iGEM 2022 — Team tudelft

{% extends "layout.html" %}
{% block title %}NOTEBOOKS{% endblock %}
{% block lead %}This page contains the notebooks of all the different Wet Lab modules, where all the experiments done are reported.
{% endblock %}
{% block page\_content %}

* [Notebook Module 1](#section2)
* [Notebook Module 2](#section3)
* [Notebook Module 3](#section4)
* [Notebook Module 4](#section5)

[![](https://static.igem.wiki/teams/4361/wiki/notebook/notebook1.jpg "Notebook module 1")](https://static.igem.wiki/teams/4361/wiki/notebook/notebook-1-production-purification-and-characterization-blcr.pdf)

## Notebook module 1

Module 1 aimed at producing and purifying BlcR, our transcription factor of interest. Furthermore, it consisted of the characterization of BlcR, to determine its optimal conditions. To find out more, you can read our digital notebook by clicking on its front page.

## Notebook module 2

The goal of Module 2 was to engineer BlcR to increase its binding affinity to its DNA sequence, by mutating teh BlcR binding domain. We first created a rational design of the amino acid substitution we wanted to carry out, we then sequenced the mutants to determine whether the mutations were successful and we lastly screened the mutants with a fluorescent assay.

[![](https://static.igem.wiki/teams/4361/wiki/notebook/notebook2.jpg "Notebook module 2")](https://static.igem.wiki/teams/4361/wiki/notebook/digital-notebook-module-2.pdf)

[![](https://static.igem.wiki/teams/4361/wiki/notebook/notebook3.jpg "Notebook module 3")](https://static.igem.wiki/teams/4361/wiki/notebook/notebook-3-engineering-blc-operator.pdf)

## Notebook module 3

With Module 3, we wanted to test the affinity of the wild type BlcR to the modified BlC operator sequence.

## Notebook module 4

Module 4 consisted of all the techniques used to immobilize DNA to the gold surface of the elctrode and then convert the BlcR dissociation to an electrical output.

[![](https://static.igem.wiki/teams/4361/wiki/notebook/notebook4.jpg "Notebook module 4")](https://static.igem.wiki/teams/4361/wiki/notebook/labbook-m4.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}ENGINEERING SUCCESS{% endblock %}
{% block lead %}In the development of our project, we faced multiple obstacles. By implementing the design → build → test → learn cycle, here we present how we overcame all the challenges.
{% endblock %}
{% block page\_content %}

* [Introduction](#section1)
* [Optimize protein production and purification](#section2)
* [Immobilization and electrical measurements](#section3)

## Introduction

To achieve the goal of our project, that being creating a rapid biosensor to detect the rape drug GHB, we went through different engineering cycles. We first divided our project into smaller subprojects which counted as separate modules. The different modules are:

* **Module 1:** we aim to produce, purify and characterize BlcR.
* **Module 2:** we try to engineer BlcR to get a better binding affinity to its DNA sequence.
* **Module 3:** we aim to modify the *blc* operator sequence to get a higher binding affinity of BlcR to its binding sequence.
* **Module 4:** we aim to optimize the immobilization technique of DNA to the gold surface of the electrode and convert the dissociation of BlcR to an electric output.

As we worked through the different modules, we faced several challenges. We utilized the design → build → test → learn cycle to overcome them and consequently improve them (**Figure 1**).

In particular, we implemented the cycle in two specific cases: to optimize protein production and purification of BlcR and to verify BlcR association and dissociation from its specific DNA sequence with Atomic Force Microscopy (AFM).

![Diagram of the stages of the engineering cycle](https://static.igem.wiki/teams/4361/wiki/engineering/figure-1-cycle-interations.png)

**Figure 1.** Diagram of the stages of the engineering cycle.

## Optimize protein production and purification

Our sensor is based on utilizing the BlcR transcription factor from Agrobacterium tumefaciens. To create our sensor different experiments with pure BlcR are performed. Because our sensor is based on this transcription factor it was important to optimize the production and purification method. Literature reports indicated that BlcR could be actively produced in *E. coli*  [[1][2]](#references) . Therefore, we designed a proper plasmid for BlcR production in BL21(DE3) cells (**Figure 2**).

![Production plasmid BlcR](https://static.igem.wiki/teams/4361/wiki/engineering/figure-2-plasmid-mad-blue2.png)

**Figure 2.** Production plasmid for BlcR. pET11a plasmid is used as a backbone and the gene coding for BlcR is used as an insert.

The chosen plasmid was a pET11a production plasmid , which could be induced with IPTG for protein production. For easier protein purification with an affinity column, a His-tag was inserted in the N protein terminal, together with a TEV protease site to remove the His-tag if needed. (**Figure 3**).

![Sketch of T7-tag, His-tag, TEV site and BlcR sequence](https://static.igem.wiki/teams/4361/wiki/engineering/figure-3-t7tag-his-tev.png)

**Figure 3.** BlcR gene upfront TEV site, his-tag, and T7-tag.

For our downstream experiments in the project, we required a highly concentrated pure protein, diluted in a suitable DNA binding buffer with low salt concentration. We went through several protein production and purification cycles to determine the best protein production and purification protocol.

#### First iteration

**Design:**

To investigate DNA/BlcR binding affinity, we utilized isothermal titration calorimetry (ITC). However, this method requires a low salt buffer to avoid interference in the DNA/BlcR binding. We found that the iGEM bielefeld team from 2015, which also worked with BlcR, utilized a low-salt storage buffer: 50 mM HEPES, pH 7.2 as the storage buffer of BlcR  [[2]](#references) . We then decided to try this buffer as both a storage and binding buffer. We produced the BlcR in *E. coli* BL21(DE3) followed by purification with a His-tag affinity column. After His-tag purification, we decided to concentrate the protein solution and dialyse it from the elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM Imidazole, 10% glycerol, pH 7.5) into the storage buffer (50 mM HEPES, pH 7.2) (**Figure 4**).

![Flowchart production and purification cycle 1](https://static.igem.wiki/teams/4361/wiki/engineering/figure-4-pp-flowchart2.png)

**Figure 4.** First production and purification process scheme of BlcR.

**Build:**

BlcR was produced in BL21(DE3) cells and purified with His-tag purification columns. 50 mM HEPES, pH 7.2, pH adjusted with NaOH was prepared as a storage buffer for BlcR.

**Test:**

After the standard protein production and purification, we obtained a stock of 98 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 05/07/2022). After the concentration and dialysis steps, we checked the protein concentration again and found a concentration of 1.6 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 08/07/2022). The protein concentration decreased by 98%.

**Learn:**

Our hypothesis for this enormous decrease in concentration is that the protein got stuck in the membrane of the concentrator. We used a concentrator tube with a 30.00 MWCO membrane. Since our protein forms 70 kDa dimers, we hypothesized that the dimers started to form in the solution and they could not pass through the membrane when concentrating the sample.

#### Second iteration

**Design:**

To maintain a high protein concentration, we decided to do the protein purification again but this time without concentrating the protein (**Figure 5**). For atomic force microscopy (AFM) measurements, we needed to dialyze BlcR into 20 mM MOPS.

![Flowchart production and purification cycle 2](https://static.igem.wiki/teams/4361/wiki/engineering/figur-5-pp-flowchart3.png)

**Figure 5.** Second production and purification process scheme of BlcR.

**Build:**

BlcR was produced in BL21(DE3) cells and purified with His-tag purification. 50 mM HEPES, pH 7.2, pH and 20 mM MOPS, 2,5 mM magnesium, pH 7, and pH adjusted with NaSO4 were prepared as storage buffers for BlcR.

**Test:**

In this trial, protein production and purification was performed without the concentration step. The protein solution was dialyzed into two different buffers; MOPS and HEPES. After dialysis, we had a lot of protein precipitation (**Figure 6**). Therefore, we again lost a high amount of the initial protein concentration. After elution, the protein concentration was 86 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 25/07/2022) and after dialysis, the protein concentration in the HEPES buffer was only 8 µM and in the MOPS buffer 17 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , date: 03/08/2022). The concentration of 17 µM of BlcR in the MOPS buffer was good enough for AFM experiments and therefore successful.

![Picture of precipitation of BlcR after dialysis.](https://static.igem.wiki/teams/4361/wiki/engineering/figure-6-precipation.png)

**Figure 6.** Protein solution after dialysis. Protein was dialyzed from the elution buffer (50 mM Tris-HCl, 300 mM imidazole, 300 mM NaCl, pH 7.2) to the storage buffer (50 mM HEPES, pH 7.2).

**Learn:**

We saw some improvements in the protein concentration without the concentration step in the purification cycle, but we got a lot of precipitation when we performed dialysis. We hypothesized that the protein precipitated due to the absence of salt, which is generally needed for protein stability. The elution buffer contained 300 mM of NaCl, but when changing buffers to a buffer with no salt, the protein started to precipitate. When performing the ITC experiments we found no activity of our protein [TU Delft,  [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D)  section]. When digging more into literature and having expert advice on protein purification and activity, we decided to drop our efforts to have a no-salt salt concentration in the storage buffer. Since these conditions do not give the ideal environment for good protein activity and stability.

With the MOPS buffer we got a higher protein concentration than with the HEPES buffer, we still wanted to look for a possible storage method in the HEPES buffer since we had limited resources of MOPS and NaSO4 to adjust the pH. MOPS was particularly necessary for electrical experiments, see  [experimental design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experimental-design%27%29%7D%7D) , therefore we wanted to save our MOPS and NaSO4 resources for those experiments.

#### Third iteration

**Design:**

From what we learned in the previous iteration, we decided to include at least 100 mM NaCl in the storage buffer. The new buffer was then: 50 mM HEPES, 100 mM NaCl, pH 7.2.

**Build:**

We made a new storage buffer: 50 mM HEPES, 100 mM NaCl, pH 7.2.

**Test:**

The protein production was performed again following the flow scheme of **Figure 5**. Once again, a lot of the protein precipitated. However, the concentration seemed to have increased compared to the second production cycle. We checked the concentration after dialysis with a Bradford assay and obtained a protein concentration of 16 uM [ [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 17/08/2022]. The concentration of protein had doubled compared to the HEPES storage buffer with no salt. After dialysis, we ran an SDS PAGE. We noticed that some impurities were present in our protein sample, as some protein bands around 83 kDa were visible (**Figure 7**).

![SDS PAGE after dialysis](https://static.igem.wiki/teams/4361/wiki/engineering/figure-7-good-good.png)

**Figure 7.** SDS PAGE to check the purity of our protein of interest. Next to the ladder (L), different concentrations of the protein solution are loaded. 1: 16 uM, 2: 8 uM, 3: 4 uM. Bands between 37 and 49 kDa could correspond to the BlcR monomer and bands between 64 and 82 could correspond to the dimer of BlcR.

**Learn:**

From this engineering cycle we confirmed that the addition of salt gives more protein stability in solution. Unfortunately, the protein concentration is still low and the SDS PAGE showed some contamination in the protein samples. Since we needed a highly pure, and more stable protein stock, we decided to continue with another iteration for protein purification.

#### Fourth iteration

**Design:**

We decided to carry out new production and purification rounds including some more steps to optimize the yield and activity of the protein (**Figure 8**). Moreover, in this iteration we also included extra purification steps for better protein stock purity and quality. We included the TEV protease digestion to avoid any His-tag interference in protein activity, plus a size exclusion purification step. Finally, we also decided to include glycerol in the storage buffer to give more stability to the protein.

![Flowchart production and purification cycle 3](https://static.igem.wiki/teams/4361/wiki/engineering/figure-8-pp-flowchart.png)

**Figure 8.** Fourth production and purification scheme for BlcR. This production cycle included also purification with size exclusion and digestion with TEV protease to get rid of the T7- and His Tag.

**Build:**

In the building phase we prepared a buffer with 50 mM HEPES, 200 mM NaCl, 10 % glycerol, and pH 7.2 for the storage of BlcR. Before size exclusion, the BlcR samples were treated with TEV protease followed by gel filtration to separate the proteins based on their size.

**Test:**

In the testing phase we checked different elution fractions from the size exclusion on SDS PAGE gel (**Figure 8**). Two bands were visible around 37 kDa which most likely indicates BlcR with T7-tag, His-tag, and TEV site (**Figure 3**), and without tags (**Figure 9, 3-5**). In some fractions contamination of other proteins around 115 kDa was visible (**Figure 9, 1-2**). The fractions with pure BlcR were combined and concentration was measured with nanodrop. A concentration of 2.9 uM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 29/08/2022) was measured.

![SDS PAGE after dialysis](https://static.igem.wiki/teams/4361/wiki/engineering/figure-9-good.png)

**Figure 9.** SDS PAGE of BlcR samples after size exclusion. Next to the ladder (L), different fractions from the size exclusion are loaded. 1: A9, 2: B5, 3: C1, 4: C2, 5: C4, 6: C8, 7: C12, 8: D1.

**Learn:**

When we used size exclusion to purify the protein we got higher purity but less concentration. Moreover, we also found that TEV protease was not 100% efficient. After purification we still saw some BlcR + tags present in the eluted sample (**Figure 9**).

#### Fifth iteration

**Design:**

Up until now we had problems in maintaining protein concentration during the purification cycle. In every purification step, we seem to lose a lot of protein. We talked to another expert in the field of protein purification, Theo van Laar, from TU Delft. He suggested eluting with TEV protease instead of imidazole. In theory, only the BlcR with the TEV site would come off the Ni-NTA beads resulting in a pure protein sample. We also decided to change the storage buffer one more time to have a higher salt concentration. The storage buffer was now 50 mM Tris, 300 mM NaCl, 0.5 mM EDTA, and pH 7.5  [[1]](#references) .

![Flowchart production and purification cycle 4](https://static.igem.wiki/teams/4361/wiki/engineering/figure-10-pp-flowchart.png)

**Figure 10.** Protein purification cycle with his tag elution by TEV digestion.

**Build:**

Tris, 300 mM NaCl, 0.5 mM EDTA, pH 7.5 buffer was prepared and His-tag purification was done with TEV digestion as an elution method.

**Test:**

Since the elution with TEV digestion was not highly successful, only a small portion of BlcR eluted from the column, a very light band is visible around 37 kDa (**Figure 11, 1**).

![SDS PAGE production and purification cycle with TEV elution](https://static.igem.wiki/teams/4361/wiki/engineering/figure-11-sds-page-tev-names.png)

**Figure 11.** SDS PAGE of purification cycle of BlcR. 1: TEV elution 1 2: TEV elution 2, 3: TEV elution 3, 4-5: column wash, 6: column wash, 7: column flow trough, 8: clear lysate.

**Build:**

We decided to do another elution round with imidazole to get all the BlcR out of the purification column.

**Test:**

The elution with imidazole and the sample after dialysis into the Tris storage buffer were checked on SDS page gel. The gel showed digested and undigested BlcR around 40 kDa (**Figure 12, 4-6**). The concentration after elution with imidazole was measured and corresponds to 49.5 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 22/09/2022). We did not notice any protein precipitation after dialyzing the protein into the Tris storage buffer. The protein concentration after dialysis was 36.1 µM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 22/09/2022), which only represented a 27% protein loss. We further purified the protein with gel filtration. We only saw contamination of a protein around 115 kDa in the first elution fractions (**Figure 13, 1-2**). Pure BlcR was present in the other elution fractions (**Figure 13, 3-9**). We measured a final protein concentration of 13.4 uM ( [Notebook 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 29/09/2022).

![SDS PAGE production and purification cycle with imidazole elution](https://static.igem.wiki/teams/4361/wiki/engineering/figure-12-sds-page-names.png)

**Figure 12.** SDS-PAGE gel showing the elution of BlcR. 1: clarified lysate, 2: column flow-through, 3: wash 3, 4-5: imidazole elution, 6: elution after dialysis.

![SDS PAGE production and purification cycle after size exclusion](https://static.igem.wiki/teams/4361/wiki/engineering/figure-13-sds-page.png)

**Figure 13.** SDS PAGE of different elution fractions after size exclusion. 1 : B1, 2 : B3, 3 : B6, 4 : B7, 6: B11, 7 : C2, 8 : C5, 9 : C7.

**Learn:**

From this iteration, we learned that TEV digestion for eluting the protein from the column is not very effective in our case. Moreover, we found that we needed at least 300 mM NaCl in the storage buffer to ensure a minimal precipitation and high protein concentration. With this final protocol, we managed to get a relatively high protein concentration with a good purity for the downstream DNA binding experiments. We used the protein sample to successfully determine the binding to engineered oligos, see results [TU Delft, section  [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%7D%7D) , module 3].

## Immobilization and electrical measurements

For our final GHB sensor , we used an interdigitated electrode with immobilized DNA (*blc* operator sequence) to the gold surface. The transcription factor, BlcR, can bind to this *blc* operator sequence. When GHB is present BlcR will dissociate from the **blc** operator sequence causing an electrical signal (**Figure 14**).

![Systematic overview of our GHB detecting sensor.](https://static.igem.wiki/teams/4361/wiki/design/figure-2-electrical-signal.png)

**Figure 14.**  Systematic overview of our GHB detecting sensor. A DNA sequence specific to the binding of the transcription factor BlcR is immobilized on an electrode that can measure capacitance differences. In the absence of GHB BlcR is bound to the DNA resulting in a more dense environment between the electrodes. In the presence of GHB BlcR will dissociate from the DNA resulting in a change in environment which can be converted to an electrical signal.

We decided to use atomic force microscopy (AFM) to first verify the DNA and BlcR binding and unbinding from in DNA-coated electrodes. With such a technique, we can scan the gold surface and read the differences in height distribution upon BlcR binding and unbinding. We immobilized ssDNA on the electrode with mercaptohexanol (MCH) to avoid nonspecific binding. We then added the complementary ssDNA to obtain a final dsDNA coated surface ready for BlcR binding and unbinding experiments. In total we want to visualize four different stages; an empty gold surface, gold surface with immobilized DNA, gold surface with immobilized DNA and BlcR, and lastly the gold surface with immobilized DNA and BlcR after addition of SSA (**Figure 15**). The conditions for the immobilization of the DNA to the gold surface are examined in different iterations of the engineering cycle.

![Different examples of the coverage of the gold surface that need to be verified with AFM](https://static.igem.wiki/teams/4361/wiki/engineering/figure-13-2-afm-stages.png)

**Figure 15.** Stages of the gold surface to verify binding and unbinding of BlcR to its DNA sequence. First empty gold plate, second DNA immobilization to the gold surface, third addition of BlcR, fourth the addition of SSA.

#### First iteration

**Design:**

For the first ssDNA immobilization trial, we wanted to immobilize the ssDNA onto the gold surface on the electrode. MCH was also immobilized on the electrode to make sure that all of the active gold was covered. We decided to immobilize MCH and ssDNA in a single-step reaction, to reduce incubation time and to have the maximum homogeneity.

**Build:**

A solution was prepared with final concentrations of 2 µM ssDNA and 1 mM MCH, that was added to the surface.

**Test:**

While attempting to immobilize the thiol-modified ssDNA and MCH, we immediately noticed that these solutions resulted in a non-soluble mixture, which is not convenient for the surface coating. See **Figure 16**.

![Photograph of two Eppendorfs with a mixture thiol modified ssDNA and MCH](https://static.igem.wiki/teams/4361/wiki/engineering/figure-14-emulsion-mch-dna.png)

**Figure 16.** Photograph of two Eppendorfs with a mixture of 2 µM thiol modified ssDNA and 1 mM MCH.

**Learn:**

From this first binding attempt, we decided to do a sequential ssDNA and MCH binding. The MCH was applied after the thiol-modified ssDNA had first been immobilized on the electrode. Additionally, we found that the electrodes were not sufficiently flat, which led us to use sonication in the subsequent iteration.

#### Second iteration

**Design:**

To make sure that both the ssDNA and the MCH can bind to the gold surface we decided to make separate solutions of ssDNA and MCH and first incubate the electrode with the thiol-modified ssDNA, followed by an incubation with MCH. In addition to sequential binding, we also decided to add a sonication step. With such, we expect to see a flatter electrode surface (**Figure 17**).

![Flowchart of stages of the immobilization of DNA and MCH on a gold plate](https://static.igem.wiki/teams/4361/wiki/engineering/figure-14-2-2.png)

**Figure 17.** Immobilization flowchart. First immobilization of thiolmodified ssDNA, second immoblization of mercaptohexanol, third addition of the second ssDNA.

In the first iteration, the electrodes were not sonicated since it was not expected that this would be necessary. In this iteration round, we decided to sonicate the electrodes to obtain a flatter surface.

**Build:**

Stock solution of 2 µM ssDNA and 1 mM MCH were prepared separately. And the immobilization of ssDNA and MCH was done in a two-step reaction.

**Test:**

To verify the binding of the DNA and BlcR, Atomic Force Microscopy (AFM) was used. AFM analyzes the surface of the electrode by scanning it with a cantilever and measuring the force needed to go through the sample.

![Two AFM pictures. One of an empty electrode and one of a electrode with DNA](https://static.igem.wiki/teams/4361/wiki/engineering/figure-15-afm.png)

**Figure 18.** Atomic Force Microscopy pictures of interdigitated electrodes (IDE). SNL-10B probe (frequency = 4.365 kHz, spring constant = 0.101 N/m). a) Empty IDE in MOPS buffer scale up to 35 nm. b) IDE with immobilized dsDNA scale up to 240 nm. See  [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D)  (27/07/2022 and 03/08/2022).

A couple of things could immediately be noticed. First, an empty interdigitated electrodes (IDE) was scanned. However, it could be seen that the surface was not flat, instead it had peaks up to 35 nm (**Figure 18a**). It would be too rough to notice protein binding or dissociation. Second of all, when 2 µM DNA (**Figure 18b**) was immobilized on the empty electrode, heights of up to 240 nm were measured (**Figure 18b**).

**Learn:**

We hypothesize that the differences in height during the measurements were clumps formed by oxidized DNA through thiol-thiol interactions. To avoid this in our next attempt, we decided to introduce a reduction step in our protocol. We also noticed that the clean electrode was not as flat as we wanted it to be. We then decided to introduce an extra cleaning step for having a flatter surface before continuing with the binding experiments.

#### Third iteration

**Design:**

To avoid oxidized-DNA clumps in the gold plate, we decided to introduce a reduction step before DNA surface binding. We treated the ssDNA with DTT for 15 minutes. Furthermore, before binding experiments, we decided to add an extra step of sonication to better clean the gold surface. This time we also decided to sonicate the electrodes as it could help in visualizing the DNA better.

**Build:**

Before immobilizing the ssDNA to the gold surface the IDE was sonicated. 100 µM stock of ssDNA was reduced with 10 mM DTT before immobilization to the gold surface. The DTT was then extracted with ethyl acetate.

**Test:**

Again, AFM was used to analyze the gold surface. We saw that extra sonication step helped with obtaining a flatter surface. (**Figure 19a**). Here, the highest point on the surface was 30.4 nM compared to 35 nm without sonication (**Figure 17a**). Moreover, we found that the DTT DNA treatment also helped. This time we did not observe the giant clumps from the last iteration. The highest point measured with reduced DNA is 63.8 nm (**Figure 19b**) compared to the 240 nm (**Figure 18b**) that we saw before.

![Two AFM pictures. One of an empty electrode and one of a electrode with DNA](https://static.igem.wiki/teams/4361/wiki/engineering/figure-16-afm.png)

**Figure 19.** Atomic Force Microscopy pictures of Interdigitated electrodes (IDE). SNL-10B probe (frequency = 4.365 kHz, spring constant = 0.101 N/m). a) Sonicated IDE in MOPS buffer. Scale up to 30.4 nm. b) IDE with 2 µM reduced thiol-modified DNA in MOPS buffer. Scale up to 63.8 nm. See  [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D)  (03/08/2022 and 10/08/2022)

**Learn:**

We concluded that the additional sonification and DTT reduction steps showed better surfaces when scanned using the AFM. We then decided to continue with this protocol for surface preparation and move on to BlcR binding and unbinding measurements.

#### Fourth iteration

**Design:**

We incubated the electrode with immobilized dsDNA with BlcR 2.45 µM for one hour. After this hour, we washed the electrode with a MOPS Mg buffer. The electrode was then analyzed with AFM in a MOPS buffer bath.

**Build:**

BlcR was purified and dialyzed into 20 mM MOPS, 2.5 mM Mg, pH 7. A stock solution of 10 µM BlcR was made and incubated on the IDE with DNA.

**Test:**

Using AFM, no clear difference in height could be seen when BlcR was added to the electrode with immobilized DNA. Only one big blot of 296 nm (**Figure 20**).

![Two AFM pictures. One of an empty electrode and one of a electrode with DNA](https://static.igem.wiki/teams/4361/wiki/engineering/figure-17-afm.png)

**Figure 20.** Atomic Force Microscopy pictures of Interdigitated electrodes (IDE). SNL-10B probe (frequency = 4.365 kHz, spring constant = 0.101 N/m). IDE with DNA and BlcR in a 20 mM MOPS buffer. Scale up to 296 nm. See  [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D)  (04/08/2022)

**Learn:**

We came to the conclusion that BlcR could unbind the DNA with the post-binding washing step that we performed in this iteration. We then decided to avoid this washing step, and take the AFM measurements in the incubation bath with BlcR.

#### Fifth iteration

**Design:**

Before going ahead with more BlcR binding experiments we decided to test atomically flat gold plates instead of interdigitated electrodes on AFM. Even though the electrodes were flatter after sonication (up to 30 nm) (**Figure 19a**), we thought that 30 nm height could hinder the BlcR binding and unbinding signal. Moreover, we also decided to take the AFM image in the same BlcR binding buffer. With this, we avoid washing away BlcR after binding onto the DNA.

**Build & Test:**

After analyzing the new flat gold plate with AFM, we saw that the surface had a maximum height of 14.7 nm (**Figure 21**). This is significantly better than the sonicated IDEs (30 nm) (**Figure 19a**).

![AFM picture of a atomically dlat surface.](https://static.igem.wiki/teams/4361/wiki/engineering/figure-18-afm.png)

**Figure 21.** QI image taken with the AFM of an empty, sonicated gold plate in MOPS buffer. Scale up to 14.7 nm. See  [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D)  (10/08/2022)

**Learn:**

Using atomically flat gold plates gives the flattest results.

#### Sixth iteration

**Design:**

Using the previously tested gold plates, we decided to test BlcR binding and unbinding. We measured the DNA-coated surface height with AFM upon (i) addition of BlcR (binding), and (ii) BlcR + SSA (unbinding).

**Build:**

1 µM of DNA was immobilized on the gold surface. The gold surface with DNA was incubated in a 10 uM BlcR solution. The plate was analyzed with AFM. 15 µM SSA was then added to the solution and the plate was analyzed with AFM once again.

**Test:**

Results show that the DNA adheres to the gold plate effectively (**Figure 22b**). Moreover, upon BlcR addition, the number of dots on the surface increases significantly, indicating an efficient DNA-protein binding (**Figure 22c**) . Finally, when SSA is added to the DNA + BlcR surface, we noticed a significant reduction in the number of AFM detected dots (a decrease of 46 % of peaks after addition of SSA) (**Figure 22d**). This could indicate that the BlcR-DNA unbinding took place effectively.

![AFM picture of the four different stages: empty gold surface, gold surface with DNA and MCH, gold surface with DNA, MCH and BlcR, gold surface with DNA, MCH and BlcR after addition of SSA](https://static.igem.wiki/teams/4361/wiki/engineering/figure-19-afm.png)

**Figure 22.** QI image of (a) an empty gold plate, (b) a gold plate immobilized with 1 µM thiol modified DNA, (c) a gold plate immobilized with 1 µM thiol modified DNA, with 3 µM BlcR and (d) a gold plate immobilized with 1 µM thiol modified DNA, with 3 µM BlcR, with 15 µM SSA. All images are scaled to a maximum value of 35 nm. See  [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D)  (10/08/2022 and 09/09/2022).

**Learn:**

Throughout the iterations of the engineering cycle we developed the best protocol to use AFM as a visualization technique for protein-DNA binding and unbinding. We changed the protocol several times to include sonication, chemical reduction, or even switching to atomically flat gold plates instead of the IDEs. All these choices helped to get the final result where we could BlcR associating and dissociating from the immobilized DNA strands (**Figure 22**).

## References

1. *Pan, Y., Fiscus, V., Meng, W., Zheng, Z., Zhang, L. H., Fuqua, C., & Chen, L. (2011). The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization. Journal of Biological Chemistry, 286(23), 20431–20440.*   [https://doi.org/10.1074/jbc.m110.196154](https://doi.org/10.1074/jbc.m110.196154%20)
2. *Gräwe, A.( March 6, 2019). A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs.*  <https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210940>
3. *Wenjing Xi and Amanda J. Haes. (February 5, 2019) Elucidation of HEPES Affinity to and Structure on Gold Nanostars. Journal of the American Chemical Society 2019 141 (9), 4034-4042.*  <https://doi.org/10.1021/jacs.8b13211>
4. *Karthika Sankar, R. Baer, Chloé Grazon, Robert C. Sabatelle, Sébastien Lecommandoux, Catherine M. Klapperich, James E. Galagan, and Mark W. Grinstaff (April 12, 2022). An Allosteric Transcription Factor DNA-Binding Electrochemical Biosensor for Progesterone. ACS Sensors 2022 7 (4), 1132-1137.*  <https://doi.org/10.1021/acssensors.2c00133>
5. *Sankar, K., Baer, R., Grazon, C., Sabatelle, R. C., Lecommandoux, S., Klapperich, C. M., Galagan, J. E. & Grinstaff, M. W. (2022, 12 april). An Allosteric Transcription Factor DNA-Binding Electrochemical Biosensor for Progesterone. ACS Sensors, 7(4), 1132–1137.*  <https://doi.org/10.1021/acssensors.2c00133>
6. *Chen, Z., Liu, X., Liu, D., Li, F., Wang, L. & Liu, S. (July 9, 2020). Ultrasensitive Electrochemical DNA Biosensor Fabrication by Coupling an Integral Multifunctional Zirconia-Reduced Graphene Oxide-Thionine Nanocomposite and Exonuclease I-Assisted Cleavage. Frontiers in Chemistry, 8.* <https://doi.org/10.3389/fchem.2020.00521>

{% endblock %}


{% extends "layout.html" %}
{% block title %}RESULTS{% endblock %}
{% block lead %}The results from each of the Wet Lab modules, including EMSA, Site-Directed-Mutagenesis, AFM and the electrical measurements are collected on this page.
{% endblock %}
{% block page\_content %}

* [Introduction](#section1)
* [Module 1](#section2)
* [Module 2](#section3)
* [Module 3](#section4)
* [Module 4](#section5)
* [Conclusion](#section6)
* [Future Prospects](#section7)

## Introduction

With SPYKE our goal is to develop a bioelectric sensor for the detection of the rape drug GHB. With our device, we try to protect the user from consuming their spiked drink. Upon GHB presence, the sensor turns on a strong light for the user to know that the drink has been spiked. The light is switched by a capacitance change from the electrode. This change comes from a transcription factor (BlcR)-DNA dissociation caused by GHB presence in the sample. More information about the biosensor design and mechanisms of action can be found  [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experimental-design%27%29%7D%7D) .

The goal of our Wet Lab experiments was to 1) test the binding and dissociation efficiency of WT BlcR to its cognate operator sequence, 2) engineer BlcR protein and/or its DNA binding sequence to obtain better protein-DNA binding and dissociation upon GHB presence, and 3) efficiently convert the protein association and dissociation into a reliable electrical signal. To achieve these three goals, we divided our project into four different modules:

* **Module 1:** we aim to produce, purify and characterize BlcR.
* **Module 2:** we try to engineer BlcR to get a better binding affinity to its DNA sequence.
* **Module 3:** we aim to modify the *blc* operator sequence to get a higher binding affinity of BlcR to its binding sequence.
* **Module 4:** we aim to optimize the immobilization technique of DNA to the gold surface of the electrode and convert the dissociation of BlcR to an electric output.

TU Delft is not in possession of an Opium act license therefore it was not possible to test with the opium drug GHB. We then decided to perform most of our Wet Lab experiments with a GHB analog: succinic semialdehyde (SSA). From literature, we know that binding of SSA to BlcR also causes dissociation of BlcR from the *blc* operator sequence [[1][2]](#references) . Nonetheless, we found our way to validate the efficacy of our biosensor with GHB. We made an appointment with the Trimbos institute, a drug and addiction center in the Netherlands [[3]](#references)  where we were able to test the dissociation of BlcR after the addition of GHB.

## Module 1: Production, Purification and Characterization of WT BlcR

In Module 1, we focused on optimization of the production and purification of BlcR. BlcR was utilized in all the experiments over the different modules therefore highly concentrated and pure BlcR samples were needed. Read in our  [engineering cycle](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D)  about the protein purification to explore how we optimized the protein production and purification cycle.

#### EMSA study for the characterization of BlcR

To characterize the binding of BlcR to its cognate DNA binding sequence (*blc* operator), we used electrophoresis mobility assay (EMSA). The concentration of the DNA was maintained constant while the concentration of BlcR was increased gradually from 0 to 1 µM (**Figure 1**).

We successfully validated binding of BlcR to the *blc* operator sequence. From the results of EMSA we determined the binding affinity of BlcR to the *blc* operator sequence, and established the degree of cooperative binding, expressed in the hill coefficient. We found a binding affinity of 390 nM, together with a hill coefficient of 1.79. These values were not far from what is currently reported in literature. Earlier research of Pan et al. reported a binding affinity of 490 nM [[1]](#references) . The 1.79 hill coefficient was further on used in modeling experiments. Read more about how we calculated the Kd and the hill coefficient in our  [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D)  section.

![EMSA study for the characterization of BlcR binding](https://static.igem.wiki/teams/4361/wiki/results/emsa-blcr-emsa-housestyle.png)

**Figure 1.** EMSA study for the characterization of BlcR binding to the 51 bp *blc* operator sequence. Concentration of Cy3 labeled DNA was maintained at 25 nM. Titration of dimeric BlcR from lane 1 to 10 . 1: 0μM, 2: 0.1μM, 3: 0.25μM, 4: 0.4μM, 5: 0.5μM, 6: 0.6μM, 7: 0.7μM, 8: 0.8μM, 9: 0.9μM, 10: 1μM.

![EMSA study for the characterization of BlcR dissociating in presence of SSA](https://static.igem.wiki/teams/4361/wiki/results/emsa-ssa-emsa-house-style.png)

**Figure 2.** EMSA study for the characterization of BlcR dissociating from to the 51 bp *blc* operator sequence in presence of SSA. Concentration of Cy3 labeled DNA and BlcR was maintained at 25 nM and 1.6 μM respectively. Titration of SSA from lane 1 to 9 . 1: 0, 2: 64 nM, 3: 320 nM, 4: 1.6 μM , 5: 08 μM, 6: 40 μM, 7: 0.2 mM, 8: 1mM, 9: 25 nM DNA only.

#### EMSA study of BlcR with SSA

With our next experiment we wanted to characterize the dissociation of BlcR from the *blc* operator in presence of SSA. We performed an EMSA study where we kept the concentration of BlcR and DNA constant at 1.6 µM and 25 nM respectively, and titrated the concentration of SSA in a range from 0 to 1 mM (**Figure 2**). With this study we successfully established the full dissociation of BlcR in presence of > 40 µM SSA.

We are creating a bioelectric sensor for the detection of GHB in drinks, aiming to fastly spot a spiked drink during the nightlife. Since many drinks contain alcohol, we wanted to make sure that association of BlcR to the *blc* operator is still possible when alcohol is present.

#### EMSA study of BlcR with ethanol

In our next EMSA study we looked at the association of BlcR in the presence of different ethanol concentrations. We found that BlcR still binds to the *blc* operator sequence in presence of ethanol concentrations of up to 20 % (**Figure 3**).

![EMSA study for the characterization of BlcR in the presence of ethanol](https://static.igem.wiki/teams/4361/wiki/results/emsa-ethanol-emsa-house-style.png)

**Figure 3.** EMSA study for the characterization of BlcR binding to the 51 bp *blc* operator sequence in the presence of ethanol. Concentration of Cy3 labeled DNA and BlcR was maintained at 25 nM and 1.6 μM respectively. Titration of ethanol from lane 1 to 6 . 1: 0, 2: 2 %, 3: 5 %, 4: 10 %, 5: 20 %, 6: 40 %.

## Module 2: Engineering of BlcR

To engineer BlcR, we used a rational design approach. Based on reported biochemical and structural data [[1][2]](#references) , we created a mutant library with amino acid substitutions that could possibly result in higher binding affinity of BlcR to the *blc* operator sequence [TU Delft, section  [library](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27site-directed-mutagenesis-library%27%29%7D%7D) ]. We successfully created 20 mutants of BlcR.

Mutants Library

**Table 1.** Sequenced verified constructed mutated plasmids from the BlcR wildtype production plasmid.

| Number | Part registry | Description |
| --- | --- | --- |
| 1 | [BBa\_K4361300](http://parts.igem.org/Part%3ABBa_K4361300) | BlcR D37R |
| 2 | [BBa\_K4361301](http://parts.igem.org/Part%3ABBa_K4361301) | BlcR D37V |
| 3 | [BBa\_K4361302](http://parts.igem.org/Part%3ABBa_K4361302) | BlcR A40V |
| 4 | [BBa\_K4361303](http://parts.igem.org/Part%3ABBa_K4361303) | BlcR S61V |
| 5 | [BBa\_K4361304](http://parts.igem.org/Part%3ABBa_K4361304) | BlcR A62V |
| 6 | [BBa\_K4361305](http://parts.igem.org/Part%3ABBa_K4361305) | BlcR A62I |
| 7 | [BBa\_K4361306](http://parts.igem.org/Part%3ABBa_K4361306) | BlcR A62K |
| 8 | [BBa\_K4361307](http://parts.igem.org/Part%3ABBa_K4361307) | BlcR A62T |
| 9 | [BBa\_K4361308](http://parts.igem.org/Part%3ABBa_K4361308) | BlcR H63T |
| 10 | [BBa\_K4361309](http://parts.igem.org/Part%3ABBa_K4361309) | BlcR H63Y |
| 11 | [BBa\_K4361310](http://parts.igem.org/Part%3ABBa_K4361310) | BlcR L66V |
| 12 | [BBa\_K4361311](http://parts.igem.org/Part%3ABBa_K4361311) | BlcR L66A |
| 13 | [BBa\_K4361312](http://parts.igem.org/Part%3ABBa_K4361312) | BlcR L66I |
| 14 | [BBa\_K4361313](http://parts.igem.org/Part%3ABBa_K4361313) | BlcR A67Q |
| 15 | [BBa\_K4361314](http://parts.igem.org/Part%3ABBa_K4361314) | BlcR A67V |
| 16 | [BBa\_K4361315](http://parts.igem.org/Part%3ABBa_K4361315) | BlcR A67H |
| 17 | [BBa\_K4361316](http://parts.igem.org/Part%3ABBa_K4361316) | BlcR V68T |
| 18 | [BBa\_K4361317](http://parts.igem.org/Part%3ABBa_K4361317) | BlcR V68K |
| 19 | [BBa\_K4361318](http://parts.igem.org/Part%3ABBa_K4361318) | BlcR V68T |
| 20 | [BBa\_K4361319](http://parts.igem.org/Part%3ABBa_K4361319) | BlcR V68S |
| 21 | [BBa\_K4361320](http://parts.igem.org/Part%3ABBa_K4361320) | BlcR L38V |

To screen the mutants, we utilized an already existing superfolded GFP expressing plasmid that was created by the Bielefeld iGEM team 2015: [BBa\_K4361115](http://parts.igem.org/Part%3ABBa_K4361115). This plasmid contains the BlcR binding sequence upstream the CDS of the superfolded GFP. Upon BlcR binding, GFP expression is repressed. Our working hypothesis for mutant screening was: the lower the fluorescence signal, the higher the percentage of BlcR bound to its operator site, the higher the binding affinity of BlcR to the *blc* operator.

Since BlcR WT can be actively produced in *E.coli*, we decided to utilize an E. coli based cell-free system to screen all 20 mutants. With this strategy, we aimed to have a robust screening platform that could bypass the conventional - often cumbersome - protein production and purification of our mutant protein candidates. We used the PURE (Protein synthesis Using Recombinant Elements) system, here PUREfrex2.0, [[4]](#references) .

As a first step to validate our screening strategy, we checked the repression of GFP expression with our purified WT BlcR protein. We prepared a cell-free reaction with 2.8 nM of GFP plasmid supplemented with 0.725 μM of purified BlcR protein. The reaction was then incubated for 6 hours at 37°C.

Furthermore, we verified the specificity of BlcR to the DNA binding sequence. From literature, we found that one single nucleotide deletion in the DNA binding cassette impairs BlcR DNA-binding (**Figure 4**) [[1][2]](#references) . We built a new GFP plasmid (GFPdel plasmid [BBa\_K4361117](http://parts.igem.org/Part%3ABBa_K4361117).) harboring the corresponding nucleotide deletion. We conducted another cell-free reaction but this time with 2.8 nM GFPdel plasmid supplemented with 0.725 μM of purified BlcR protein.

![51-bp <i>blc</i> operator sequence](https://static.igem.wiki/teams/4361/wiki/results/pure-1.png)

**Figure 4.** 51-bp *blc* operator sequence. When the deletion of the nucleotide displayed in blue is introduced, BlcR binding to the DNA sequence is hindered.

End-point measurements of GFP fluorescence showed that BlcR leads to a 55% decrease of GFP signal with the reporter plasmid containing the correct *blc* operator sequence (**Figure 5a**). This result suggests that BlcR can bind to the blc operator and partially represses GFP expression. In contrast, the use of GFPdel plasmid did not result in a decrease of fluorescence, demonstrating that BlcR-induced transcription inhibition is specific to the presence of the cognate *blc* operator site (**Figure 5b**).

![Purified BlcR represses cell-free expression of a reporter gene harboring the cognate <i>blc</i> operator sequence](https://static.igem.wiki/teams/4361/wiki/results/pure-2.png)

**Figure 5.** Purified BlcR represses cell-free expression of a reporter gene harboring the cognate *blc* operator sequence. (**a**) 2.8 nM GFP plasmid ([BBa\_K4361115](http://parts.igem.org/Part%3ABBa_K4361115)) with or without 0.725 µM BlcR. (**b**) 2.8 nM GFPdel plasmid with altered ([BBa\_K4361117](http://parts.igem.org/Part%3ABBa_K4361117)) with or without 0.725 µM BlcR. End-point fluorescence measurements (Excitation: 485 nm , Emission: 528 nm ) after six hours incubation at 37°C ( [Notebook 2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 06/09/2022).

From the EMSA results, we saw that BlcR dissociates from the operator sequence in presence of SSA (**Figure 2**). We then decided to perform a dissociation experiment in the cell-free PURE system by analyzing the end-point fluorescence of the *blc*-containing GFP reporter plasmid in the presence or absence of SSA.

![20 uL PURE reaction](https://static.igem.wiki/teams/4361/wiki/results/pure-3.png)

**Figure 6.** 20 uL PURE reaction: GFP: 2.8 nM GFP plasmid ([BBa\_K4361115](http://parts.igem.org/Part%3ABBa_K4361115)) BlcR : 0.725 μM BlcR, SSA: (**a**) 700 μM (**b**) 7 mM. End point fluorescence measurement (Excitation: 485 nm, Emission: 528 nm ) after six hours incubation at 37°C ( [Notebook 2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 19/09/2022, 21/09/2022).

While GFP intensity consistently decreased (about 63% lower) when purified BlcR was added to the reaction, no significant increase (only 12%) was observed upon addition of SSA (**Figure 6a**). We had expected to recover a similar GFP intensity as in the condition without BlcR. We performed another experiment with a higher concentration of SSA (7 mM vs 0.7 mM), but this did not result in an increased GFP signal (**Figure 6b**).

We hypothesized that SSA could be directly interfering with the expression of GFP in PURE reactions. However, we performed GFP expression experiments with varying SSA concentrations and did not see a strong inhibition. We then decided to continue with this cell-free PURE expression strategy to screen for BlcR-binding DNA variants and resorted to other experimental approaches for assaying SSA-mediated dissociation of BlcR-DNA complexes.

Prior to screening our library of BlcR-binding DNA sequences, we decided to validate the production of WT BlcR in PURE reactions. To this end, we expressed 4 nM plasmid encoding for BlcR and supplemented PURE with GreenLys, a reagent used for co-translational fluorescent labeling of the synthesized products. The reaction solution was analyzed by SDS PAGE and the fluorescently labeled proteins were visualized with the Typhoon instrument. Compared to the control reaction that did not contain DNA, two protein bands corresponding to the expected size of the BlcR dimer (~70 kDa) and the BlcR monomer (~35 kDa) could be detected (**Figure 7**). These results demonstrate that BlcR can efficiently be expressed in the PURE system, where it can oligomerize in dimers.

![Production plasmid BlcR](https://static.igem.wiki/teams/4361/wiki/results/pure-4.png)

**Figure 7.** SDS PAGE showing that BlcR is cell-free expressed in PURE system supplemented with GreenLys. 1: PURE without plasmid. 2: PURE with BlcR WT production plasmid [BBa\_K4361106](http://parts.igem.org/Part%3ABBa_K4361106). 4 hours incubation 37°C ( [Notebook 2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 06/09/2022).

In light of these results with the WT BlcR we decided to investigate if our designed BlcR mutants could be expressed in the PURE system. All 20 plasmids coding for different mutants were individually expressed in PURE reactions supplemented with GreenLys. We found that not all the mutants could be produced in the PURE system. For the mutants D37R, A62V, L66V, and A67H, no protein bands corresponding to the monomeric BlcR were visible. Expression of all the other mutants led to a protein band that is not detectable in the negative control, corresponding to the monomer of BlcR. An upper (fainter) band that does not appear in the negative control and that may correspond to the dimer of BlcR can also be seen in most of these mutants. In conclusion, most of the selected mutants can effectively be produced in an E. coli based cell-free system (**Figure 8**).

![SDS PAGE showing the translation products of 20 different plasmids](https://static.igem.wiki/teams/4361/wiki/results/pure-6.png)

**Figure 8.** SDS PAGE showing the translation products of 20 different plasmids coding for BlcR mutants. PURE reaction solution was supplemented with GreenLys and the synthesized proteins were imaged with Typhoon using a 488-nm laser. C: negative control with no plasmid. WT: positive control with expressed wildtype BlcR. Numbers corresponding to the mutants as displayed in **Table 1**. ( [Notebook 2](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 10/09/2022).

We finally examined whether the cell-free expressed BlcR was able to repress transcription of the GFP reporter gene containing a *blc* site. Unfortunately, protein activity could not unambiguously be demonstrated, most probably because the buffer composition is not compatible for functional BlcR folding. Therefore, we decided to not use the PURE system as a platform to test the activity of the different BlcR mutants. For activity assays, we recommend to use purified BlcR in combination with the *blc*-containing GFP reporter plasmid.

## Module 3: Engineering of the BlcR operator

To complement the protein engineering approach taken in module 2, this module focuses on the design of novel *blc* operator sequences to which BlcR binds. To improve the affinity between BlcR and DNA, we designed and screened variations of the known operator sequence and evaluated their ability to bind to BlcR. You can find an overview of all the different DNA sequences in **Table 2**. We used automated gel electrophoresis to determine the fraction of BlcR bound to the engineered oligos.

Moreover, we performed DNA binding measurements with isothermal titration calorimetry (ITC). However, we did not obtain conclusive results. ITC is highly sensitive to contaminants and we could not reach the required purity and protein concentration needed to avoid background noise.

To validate our screening method by automated gel electrophoresis, a control experiment was first conducted with samples of the WT operator sequence and a random DNA sequence, both with or without addition of BlcR. Here, we aimed to determine the positions at which the free DNA and DNA bound by BlcR migrate. As **Figure 9a** shows, DNA is significantly retained in the samples containing BlcR and appears as a band in the region of 250 base pairs, while free DNA appears clearly as a band in the region of around 100 base pairs.

![Probing DNA-BlcR binding with automated gel electrophoresis](https://static.igem.wiki/teams/4361/wiki/results/3-1.png)

**Figure 9.** Probing DNA-BlcR binding with automated gel electrophoresis. (**a**) ScreenTape D5000 assay of the wild type 140 nM *blc* operator sequence (0) and the negative control 140 nM scrambled sequence (00), with (+) and without (-) 8 µM BlcR. (**b**). Fraction of DNA bound to BlcR calculated by electropherogram peak integration ( [Notebook 3](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 04/10/2022).

Using this same method, a library of 20 operator sequence variants was screened to evaluate their affinity to BlcR. Read why we chose those operator sequences on the  [library](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27site-directed-mutagenesis-library%27%29%7D%7D) page.

Oligonucleotides Library

**Table 2.** Engineered *blc* operator sequences

| Number | Sequence | Description | Part registry |
| --- | --- | --- | --- |
| 0 | ATACTGCAATGTACTTAATAGATCA CCGCTGCTTGCACTAGTGGTTATAAT | Scrambled sequence (51 bp) | [BBa\_K4361000](http://parts.igem.org/Part%3ABBa_K4361000) |
| 1 | CCATAGTTC**ACTCTAATgATTCAAGT** TCA**ATTAGttgaactCTAAT**GCGGG | Original (51 bp) | [BBa\_K4361001](http://parts.igem.org/Part%3ABBa_K4361001) |
| 2 | CCATAGTTC**ACTCTAATgATTCAAGT** TCA**ACTCTAATgATTCAAGT**GCGGG | IR1 repeated (51 bp) | [BBa\_K4361002](http://parts.igem.org/Part%3ABBa_K4361002) |
| 3 | CCATAGTTC**ATTAGttgaactCTAAT** TCA**ATTAGttgaactCTAAT**GCGGG | IR2 repeated (51 bp) | [BBa\_K4361003](http://parts.igem.org/Part%3ABBa_K4361003) |
| 4 | CCATAGTTC**ACTCTAATgATTAGAGT** TCA**ATTAGttgaactCTAAT**GCGGG | IR1 perfect RV 1 + IR2 (51 bp) | [BBa\_K4361004](http://parts.igem.org/Part%3ABBa_K4361004) |
| 5 | CCATAGTTC**ACTTGAATgATTCAAGT** TCA**ATTAGttgaactCTAAT**GCGGG | IR1 perfect RV 2 + IR2 (51 bp) | [BBa\_K4361005](http://parts.igem.org/Part%3ABBa_K4361005) |
| 6 | CCATAGTTC**ATTAGttgaactCTAAT** TCA**ACTCTAATgATTCAAGT**GCGGG | IR2 + IR1 (51 bp) | [BBa\_K4361006](http://parts.igem.org/Part%3ABBa_K4361006) |
| 7 | CCATAGTTC**ACTCTttgaactCAAGT** TCA**ATTAGttgaactCTAAT**GCGGG | R1 outer 5 + IR2 (51 bp) | [BBa\_K4361007](http://parts.igem.org/Part%3ABBa_K4361007) |
| 8 | CCATAGTTC**ACTCTttgaactAGAGT** TCA**ATTAGttgaactCTAAT**GCGGG | IR1 perfect RV 1 outer 5 + IR2  (51 bp) | [BBa\_K4361008](http://parts.igem.org/Part%3ABBa_K4361008) |
| 9 | CCATAGTTC**ACTTGttgaactCAAGT** TCA**ATTAGttgaactCTAAT**GCGGG | IR1 perfect RV 2 outer 5 + IR2 (51 bp) | [BBa\_K4361009](http://parts.igem.org/Part%3ABBa_K4361009) |
| 10 | CCATAGTTC**ACTTGAATcATTAGAGT** TCA**ATTAGttgaactCTAAT**GCGGG | IR1 flip + IR2 (51 bp) | [BBa\_K4361010](http://parts.igem.org/Part%3ABBa_K4361010) |
| 11 | CCATAGTTC**ACTCTAATgATTCAAGT** TCA**ATTAGagttcaaCTAAT**GCGGG | IR1 + IR2 flip (51 bp) | [BBa\_K4361011](http://parts.igem.org/Part%3ABBa_K4361011) |
| 12 | CCATAGTTC**ACTTGAATcATTAGAGT** TCA**ATTAGagttcaaCTAAT**GCGGG | IR1 flip + IR2 flip (51 bp) | [BBa\_K4361012](http://parts.igem.org/Part%3ABBa_K4361012) |
| 13 | CCATAGTTC**ATTAGagttcaaCTAAT** TCA**ACTTGAATcATTAGAGT**GCGGG | IR2 flip + IR1 flip (51 bp) | [BBa\_K4361013](http://parts.igem.org/Part%3ABBa_K4361013) |
| 14 | CCATAGTTC**ACTCTAATgATTCAAGT** TCAGCGGG**ATTAGttgaactCTAAT** GCGGG | IR1 + 5 bp linker + IR2 (56 bp) | [BBa\_K4361014](http://parts.igem.org/Part%3ABBa_K4361014) |
| 15 | CCATAGTTC**ACTCTAATgATTCAAGT** TCA**ATTAGttgaactCTAAT**TCA **ACTCTAA TgATTCAAGT**GCGGG | IR1 + IR2 + IR1 (71 bp) | [BBa\_K4361015](http://parts.igem.org/Part%3ABBa_K4361015) |
| 16 | CCATAGTTC**ACTCTAATgATTCAAGT** TCA**ATTAGttgaactCTAAT** TCA **ACTCTAATgATTCAAGT** TCA **ATTAGttgaactCTAAT**GCGGG | IR1 + IR2 + IR1 + IR2 (91 bp) | [BBa\_K4361016](http://parts.igem.org/Part%3ABBa_K4361016) |
| 21 | CCCGCACCATAGTTCACTCTAATGAT TCAAGTTCAATTAGTTGAACTCTAAT GCGGG | Consensus Sequence found by DTU | [BBa\_K4361021](http://parts.igem.org/Part%3ABBa_K4361021) |
| 22 | CCCGCACTATAGTTCAGCTAATTGAA CTTGAATCATTAGAGTGAACTAT | Strain variant found by DTU | [BBa\_K4361022](http://parts.igem.org/Part%3ABBa_K4361022) |

As shown in **Figure 10**, the DNA variants appear to have a wide range of affinities for BlcR. Some variants (04, 11, 12, 13, 14) show as little binding as the negative control, suggesting no specific affinity at all. Interestingly, the apparent affinity of other variants (07, 08, 09, 21, 22) matches or even exceeds the reference value of the wildtype sequence.

![ScreenTape D5000 assay of <i>blc</i> operator sequence variants with BlcR](https://static.igem.wiki/teams/4361/wiki/results/3-2.png)

**Figure 10.** Screening DNA-BlcR binding with automated gel electrophoresis for a library of operator sequences. (**a**) ScreenTape D5000 assay of *blc* operator sequence variants with BlcR. In every sample 140 nM of DNA variant and 8 µM BlcR were incubated. (**b**) Quantity of DNA seen in the protein-bound band normalized to the sum of free and protein-bound DNA, as calculated by electropherogram peak integration. The reference values of the negative control (00) and wildtype (01) are highlighted ( [Notebook 3](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 04/10/2022)

The 5 variants exhibiting high affinity were selected for a second round of screening, both for confirmation of the previous result and to evaluate the BlcR-DNA unbinding effect in the presence of SSA. **Figure 11** shows that all of the selected variants have similarly increased binding to BlcR with a bound fraction of around 0.83 compared to the wildtype at 0.73. Moreover, the apparent binding affinity of each variant drops when SSA is present, as expected for a functional drug-responsive system. This is evidenced as a decrease in the fraction of DNA bound from an average value of 0.83 to 0.64. This drop is similar amongst the 5 variants, but slightly less than the drop from the wild type DNA sequence. These results suggest that all DNA variants have a higher binding affinity to BlcR compared to the wildtype sequence, without a significant loss of the SSA-dependent dissociation mechanism.

![electropherogram peak integration](https://static.igem.wiki/teams/4361/wiki/results/second-screening-module-3.png)

**Figure 11.** Fraction of DNA in the protein-bound band of *blc* operator sequence variants, as calculated by electropherogram peak integration. Fraction of DNA bound to BlcR is determined before and after addition of SSA. In every sample 140 nM DNA variant, 8 µM BlcR and 25 µM SSA were incubated ( [Notebook 3](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) , 05/10/2022).

Since all 5 variants show a similar increase in affinity, we investigated what their common DNA features are and what the differences are compared to the sequences with low binding affinity. **Figure 12** shows a multiple sequence alignment of the 5 selected variants with the highest apparent affinity (upper five) and 5 variants with the lowest apparent affinity (lower 5). Interestingly, we found that the IR1 region described by Pan et al. as the possible binding site of BlcR [[1][2]](#references) , is not conserved among the high-affinity variants (**Figure 11**).

![Inverted repeats described by Pan et al.](https://static.igem.wiki/teams/4361/wiki/results/3-4-2.png)

**Figure 12.** Inverted repeats described by Pan et al. IR1 displayed in purple and IR2 in blue [[1]](#references) .

The most striking difference between high- and low-affinity variants is that an 8-bp sequence is almost entirely conserved in all high-affinity variants and lacking in almost all low-affinity variants. This suggests that this 8-bp sequence is essential for BlcR-DNA binding. Remarkably, the exact reverse complement of this sequence occurs at the other end of the site, forming a set of inverted repeats (pink outlines in **Figure 13**). Two 5-bp inverted repeats (blue outlines in **Figure 13**) can also be recognized in between the larger ones, although there is no suggestion that these are important for BlcR-DNA binding, as one of them is not conserved throughout the high-affinity variants.

![Multiple sequence alignment](https://static.igem.wiki/teams/4361/wiki/results/3-5.png)

**Figure 13.** Multiple sequence alignment of selected *blc* operator variants showing exceptionally high affinity to BlcR (top: 01-22) or exceptionally low affinity to BlcR (bottom: 00-14). Highlighted in the alignment are the highly conserved outer inverted repeat (pink) and the lesser conserved inner inverted repeat (blue). Wildtype operator sequence 01, with the previously reported inverted repeat regions highlighted, is aligned at the bottom for reference.

This set of inverted repeats does not coincide with the inverted repeats reported in literature (**Figure 12**). Nonetheless, our partner, the DTU iGEM team, compared the analogous operator sequences from different strains of A. tumefaciens, and reported the existence of these other sets of inverted repeats. More of it can be found on our  [partnership](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27partnership%27%29%7D%7D)  page. If we highlight these inverted repeats on a model of the wildtype operator, it shows that the regions are not only symmetrical in sequence, but also in structure (see **Figure 14**). This might suggest that BlcR binds to this site through symmetrical tetramerization.

![The highly conserved outer inverted repeat and the lesser conserved inner inverted repeat visualized](https://static.igem.wiki/teams/4361/wiki/results/3-8.png)

**Figure 14.** The highly conserved outer inverted repeat (purple and pink) and the lesser conserved inner inverted repeat (blue and cyan) visualized in a double-stranded sequence and in modeled double-stranded DNA.

From screening different operator sequences we found that five operator sequences (7, 8, 9, 21, 22) bind BlcR stronger than the original WT sequence. We also found that the similarities between those sequences are in a set of inverted repeats found by the DTU iGEM team, suggesting a different BlcR binding site than previously described in the literature [[1][2]](#references) .

## Module 4: Immobilization and Electrical measurements

The first goal of module 4 was to verify the binding of the thiol-modified BlcR DNA binding sequence on the gold surface of our electrode. We used the  [engineering cycle](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D)  to establish the best immobilization conditions. With Atomic Force Microscopy (AFM), surfaces can be analyzed and displayed in a height distribution pattern. We used AFM to confirm the immobilization of DNA and BlcR on a gold surface. In addition, we conducted a DNA-BlcR complex dissociation experiment to see the difference in height distribution after adding SSA. To reach these goals, we first used AFM to scan an empty gold plate (**Figure 15a**), a gold plate with immobilized *blc* operator (**Figure 15b**), a gold plate with immobilized *blc* operator and added BlcR (**Figure 15c**), and a gold plate with immobilized *blc* operator, added BlcR plus SSA, the GHB analog (**Figure 15d**). To make the pictures comparable across the different conditions, all the height distribution patterns of **Figure 15** are scaled from 0 to 35 nm.

By calculating the number of peaks in the images (using ImageJ) for all resulting AFM scans (**Table 3**), we confirmed that 1) the DNA could evenly bind to the gold plate surface (**Figure 14b**), 2) BlcR is able to bind an already immobilized cognate binding sequence and 3) SSA addition causes some BlcR to unbind the immobilized DNA.

![AFM images](https://static.igem.wiki/teams/4361/wiki/results/m4-1.png)

**Figure 15.** AFM image of (**a**) an empty gold plate, (**b**) a gold plate immobilized with 1 µM thiol-modified DNA, (**c**) a gold plate immobilized with 1 µM thiol-modified DNA, with 3 µM BlcR and (**d**) a gold plate immobilized with 1 µM thiol-modified DNA, with 3 µM BlcR and 15 µM SSA. All images are scaled to a maximum value of 35 nm ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D), 10/08/2022 and 09/09/2022).

 **Table 3.** The amount of peaks in the height distribution patterns visualized with AFM after image analysis.

|  | Empty (dots) | DNA (dots) | BlcR (dots) | SSA (dots) |
| --- | --- | --- | --- | --- |
| With smoothing | x | 9 | 366 | 197 |

With AFM we successfully confirmed the immobilization of thiol-modified *blc* operator onto a gold surface. Moreover, we showed that the already immobilized DNA is accessible for BlcR binding and unbinding. Our next step was to convert BlcR-DNA complex dissociation events into a capacitance change. More information about this can be found in our  [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experimental-design%27%29%7D%7D)  page. To test whether this was possible, we immobilized the *blc* operator sequence (electrode A), and a scrambled DNA sequence (electrode B) to the gold surface of an interdigitated electrode. We measured the capacitance change with vector impedance at 1 kHz after addition of BlcR and SSA.

![Average capacitance values during the vector impedance measurements](https://static.igem.wiki/teams/4361/wiki/results/m4-2.png)

**Figure 16.** Average capacitance values during the vector impedance measurements. (**a**) Electrode A with 1 µM immobilized *blc* operator. (**b**) Electrode B with 1 µM immobilized scrambled DNA. Capacitance was measured without BlcR or SSA addition, labeled ‘DNA’, after incubation with 4 µM BlcR, labeled ‘BlcR’, and after incubation with 150 µM SSA, labeled ‘SSA’ ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D), 23/08/2022).

In **Figure 16**, one can see that with electrode A, the capacitance decreases after addition of BlcR and increases again after incubation with SSA. This specific response is not visible with electrode B, where the capacitance stays in the same range after addition of BlcR and SSA. These results support our hypothesis: when BlcR is added it binds to the *blc* operator sequence replacing the water molecules between the electrodes. Upon the removal of the solvent, the permittivity decreases, which leads to a drop in the capacitance. When SSA is added, BlcR dissociates from the DNA and water molecules can diffuse back between the plates, which in turn increases the permittivity and results in a capacitance increase.

From vector impedance, we moved on to the universal transducer interface (UTI) to measure capacitance. More information about UTI can be found here. The results of the UTI and the vector impedance were comparable to our previous results: we saw a decrease in capacitance when BlcR is added and a subsequent increase after SSA addition. Experiments with scrambled DNA did not show capacitance changes after BlcR and SSA addition, as expected (**Figure 17**).

![Visualization of the average capacitance during UTI measurements](https://static.igem.wiki/teams/4361/wiki/results/m4-3.png)

**Figure 17.** Average capacitance values during UTI measurements. (**a**) Electrode A with 1 µM immobilized *blc* operator. (**b**) Electrode B with 1 µM immobilized scrambled DNA. Capacitance has been measured without BlcR or SSA addition, labeled ‘DNA’, after incubation with 4 µM BlcR, labeled ‘BlcR’, and after incubation with 150 µM SSA, labeled ‘SSA’ ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D), 24/08/2022)

To make our device more accessible, we created an Arduino circuit to measure capacitance. All the information about our circuit can be found in the hardware page. With Arduino, we aimed to turn on a light when a difference of 1.6\*10-2 pF is measured; read our hardware section to understand why we chose this specific threshold. We used Arduino to measure the capacitance after incubating the electrode with 1 µM DNA (*blc* operator: electrode A, scrambled DNA: electrode B) and 4 µM of BlcR. We set the measured capacitance value as a baseline to measure the difference in capacitance following the addition of 150 µM SSA (**Figure 18**). After addition of SSA we measured a capacitance difference 5.4\*10-2 pF with electrode A and 0.0 pF with electrode B (**Figure 18**). The results confirm a possible change of >1.6\*10-2 pF in the presence 150 µM SSA.

![Production plasmid BlcR](https://static.igem.wiki/teams/4361/wiki/results/m4-4.png)

**Figure 18.** Capacitance measurements with Arduino circuits. Baseline electrode with immobilized 1 µM DNA; electrode A: immobilized *blc* operator sequence, electrode B: immobilized scrambled DNA, and 4 µM BlcR. Capacitance difference is measured in the presence of 150 µM SSA ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D), 27/09/2022).

Up until here, all experiments were performed with the GHB analog SSA. We managed to visit the Trimbos Institute, where we could perform capacitance measurement using GHB. For this assay, we decided to use a UTI device due to its accuracy: it can measure the capacitance differences with a standard deviation of 3\*10-4 [[5]](#references) . We incubated different electrodes with 1 µM DNA; *blc* operator: electrode A, scrambled DNA; electrode B and 4 µM BlcR. We aimed to measure the capacitance difference after addition of GHB to a 55 mM final concentration (**Figure 19**).

Results showed an increase in capacitance of 2.5\*10-2 pF after GHB addition on an electrode with immobilized *blc* operator sequence (**Figure 19a**). This increase in capacitance is not shown when GHB was added to the electrode coated with the scrambled DNA (**Figure 19c**). With these final results, we successfully validated that our sensor can measure an > 1.6\*10-2 pF increase in capacitance when GHB is present at a concentration of 55 mM, which corresponds to the typical GHB concentration used to spike drinks [[6]](#references) . This specific change in capacitance could turn on the light of our Arduino circuit. This light is our main biosensor output to warn people that their drink has been spiked.

![Production plasmid BlcR](https://static.igem.wiki/teams/4361/wiki/results/m4-5.png)

**Figure 19.** Average capacitance values during UTI measurements. (**a**) Electrode A with 1 µM immobilized *blc* operator. (**c**) Electrode B with 1 µM immobilized scrambled DNA. Capacitance was measured after incubation with 4 µM BlcR: ‘BlcR’ and after addition of GHB to a final concentration of 55 mM: ‘GHB’ ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D), 04/10/2022)

## Conclusion

The goal of our Wet Lab experiments was to 1) test the binding and dissociation efficiency of WT BlcR to its cognate operator sequence, 2) engineer BlcR protein and/or its DNA binding sequence to obtain improved protein-DNA binding and dissociation upon GHB presence, and 3) efficiently convert the protein association and dissociation into a reliable electrical signal.

With EMSA studies we successfully characterized the binding between BlcR and the *blc* operator sequence and established that protein-DNA interaction occurs in an environment up to 20% of ethanol. Twenty mutants of BlcR were created and production of 16 mutants in an *E. coli*-based cell-free system was verified. By screening the different operator sequences for BlcR binding, we found five oligos that bind BlcR stronger. After sequence analysis we discovered together with the DTU iGEM team another possible binding site for BlcR that has not been described in literature.

Our overall project's objective was to create a functional device that could accurately detect GHB in beverages by turning on a light. From electrical experiments we could conclude that the capacitance signal generated by the dissociation of BlcR to its DNA binding sequence is strong enough to turn on a light to warn the user.

## Future Prospects

#### Engineering of BlcR

We found out that the PURE system is not the optimal environment to screen the cell-free expressed mutants on their activity. Future experiments could either focus on optimizing the PURE system for protein mutant screening, or on a purified-protein screening strategy. The mutants that could successfully be expressed in the PURE system can be produced in E. coli following the optimized protein production protocol we developed through different iterations of the  [engineering cycle](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%7D%7D) . The activity of the mutants can then be determined electrically with Arduino or UTI measurements.

#### Engineering of the BlcR operator

From the results of screening of the different oligos and the sequence analysis performed  [by DTU](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27partnership%27%29%7D%7D)  we concluded that BlcR could possibly bind to different inverted repeats than the ones proposed by Pan et al [[1]](#references). The crystal structure of dimeric BlcR has been reported [[1]](#references), but a crystal structure of tetrameric BlcR bound to the *blc* operator sequence is still missing. For future experiments we propose crystallizing BlcR bound to DNA to better visualize the tetrameric-DNA binding structure of BlcR. This information could help to optimize the bioelectrical sensor:

* When the position where BlcR binds on the DNA is known, amino acids involved in the interaction with DNA can be targeted for site-directed mutagenesis. Mutants can therefore be created more specifically to increase the binding affinity of BlcR to its DNA sequence.
* Once the binding site of the DNA is revealed, we could engineer longer oligos with multiple binding sites to implement in our sensor. From literature we know that increasing the amount of transcription factors bound to one oligo could improve the sensitivity of electrical biosensors [[7]](#references).

#### Immobilization and Electrical measurements

From the EMSA results we learnt that BlcR is still active with ethanol concentrations up to 20% (**Figure 3**). When the bio-electrical system was tested with 10% ethanol during the UTI measurements we saw that the signal became very unstable ( [Notebook 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D) ). Because of this instability in the electrical output we were not able to detect an increase of capacitance after addition of GHB. Our sensor is based on drinks, often alcoholic drinks, therefore it is of great importance to make it work in an environment with alcohol up to 20%. Further experiments are needed to determine the cause of the system’s instability, and subsequently tackle the issue to ensure the correct functioning of our product.

## References

1. *Pan, Y., Fiscus, V., Meng, W., Zheng, Z., Zhang, L.-H., Fuqua, C. and Chen, L. (2011). The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization. The Journal of Biological Chemistry, [online] 286(23), pp.20431–20440.*   doi:10.1074/jbc.M110.196154
2. 2015.igem.org. (n.d.). Team:Bielefeld-CeBiTec - 2015.igem.org. [online] Retrieved on 10 June 2022. Available at:  [https://2015.igem.org/Team:Bielefeld-CeBiTec](https://2015.igem.org/Team%3ABielefeld-CeBiTec)
3. GHB. (2020, 31 December). Jellinek. Consulted on 8 October 2022. Retrieved 25 August 2022. Available at: <https://www.jellinek.nl/english/drugs/ghb/>
4. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
5. Smartec Sensors - UTI Interface. (z.d.). Retrieved on 8 October 2022. Available at:. <https://smartec-sensors.eu/cms/pages/products/uti-interface.php>
6. Castagna, G. (2022, 3 October). Trimbos-instituut – Voor mentale gezondheid (home). Trimbos-instituut. Retrieved on 8 October 2022. Available at:  [https://www.trimbos.nl/](https://www.trimbos.nl/%20)
7. Sankar, K., Baer, R., Grazon, C., Sabatelle, R. C., Lecommandoux, S., Klapperich, C. M., Galagan, J. E. & Grinstaff, M. W. (2022, 12 april). An Allosteric Transcription Factor DNA-Binding Electrochemical Biosensor for Progesterone. ACS Sensors, 7(4), 1132–1137.  [https://doi.org/10.1021/acssensors.2c00133](https://doi.org/10.1021/acssensors.2c00133%20)

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