EXPERIMENTAL DESIGN

Introduction

With project SPYKE, we aim to develop a sensitive bioelectric sensor to detect the rape drug gamma-Hydroxybutyric Acid (GHB). Our sensor measures the GHB concentration in drinks and subsequently prevents the user from consuming them if they have been spiked. Inside the sensor, electrical and biological components are incorporated. The three major parts are: an electrode, a transcription factor (TF), and a TFs-specific binding sequence. The protein that we are utilizing is the allosteric transcription factor BlcR from the bacterium Agrobacterium tumefaciens. This plant bacterium is able to use GBL, a precursor of GHB, as an energy source. In the absence of GBL, GHB or SSA, BlcR will bind to the blc operator and acts as a repressor for the transcription of the blc genes. When GBL, GHB, or SSA binds to BlcR, it is released from the DNA and the BlcA, BlcB and BlcC proteins are transcribed and digest GBL to succinate (SA)(Figure 1) [1][2] . A more detailed description of the BlcR pathway can be found here.

We have designed a novel bioelectronic sensor for the detection of GHB in drinks by combining the specificity of the BlcR regulatory mechanism with the reliability of electronics. BlcR is tethered by dsDNA oligonucleotides carrying the blc operator sequence to the surface of a gold interdigitated electrode (IDE). We measure the capacitance of the IDE, which is influenced by the protein molecules near the surface of the electrode. When GHB enters the system, it binds BlcR, causing the TF to dissociate from the electrode which leads to an increase in capacitance. The change in capacitance is then interpreted by the hardware and translated into an output signal to warn the user (Figure 2).

The goal of our Wet Lab experiments is: 1) to optimize the association and dissociation of BlcR to the blc operator and 2) convert the dissociation of BlcR from the blc operator sequence into a capacitance change. To achieve these goals, we divided our project into four different modules:

• Module 1: we aim to produce, purify and characterize BlcR.
• Module 2: we aim 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.

Different lab techniques were used to characterize association and dissociation of BlcR to the blc operator sequence in the absence and presence of SSA or GHB (Figure 3).

TU Delft is not in possession of an Opium act license. Therefore, it was not possible to assay with the opium drug GHB. We decided to perform most of our Wet Lab experiments with a GHB analog: succinic semialdehyde (SSA). Nonetheless, we found a 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 [5], where we were able to confirm the dissociation of BlcR after the addition of GHB.

Module 1: Production, Purification, and characterization of BlcR

Module 1 is aimed at producing, purifying, and characterizing our protein of interest, BlcR. The produced protein was consequently used for further experiments in module 3 and module 4.

Production and Purification of BlcR

For the production of BlcR, an expression vector containing the blcR gene has been designed (Figure 4). A pET-11a expression system in E. coli is popular because it combines a high protein yield with good regulation over the expression. The T7 RNA polymerase has a lacUV5 promoter that is Isopropyl 𝛃-D-1-thiogalactopyranoside (IPTG) inducible. With the addition of IPTG, the gene downstream of the T7 promoter can be transcribed.

The vector is transformed into the BL21(DE3) strain as a first step of the protein production and purification cycle. During our project, we performed different protein production and purification cycles to optimize the process. More information about the optimization can be found on our engineering successes page.

Characterization of BlcR

To characterize the binding of the transcription factor BlcR to its DNA binding sequence, we used Electrophoretic Mobility Shift Assay (EMSA). EMSA's are commonly employed for the analysis of nucleic acid/protein interactions with a native gel system. This gel shift assay allowed us to determine the binding affinity of BlcR to the DNA. Since our protein of interest is an allosteric transcription factor, we also determined the binding affinity of BlcR to its effector SSA.

During a gel shift assay, the bands of free DNA will migrate further in the gel whereas the bound fraction will migrate slowly, resulting in two different layers of bands. Furthermore, if the protein concentration varies across the samples, a change in the intensity of the visible bands is expected. The lower the concentration of BlcR in the sample is, the higher the intensity of the free DNA fragment. However, the opposite is valid concerning the bound fraction bands: the higher the concentration of protein in the sample, the higher the intensity of the bound fraction band.

Furthermore, to facilitate the visualization of this experiment, a fluorescent tag is added to the blc operator sequence DNA. In our case, we used a Cy3 tag, an orange-fluorescent dye. We used the 532 nm laser line and visualized the Cy3 tag with TRITC (tetramethylrhodamine) filter sets. With EMSA, we first aimed to characterize the binding of BlcR to the blc operator sequence by calculating the binding affinity. Second, we aimed to characterize the binding between the effector molecule SSA and BlcR. And finally, we aimed to characterize the binding between the blc operator and BlcR in an alcoholic environment.

Module 2: Engineering BlcR

Module 2 aimed at increasing the binding affinity between the transcription factor BlcR and the the blc operator. To do this, we modified the DNA binding site on the protein.

Increasing binding affinity

Agrobacterium tumefaciens produces the IclR-type family member BlcR, an allosteric transcription factor. BlcR contains two distinct binding sites; the DNA binding domain (DBD), which interacts with the blc operator sequence, and the effector binding domain which detects the effector molecule gamma-Hydroxybutyric Acid (GHB) or the GHB analog succinic semialdehyde (SSA), that allow this protein to bind to both DNA and GHB (or an analog of GHB such as SSA) [2]. (Video 1) . We ran simulations to determine how to make our sensor as accurate as possible, taking the surrounding conditions and the required BlcR concentration into account. Both the interaction of the transcription factor with DNA and GHB were examined. We discovered the endogenous concentration of GHB in certain alcoholic drinks could cause the sensor to disfunction as the required BlcR concentration under these conditions would be too high and the signal-to-noise ratio too low. To improve our sensor we want to maximize the signal-to-noise ratio to exclude false positives and negatives and to and minimize the required BlcR concentration. The low signal-to-noise in our sensor is caused by a high concentration of free BlcR proteins between the capacitor plates. Increasing the binding affinity of BlcR to the blc operator will reduce the amount of free BlcR between the capacitor plates and lower the required BlcR concentration. We concluded that altering BlcR's DBD would enhance the binding affinity, improving the sensor’s performance [TU Delft, section model ].

Modifying BlcR

To increase the binding affinity between BlcR and the DNA binding sequence, we wanted to mutate the BlcR DNA binding domain. We did this by making a rational design of amino acid substitutions in the DNA binding site of BlcR. See the library page to read how we constructed the SDM library.

Site-Directed Mutagenesis

Once the library was created, we used Site-Directed Mutagenesis ( protocols ) to modify and implement the single amino acid substitutions. We based our Site-Directed Mutagenesis technique on the Q5® Site-Directed Mutagenesis Kit from New England Biolabs [6] (Figure 5). This technique is typically used to deliberately and precisely alter the DNA sequence of a gene and its gene products. A DNA primer must be created, which has the desired mutation and is complementary to the template DNA surrounding the mutation site in order to hybridize with the DNA in the target gene. The reverse primer is designed in a back-to-back way with respect to the forward primer. Like this, different forward primers can be combined with one reverse primer [7] . A DNA polymerase was subsequently used to lengthen the single-strand primer, by copying the remaining portions of the gene. After performing Phusion Polymerase PCR, we conducted a Kinase, Ligase, DpnI (KLD) mix reaction. The KLD mix contains three enzymes: kinase, for phosphorylation, ligase for ligation, and DpnI for removal of the plasmid template (Figure 5). DNA sequencing was used to identify mutants and determine whether they carry the desired mutation. To know more about the methods we used, see the protocols page.

Fluorescence assay

A fluorescence assay was designed, in which the fluorescence signal represents the binding of BlcR to its binding sequence. The Bielefeld iGEM team of 2015 created a biosensor for GHB with a fluorescence output [2] . To accomplish this, the team designed a plasmid with the GFP gene upfront of the blc operator (Figure 6).

With this plasmid, the GFP expression is controlled by BlcR. The transcription of GFP is interrupted when BlcR attaches to the blc operator sequence on the plasmid. This plasmid was used by us to characterize the binding of WT BlcR to the blc operator. GFP is expressed using the PURE (Protein synthesis Using Recombinant Elements) system, here PUREfrex2.0 [8] . With the PURE system, we conducted association experiments with the GFP plasmid and already purified BlcR and dissociation experiments with the GFP plasmid, purified BlcR, and SSA (Video 2).

To validate the production of our created mutants in an E. coli based cell-free system, we produced our created BlcR mutants could be expressed in the PURE system.

Module 3: Engineering of the Blc operator sequence

To complement the protein engineering approach in module 2, this module focuses on the DNA sequence 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.

Designing different blc operators

We took a rational approach to engineer the operator sequence, due to the very limited screening power of the methods we intended to use. The sequence described by Pan et al. [4] is 51 bp and contains two inverted repeats (IR1 and IR2) of 17 bp each. We designed variations of the sequence in which the framework of this structure is conserved, by interchanging, inverting, mirroring, and duplicating the existing inverted repeats [TU Delft, section library ].

Additionally, the DTU iGEM team assisted in the design of variations. Through database searching, they discovered analogous operator sequences from different strains of A. tumefaciens and recommended a number of them for screening. You can read more about this research on the Partnership page .

Screening different operators

To observe binding and unbinding, we used isothermal titration calorimetry (ITC), which measures the temperature changes that occur when molecules bind. By titration of one of the two interacting molecules, we can observe how much energy is released or consumed by each titration step. As a larger fraction of the molecules are bound, the solution comes closer to saturation and the energy difference caused by titrations decreases. Plotting the surface area of every peak against the molar ratio results in a curve that shows the process of saturation. From this curve, we can derive parameters like the association/dissociation constants and the molar ratio at which saturation is achieved.

As an alternative method for screening the affinity of various operator sequences for BlcR we used EMSA, a method that we also used for the characterization of BlcR/DNA association in module 1. With this method, free DNA and protein-bound DNA are separated based on their mass by electrophoresis, and we can quantitate how much of the DNA is bound by comparing the two fractions. While in module 1 non-denaturing PAGE was used for EMSA, we opted for automated electrophoresis because it can quickly run multiple samples and DNA is visualized immediately without the need for separate staining or fluorescent labeling.

Module 4: Immobilization, Electrical measurements and Hardware

In module 4, the ultimate goal was to immobilize thiol-modified oligonucleotides on interdigitated electrodes and measure a change in capacitance, caused by the unbinding of BlcR when GHB or SSA, a GHB analog, is added to the solution.

We wanted to verify the attachment of the DNA to the gold surface of the electrode, the binding of BlcR to its specific sequence, and the dissociation of BlcR from DNA in presence of GHB or SSA. Different verification techniques have been used, namely Atomic Force Microscopy (AFM), vector impedance, Universal Transducer Interface (UTI), and a novel Arduino measurement.

Atomic Force Microscopy

With our first verification technique, Atomic Force Microscopy (AFM), the surfaces of samples can be analyzed. The surface of a sample is scanned with a tip attached to a cantilever. The bending of the cantilever is measured by the position of the reflection of a laser beam (Figure 7). The output of an AFM measurement is a height distribution pattern over the sample. The height difference can indicate immobilized molecules on a surface.

With AFM, we aimed to verify the immobilization of the DNA to the gold surface and the binding and unbinding of BlcR to its specific DNA sequence in the absence and presence of SSA. To verify this, we aimed to measure four different height distribution patterns: an empty plate, a plate with immobilized DNA, a plate with immobilized DNA and BlcR, and a plate with immobilized DNA, BlcR, and SSA (Figure 8). To obtain the best immobilization technique, we went through different engineering cycles. Read about the different iterations we went through to obtain the AFM pictures here .

Capacitance measurements

The output of our bioelectric sensor relies on a change in the electrical signal which corresponds to a capacitance change. Capacitance describes the change in electric charge with respect to the change in potential. In other words, it is the ability of a system to store electrical charge. In a system with two plates, the capacitance is dependent on three factors: the permittivity of the solution between electrodes, the area of the electrodes, and the distance between the electrodes. The relation between these parameters is formulated in Formula 1a.

When the area and the distance between two plates are constant, the capacitance change is dependent on the change in permittivity. The permittivity describes the ability of a solution to redistribute charge between the plates in response to an electric field. The relative permittivity (ε_r) is dependent on the absolute permittivity (ε) and the permittivity in vacuum (ε₀), see Formula 1b.

Our sensor consists of an interdigitated electrode (IDE) to measure the change in capacitance. On the gold surface, we immobilized a DNA sequence specific for the binding of the transcription factor BlcR. If GHB or SSA is not present, BlcR is immobilized to the DNA. The surface is now densely covered, making it difficult for water molecules to be present near the electrode. In this scenario, the average distance between the water molecules and the working electrode is high. When GHB is present, BlcR will dissociate from its DNA sequence. This allows water to move near the electrode. Since water molecules have one of the highest relative permittivities, this will increase the relative permittivity. An increase in the permittivity will increase the capacitance, as seen in Formulas 1a and 1b.

Vector Impedance

With a vector impedance meter, the capacitance of an electrode can be measured over a broad range of frequencies. This machine measures complex electrical impedance as a function of frequency.

Universal Transducer Interface

Our second electrical verification method was the universal transducer interface (UTI). It can be used to measure the capacitance more precisely. The UTI measures the unknown capacitance by using two reference capacitances that are precisely known. A picture of the UTI board is shown in Figure 9. The UTI can measure capacitance very accurately with a standard deviation of 3*10^-4 pF [10] .

The system first converts the capacitance to a voltage using a capacitance-to-voltage converter (CVC). This CVC builds up charge over the capacitor and unloads it, building up a square wave voltage signal. This voltage signal is converted into a specific period using a voltage-to-period convertor (VPC). These conversions are done for the unknown and the two known reference capacitors, guided by the multiplexers. A digital divider is used to change the frequency to the desired data acquisition frequency. An external control system is used.

Arduino

The last verification method and the system that was implemented in the final design is a novel Arduino circuit. We created an Arduino circuit that can measure the capacitance change between two electrodes and can turn on a light when the capacitance change is caused by the dissociation of BlcR in the presence of GHB. More information about the circuit can be found here .