10 µM) | 30 | 0.3 µM | 10 µM |
| Template | Vary |  | 20 ng DNA |
| Total | 1000 |  |  |

##### Thermocycler Program

| Step | Temp (°C) | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 2 min |  |
| Denaturation | 98 | 15 sec |  |
| Annealing | 60 | 20 sec | 37 cycles |
| Elongation | 72 | 20 sec |  |
| Final Elongation | 72 | 5 min |  |
| Hold | 4 or 12 | Infinite |  |

### PCR Clean-up (NucleoSpin®)

The following protocol (NucleoSpin®) is suitable for PCR clean-up, DNA concentration, and removal of
salts, enzymes, etc. from enzymatic reactions (SDS < 0.1%)NucleoSpin Gel and PCR Clean-up [[5]](#ref5)

#### *Materials*

* DNA binding buffer
* Elution buffer
* Column

#### *Procedure*

##### Adjust DNA binding condition

For very small sample volumes < 30 μL, adjust the volume of the reaction mixture to 50–100 μL with
water. It is not necessary to remove mineral oil.

Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix 100 μL PCR reaction and 200 μL Buffer
NTI).

*Note:* For removal of small fragments like primer dimers, dilutions of Buffer NTI can be used
instead of 100% Buffer NTI.

##### Bind DNA

Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL
sample. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the
collection tube.

*Note:* Load remaining sample if necessary and repeat the centrifugation step.

##### Wash silica membrane

Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x
g. Discard flow-through and place the column back into the collection tube.

*Recommended:* Repeat the previous washing step to minimize chaotropic salt carry-over and
improve A260/A230 values.

##### Dry silica membrane

Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does
not come in contact with the flow-through while removing it from the centrifuge and the collection
tube.

*Note:* Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of
ethanol can be achieved by incubating the columns for 2–5 min at 70°C prior to elution.

##### Elute DNA

Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube (not
provided). Add 15–30 μL Buffer NE and incubate at room temperature (15–25°C) for 1 min. Centrifuge
for 1 min at 11,000 x g.

*Note:* DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elution
steps with fresh buffer, heating to 70°C, and incubation for 5 min.

### USER Cloning

Using the specific design primers, we can perform User cloning attached parts in a customize
manner [[6]](#ref6).

#### *Materials*

Reagents:

+ USER reaction targets (PCR products)
+ CutSmart buffer
+ USER enzyme (keep on ice)
+ DpnI enzyme (keep on ice)
+ MQ water

#### *Procedure*

##### Prepare the mix:

| Reagent | Volume (μL) |
| --- | --- |
| MQ water | 11 μL |
| PCR product | 2 μL each |
| CutSmart buffer | 1 μL |
| USER enzyme | 1 μL |
| DpnI enzyme | 1 μL |
| Total | 20 μL |

##### Thermocycler Settings

| Temperature | Time |
| --- | --- |
| 37°C | 20 min |
| 25°C | 20 min |
| 4-12°C | Hold |

### Monarch Plasmid Miniprep Kit (For Purification of Plasmids from E. coli)

Follow this protocol to purify the desired plasmid which was inserted in the *E.coli*cells.
For more detailed instructions, please refer to the protocol [[7]](#ref7).

#### *Reagents*

* Plasmid Resuspension Buffer (B1) (pink)
* Plasmid Lysis Buffer (B2) (blue/green)
* Plasmid Neutralization Buffer (B3) (yellow)
* Plasmid Wash Buffer 1
* Plasmid Wash Buffer 2
* DNA Elution Buffer or Nuclease-Free Water

#### *Consumables*

* Centrifuge tubes from the kit
* 1.5 mL Eppendorf tubes
* Maybe 2 mL Eppendorf tubes

#### *Equipment*

* Microcentrifuge

#### *Procedure*

##### Pellet Bacterial Culture

Pellet 4 mL bacterial culture by centrifugation for 30 seconds. Discard supernatant.

##### Resuspend Pellet

Resuspend pellet in 200 μL Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are
completely resuspended.

##### Lyse Cells

Lyse cells by adding 200 μL Plasmid Lysis Buffer (B2). Invert the tube immediately and gently 5–6
times until the color changes to dark pink and the solution is clear and viscous. Do not vortex!
Incubate for one minute.

##### Neutralize Lysate

Neutralize the lysate by adding 400 μL of Plasmid Neutralization Buffer (B3) (yellow). Gently invert
the tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2
minutes.

##### Clarify Lysate

Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.

##### Transfer Supernatant

Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.

##### Wash Column

Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Centrifuge for 1
minute. Discarding the flow-through is optional.

Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

##### Elute DNA

Transfer column to a clean 1.5 mL microfuge tube. Add ≥ 30 μL DNA Elution Buffer to the center of the
matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

### DNA Digestion with Restriction Enzyme

In order to validate the correct assembly we digest our constructs with Restriction Enzymes.For
restriction enzyme analysis and to visualize cutting sites, use the [[8]](#ref8).

#### *Materials*

* DNA template
* Restriction Enzyme(s)
* PCR grade water (Nuclease-free water)
* Tubes (PCR tubes can work)
* Buffer (depends on the Enzyme(s) used)

#### *Procedure*

According to the restriction enzyme used, different buffers are required. Check the NEBcloner
Restriction Enzyme Digestion for specific buffer recommendations.

##### Reaction Setup

| Component | Amount |
| --- | --- |
| DNA | 0.5 µg |
| Buffer | 2.5 µL (1X) |
| Enzyme 1 | 1.0 µL |
| Enzyme 2 (optional) | 1.0 µL |
| Nuclease-free Water | to 25 µL |

##### Incubation

Incubate at 37°C for 1 hour.

In-cell assay

### In-Cell β-galactosidase Assay

This assay is based on a paper about a YES/YAS assay [[1]](#ref1).Though this, we
can testify the presence or not of the EDCs using yeast cells are scaffolds. The protocol reference
also describes how to calculate the LacZ values, creating the standard curves, and calculating EEQ
values.

#### *Reagents*

* Yeast strain carrying the pRR plasmids
* Glucose media broth (trp-)
* Galactose media broth (trp-)
* Sodium carbonate (105.9 g sodium carbonate in a final volume of 1 L)
* 1 M dithiothreitol (DTT). Should be frozen in 25 µL aliquots. CAUTION: use gloves and fume hood.
* 9.75 nM estradiol standard
* 227.5 nM estradiol standard
* LacZ buffer with ONPG
* 50% ethanol

#### *Consumables*

* Filters for sterilization of media
* Sterilized 96-well polypropylene plates (done by autoclaving stacks wrapped in foil)
* Sterile, adhesive, porous film for sealing the plates
* Non-sterilized 96-well polypropylene plates
* Sterile pipette tips (all sizes)
* Gloves

#### *Equipment*

* 250 ml Erlenmeyer flask
* Fume hood
* Incubator (set to 30 °C and 200 rpm)
* Spectrophotometer
* Plate spectrophotometer
* Sterile beakers
* 1000 µL multipipette
* 100 µL multipipette
* 1000 µL pipette
* 100 µL pipette
* 10 µL pipette

#### *Procedure*

##### Before Assay

1. Maintain active yeast cultures (with the pRR plasmids) by incubating yeast in filter-sterilized
   glucose media (trp-) at 30 °C. Subculture yeast weekly in fresh filter-sterilized glucose media.
2. Two nights (about 42 h) prior to preparing the 96-well assay plates, subculture yeast by adding
   0.1 mL of active yeast cultures to 10 mL of filter-sterilized glucose media using sterile
   technique. Incubate at 30 °C for two nights. Shaking is not required if yeast are grown as
   shallow cultures in sterile 250 mL Erlenmeyer flasks.

##### Day 1 (Prepare the Assay Plates)

1. In a sterile beaker and using sterile technique, dilute yeast from step 3.2 to an optical
   density of 0.065 ±0.005 at 610 nm (OD610) in filter-sterilized galactose media.
2. Confirm optical density by pipetting 120 μL of each yeast sample in triplicate along with
   triplicate galactose blanks into a clear polystyrene plate (the plate does not need to be
   sterile). Use a plate spectrophotometer to verify that the diluted yeast culture has a net OD610
   of 0.065 ±0.005. Subtract OD610 readings of galactose blanks from yeast OD610 readings to
   determine the net OD610 of the yeast. Prepare a final volume of at least 30 mL diluted yeast for
   each 96-well plate. NOTE: Spectrophotometer filters measuring absorbance between 595 and 610 nm
   can be used for this measurement. An approximate 1:6 v:v ratio of yeast:media typically yields
   an OD610 close to 0.065, so start by adding 4.5 mL yeast to 30 mL media and add more yeast or
   media as appropriate to achieve a net OD610 between 0.060 and 0.070.
3. Using sterile technique, add this diluted yeast suspension to the wells of a sterile,
   polypropylene, 96-well microplate according to the plate layout (see figure below). White wells
   are for the standard curve and light gray wells are for the controls.
4. During pipetting, keep the source yeast suspended by continuously swirling the container or
   mixing with the pipette. For this step, it is helpful to use a repeating pipettor with a sterile
   syringe tip.
5. Add 120 µL filter-sterilized galactose media to 3 additional wells (D4 - D6) according to the
   plate layout. These wells serve as media controls to account for absorbance contributed by the
   media alone.
6. Add 5 µL of vehicle (50% ethanol) to vehicle control wells containing the yeast suspension (D1 -
   D3) according to the plate layout. NOTE: Vehicle control wells are used to quantify background
   absorbance associated with yeast cells and media. Additionally, cytotoxicity or growth-promoting
   effects of test samples can be detected by comparing the OD610 values of test wells with those
   of vehicle control wells.
7. Construct a standard curve in each plate by serially diluting triplicates of hormone working
   standards in wells containing the yeast suspension according to the plate layout:
   ![plate layout in-cell assay](https://static.igem.wiki/teams/5107/platelayout.webp)
8. Begin by adding 5 µL of hormone standards to wells A1 through C1. Mix microwell contents by
   pipetting, and then serially dilute this suspension by moving 205 µL from wells A1 - C1 into
   wells A2 - C2. Repeat the mixing and transfer of 205 µL through column 12. Note: After the
   serial dilution is completed, discard 205 µL from wells A12 - C12. At the end of this step, all
   standard wells should contain 120 µL. For the serial dilution steps, it is helpful to use a
   multichannel pipettor with sterile tips.
9. Mix the contents of the sample, extraction control, and vehicle control wells by pipetting.
   Adjust volumes of these wells to 120 µL by removing 205 µL from each as described in the legend
   for. This can be done using a multichannel pipette.
10. Seal the plate(s) with a sterile, adhesive, porous film. Label plates and incubate for 17 h at
    30 °C. The plate does not need to be shaken during incubation.

##### Day 2 (Processing of the Assay Plates)

1. After the 17 h incubation at 30 °C, remove plates from the incubator. Use a multichannel
   pipettor to mix the contents of each row of wells, and then transfer 50 µL of yeast suspension
   from each well to the corresponding well of a clear, polystyrene, 96-well microplate. Note:
   Polystyrene plates do not need to be sterile but should have a lid. Use new pipet tips for each
   row of wells to avoid cross-contaminating wells, although if pipetting across triplicates with
   an 8-tip multichannel pipette, tips only need to be changed between triplicate sets.
2. Add 20 µL of 1 M DTT to 20 mL of thawed, room-temperature LacZ buffer for a final concentration
   of 1 mM DTT. Mix this LacZ buffer well. CAUTION: Wear gloves and work with DTT in a hood.
3. Using either a multichannel pipette or repeating pipettor, add 200 µL of LacZ buffer containing
   1 mM DTT to all wells of the polystyrene plate, and immediately measure and record the OD610 of
   all wells using a plate spectrophotometer.
4. Cover plates with a lid. Incubate plates containing yeast expressing ERα at 30 °C for 40 min and
   plates containing yeast expressing ERβ at 30 °C for 70 min. You can use another plate as the lid
   if you don't have one.
5. After incubating the plates, use a multichannel pipette or repeating pipettor to add 100 µL of
   sodium carbonate to each well. Sodium carbonate raises the pH and halts the β-galactosidase
   reaction.
6. Measure and record the OD405 (for ONPG) of all wells using a plate spectrophotometer.

Cell-free assay

This protocol is based on the Rosalind System Manual [[9]](#ref9). It is used to
validate that our DNA constructs can be transcribed into an aptamer that in complex with the
fluorophore DFHBI-IT emits fluorescence at the appropriate wavelength.

#### *Materials*

* 10x IVT buffer
* 100 mM Tris-buffered NTPs
* 2 U/uL inorganic pyrophosphatase
* 1 M IVT linear DNA template
* 40 mM DFHBI-IT
* H2O
* 1 mg/mL T7 RNAP

#### *Equipment*

* 384 plate reader
* Nuclease-free PCR strips

#### *Procedure*

##### Setting the Microplate Reader

* the temperature to 37°C. The wavelengths should be set to 488 nm for excitation and 530 nm for
  emission.
* kinetic measurement, take reads every 3 minutes over the course of a 4-hour reaction.

##### Preparing the Master Mix

Mix the components to make a 15 µL master mix for each reaction:

| Component | Final Conc. | Volume per reaction (µL) |
| --- | --- | --- |
| 10x IVT buffer | 1x | 2 |
| 100 mM Tris-buffered NTPs | 11.4 mM | 2.28 |
| 0.1 U/uL inorganic pyrophosphatase | 0.015 U/uL | 3 |
| 1 M IVT linear DNA template | 5-50 nM | Vary (max is 7.68 µL) |
| 40 mM DFHBI-IT | 0.2 mM | 0.1 |
| H2O | - | Up to 15 µL |

Note: The parameters should be optimized, as the concentration of DFHBI-IT greatly impacts
transcription efficiency.

##### Preparing the Activator Mix

Incubate the master mix at 37°C while preparing the activator mix. Mix the components listed in a
separate tube:

| Component | Final Conc. | Amount per reaction (µL) |
| --- | --- | --- |
| 1 mg/mL T7 RNAP | 10 ng/uL | 2 µL |
| H2O | - | 2.8 µL (up to 5 µL) |

##### Reaction Setup

Ensure the microplate reader is ready, then add 5 µL of the activator mix to 15 µL of the master
mix. Gently pipette up and down several times to mix, avoiding the introduction of air bubbles.

If setting up multiple reactions simultaneously, use a multichannel pipette to add the activator mix
to the master mix to ensure that each reaction activates at the same time.

Immediately transfer the reaction onto a 384-well plate, seal the plate to prevent evaporation, and
start the microplate reader run.

## Protein Concentration

Because the protein concentration is low, we use protein concentrators [[10]](#ref10)
with a cutoff of 10K. Since our protein is 72 kD, we expect everything smaller than this size to
pass through the filter in the column.
This process allows us to reduce the volume of the protein sample used in our assay and switch to a
buffer that is more compatible with the assay.

#### *Materials*

* PBS 1X
* Column[[10]](#ref10)
* Centrifuge

#### *Process*

1. Place the sample into the concentrator sample chamber.
2. Cap, then place the concentrator assembly into the rotor with proper counterbalance.
3. Centrifuge the sample at 15,000 g for up to 7 minutes. A good strategy is to load the column
   with up to 500 μL of sample, then centrifuge until the volume is reduced to 50 μL.
4. Load an additional 450 μL of the desired buffer (PBS in this case) and centrifuge again.
5. Repeat this process as many times as possible to increase the efficiency.
6. Use a pipette tip to gently aspirate the retentate from the bottom and center of the sample
   chamber.

### Cell Free assay with Receptor

In this experiment we test the capabilty of our desing to sence the presence of hormone in the
solution

#### *Materials*

* Androgen receptor (0.14 μg/μl), MW = 72kDa
* IVT buffer
* NTPs
* 2 U/μl inorganic pyrophosphatase
* 40 mM DFHBI-IT
* Nuclease-free Water
* 1 mg/mL T7 RNAP

#### *Procedure*

1. Setting the microplate reader:

* Set the temperature to 37°C.
* Wavelengths should be set to 488 nm for excitation and 530 nm for emission.
* For kinetic measurement, take reads every 1 min over the course of a 2-h reaction.

2. Mix the components to make a 15 µL master mix for each reaction:

* Note: The parameters should be optimized, as the concentration of DFHBI-1T greatly impacts
  the transcription efficiency.

3. Incubate the master mix at 37°C for 1h 30 mins while preparing the activating reaction.
4. Mix the components listed below in a separate tube to make an activator mix:

| Component | Concentration | Volume |
| --- | --- | --- |
| 1 mg/mL T7 RNAP | 10 ng/μL | 2 μL |
| H₂O | - | up to 5 μL |

5. Ensure the microplate reader is ready to read, then add 5 μL of the activator mix to 15 μL of
   the master mix.
6. Gently pipette up and down several times to mix, avoiding the introduction of air bubbles.
7. If setting up multiple reactions simultaneously, use a multichannel pipette to ensure each
   reaction activates at the same time.
8. Immediately transfer the reaction onto a 384-well plate, seal the plate to prevent evaporation,
   and start the microplate reader run.

Protein analysis

### SDS-PAGE

This protocol describes the SDS-PAGE method for visualizing protein expression and purification,
particularly for the Androgen Receptor. The method applies to other SDS-PAGE samples as well.
**Note:** Acrylamide is carcinogenic, so always wear gloves when handling gels or
buffers.

#### *Materials:*

##### Reagents:

+ 4X Laemmli loading buffer
+ 500 mM DTT
+ Precision Plus Standard (Unstained)
+ Pre-cast SDS-PAGE gel (12% acrylamide)
+ 1X Tris/Glycine/SDS running buffer
+ Bio-Safe Coomassie stain

##### Consumables:

+ ThermoMixer at 95°C
+ Staining tray

#### *Sample Preparation:*

| Sample | Sample Volume/Amount | 4X Laemmli Loading Buffer | 500 mM DTT |
| --- | --- | --- | --- |
| Culture | 500 μL | 200 μL | 2 μL |
| Crude protein (after centrifugation) | 50 μL | 200 μL | 2 μL |
| Pellet (after centrifugation) | Approx. 2 μL | 100 μL | 2 μL |

#### *Procedure:*

1. Prepare the SDS-PAGE sample mixes according to the table above.
2. Boil the samples at 95°C and 300 RPM for 10 minutes to ensure full protein denaturation.
3. Prepare a pre-cast gel.
4. Pour 1X SDS running buffer in the tank.
5. Load the gel: 5 μL unstained standard and 10 μL of each SDS sample.
6. Run the gel at 180V for 45 minutes.
7. Once the run is complete, open the gel casing with a gel opener and transfer the gel to a
   staining tray.
8. Wash the gel 3 times for 5 minutes each with demineralized water, using slow agitation.
9. Stain the gel for 1 hour with Coomassie stain.
10. Destain for approximately 1 hour with frequent water changes.
11. Once destained, place the gel in a plastic sheet and scan it.

### Western Blotting with Turbo Blotter

#### *Introduction:*

This protocol describes Western blotting using a Turbo blotter. While other methods can be used, this
is the fastest and simplest method.

#### *Materials:*

* SDS-PAGE gel (unstained) using Dual color protein standard.
* Mini transfer pack.
* Turbo blotter.
* Skim milk buffer (2% skim milk powder, 1X TBS).
* Anti-His antibodies (HRP-conjugated).
* 15 mL Falcon tube.
* AEC staining kit.

#### *Procedure:*

##### Blotting:

1. Load the gel as with SDS-PAGE but use Dual color protein standard. Run the gel until the blue
   color exits completely.
2. Place the bottom stack (with membrane) on the cassette base, followed by the gel, then the top
   stack.
3. Close and lock the cassette lid. Insert cassette into the instrument and start transfer.
4. Run the block that is loaded.

##### Washing the Membrane:

1. Transfer the membrane to a fitting container without color stains and block it in skim milk
   buffer for 1 hour with slow agitation.
2. Discard the buffer and incubate the membrane in skim milk buffer with antibodies for 1 hour,
   again with slow agitation.
3. Discard the buffer and wash the membrane 3 times for 15 minutes each with slow agitation in skim
   milk buffer.
4. Rinse the membrane briefly in demineralized water.

##### Staining:

1. Prepare the AEC staining solution using the AEC staining kit:

* Add 4 mL demineralized water to a 15 mL Falcon tube.
* Add 2 drops (60 μL) of acetate buffer.
* Add 1 drop (30 μL) of AEC chromogen.
* Add 1 drop (30 μL) of hydrogen peroxide solution.

2. Mix gently by inverting a few times.
3. Pour the staining solution over the membrane, ensuring it fully covers the upward side.
4. Leave the membrane to stain for 5-10 minutes (or until sufficient staining is achieved) under
   tinfoil in the fume hood.
5. Pour the solution out and rinse with excess demineralized water.
6. Scan the membrane in the same way as for gels.

References

1. Edwards, T. M., Morgan, H. E., Coralia Balasca, Chalasani, N. K., Yam, L., & Alison McCombe Roark.
   (2018). Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for
   the Undergraduate Teaching Laboratory. Journal of Visualized Experiments, 131. <https://doi.org/10.3791/55754>.
2. Mix & Go E. coli Transformation Kit & Buffer Set (2021). Zymo Research. Retrieved from
   <https://files.zymoresearch.com/protocols/_t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf>.
3. Thermo Scientific Phusion U Hot Start DNA Polymerase (2018). Thermo Scientific. Retrieved
   from <https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012916_PhusionUHotStart_DNAPolymerase_F555L_UG.pdf>.
4. AQ97 High Fidelity DNA Polymerase 2x Master Mix. Ampliqon. Retrieved September 22, 2024, from <https://ampliqon.com/en/pcr-enzymes/pcr-enzymes/high-fidelity/aq97-high-fidelity-dna-polymerase-2x-master-mix/>.
5. USER manual. NucleoSpin Gel and PCR Clean-up (2023). Macherey-Nagel. Retrieved from <https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf>.
6. Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T., & Halkier, B. A. (2007). USER fusion: a rapid
   and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Research,
   35(7), e55. <https://doi.org/10.1093/nar/gkm106>.
7. Monarch Plasmid DNA MiniPrep Kit Protocol (2015). New England Biolabs. Retrieved from
   <https://www.neb.com/en/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010>.
8. NEB cutter 3.0. New England Biolabs. Utilized September 22, 2024, from <https://nc3.neb.com/NEBcutter/>.
9. Jung, J. K., Alam, K. K., & Lucks, J. B. (2022). ROSALIND: Rapid Detection of Chemical
   Contaminants with In Vitro Transcription Factor-Based Biosensors. Methods in Molecular Biology, 325–342. <https://doi.org/10.1007/978-1-0716-1998-8_20>.
10. Pierce™ Protein Concentrators, PES (2019). Thermo Fisher. Retrieved from <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011811_Pierce_Concent_PES_3K_10K_30K_100K_MWCO_0.5mL_UG.pdf>.

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# Results

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The aim of EndoSense was to develop and test a cell-free EDC biosensor using human nuclear receptors.
This was achieved with the androgen receptor using multiple different DNA templates.
The tests were done using the natural hormones of the receptors. Additionally, we aimed to test an in-cell biosensor for proof of concept. This was successfully tested using the natural hormones of the receptors and water samples from various sources.

##### Table of Contents

* [In-cell Assay: Test of EDCs in water samples](#In-cell assay)
* [Cell-free Biosensor: EndoSense Proof of Concept on EDCs detection](#Cell-free biosensor)

# In-cell assay: Test of EDCs in water samples

The initial test of the in-cell biosensor consisted of creating **dose response curves using the natural hormones** for the receptors. The LacZ values were converted to a percentage of the maximum value for easy comparison between the curves. Unfortunately, we were not able to obtain progesterone to test the PR, and for MR we saw very high standard deviations and no significant response (data not shown). The four functional dose-response curves are shown in [Figure 1](#fig1) below.

![Dose response curves](https://static.igem.wiki/teams/5107/eng-incellresults1.webp)

Figure 1: Dose-response curves for the in-cell assay. Percent maximal response is shown for easy comparison. The concentration of the added hormone is shown (not the concentration in the reaction). All measurements were done in triplicate and standard deviations are represented by error bars. The sample positive controls are shown from left to right; ERb, ERa, AR, GR.

Finally, the system was tested on four water samples. We tested two different types of bottled water, tap water, and a lake sample from a local lake. As a positive control, the natural hormones were added to the tap water sample to show that we can replicate the data from the dose-response curves. These positive controls are shown in [Figure 1](#fig1) and fit the dose-response curves. All data from the test of the water samples is shown in [Table 1](#table1) below:

Table 1: Overview of percent maximal response
for the in-cell assay on water samples. All measurements were done in triplicates and standard
deviations are represented by error bars. Colors correspond to the receptors shown in [Figure 1](#fig1).

|  | Bottled water #1 | Bottled water #2 | Tap water | Lake water |
| --- | --- | --- | --- | --- |
| ERa | 1.3% ± 5.9% | 5.3% ± 3.7% | 5.3% ± 3.6% | -0.8% ± 3.0% |
| ERb | 2.4% ± 16.6% | 1.4% ± 6.0% | 5.3% ± 4.9% | -1.8% ± 1.8% |
| GR | -13% ± 50.9% | 9.4% ± 11.3% | 10.5% ± 10.8% | -2.2% ± 31.5% |
| AR | 15.7% ± 53.4% | 28.9% ± 24.7% | 28.3% ± 24.0% | 33.7% ± 53.0% |

None of the measurements displayed in  [Table 1](#table2) are significantly different from the lowest value on the corresponding dose-response curve in [Figure 1](#fig1). Therefore we can conclude that **none of these water samples contain EDCs binding the tested receptors** above our measurement threshold.

# Cell-free Biosensor: EndoSense Proof of Concept on EDCs detection

During the development of the biosensor, we started by optimizing the DNA quantity, buffer conditions, and detection methods. Then we proceeded to test the ligand's influence and inhibitory effects. We observed that the ligand affected the fluorescence output even in the absence of the receptor. We accounted for this influence when testing the complete biosensor system with the receptor included, by relatively increasing the signal when the ligand was added.

We demonstrated the capability of detection of the EndoSense biosensor as **proof of concept** by using the androgen receptor, since it was the most recommended by our interactions in the human practices and our own custom DNA template: T7-HREmin-sB-T. We used the hormone, testosterone, a natural ligand of the receptors to prove the functioning.

The detection of the EDC is recognizable by the difference in the adjusted signal between the presence and absence of the ligand ([Figure 2](#fig2)). The output signal is expressed as a standardized value as we included in each measurement a serial dilution of Fluorescein Sodium Salt (FSS), from the distribution kit, and normalized the values as Micromolar Equivalent Fluorescein (MEF).

![Figure 2: Statistical significance of ligand detection](https://static.igem.wiki/teams/5107/engineering/proof-of-concept-motherfucker3.webp)

Figure 2: Difference in signal between presence and absence of the testosterone hormone (indicated as *H*, at 4 µM) with the androgen receptor (*R*), and the DNA template (at a concentration of 10 nM). For each time point, a statistical t-test was performed between the two shown measurements. The p-value for each test is shown as stars symbols above the top point.

As we saw an increase in the signal, opposite to the decrease we were expecting instead, we reconsidered the working mechanism of the biosensor. As our data would suggest, the EndoSense biosensor seems to behave in an OFF/ON way, meaning that without the EDC, there is a low output. On the other hand, the presence of the ligand will result in an increase of the signal. This working mechanism is shown in the [Figure 3](#fig3) below.

![Figure 3: Statistical significance of ligand detection](https://static.igem.wiki/teams/5107/results/actual-working-mechanism.webp)

Figure 3: OFF/ON behavior of the EndoSense biosensor. When the water sample does not contain EDCs, the fluorescence signal is inhibited. When EDCs are present, this will result in the transcription of the broccoli aptamer and ultimately a fluorescence signal.

From this analysis, **we show how EndoSense can detect an EDC in the form of testosterone**, with a high statistical significance. This proof of concept shows great improvement from the current detecting methods, as the EndoSense biosensor can reduce EDC testing time from days to hours.

From our modeling analysis, we predicted the biosensor's **Limit of Detection** (LOD). We found an upper LOD of 8.39 µM and a lower LOD of 0.1 µM. While the current detection limit may be higher than ideal for real-world sample analysis, it has not yet been fully tested. Consequently, there is potential for improvement as we refine the system, since we have only done a few tests.

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