Introduction
Synthetic biology is a multidisciplinary area which involves a whole range of disciplines such as molecular biology and engineering in order to create products that can solve many health, agricultural, and environmental challenges. For instance, in the agriculture sector, there is a growing need to solve issues such as food security, nutrition, and crop production. The solution may lie in engineering synthetic metabolic routes to produce crops that are tolerant to climate change, more nutritious, and less reliant on fertilizers [1].
Surface plasmon resonance (SPR) is a real-time, label-free method to obtain quantitative information about the interactions between DNA-protein, protein-protein, and protein-small molecule. Such quantitative information includes the dissociation equilibrium constant (KD), the rate of association (kon), and rate of dissociation (koff) values. The determination and tuning of these parameters can lead to optimization of synthetic biological pathways to make engineered products with desired characteristics. Examples include studying transcriptional control, screening synthetic zippers, and designing biosensors for different applications. This blog will highlight these examples to showcase the power of SPR to perform rapid, quantitative verification of these interactions to provide a clearer picture of the binding kinetics and affinities of the biomolecules involved. Then, research examples demonstrated by Affinite’s P4SPR™ will also be shown.
SPR in Synthetic Biology Research
SPR was used to collect KD, kon, and koff, to elucidate a clear picture of how a transcription factor called LEAFY (LFY) affects the AGAMOUS (AG) gene expression by performing multiple mutations along the second intron of AGAMOUS (AG) to produce two different intron variants (Figure 1). The mutations led to an increase in KD for both intron variants compared to the wild type intron. Using data from SPR analysis, the researchers were able to verify the binding positions of LFY to AG and compare the binding affinities of the transcription factor to multiple sites of a long DNA molecule [2].
Figure 1. SPR setup to study the binding affinities and kinetics of LFY to multiple sites for various intron mutants of AG. Double-stranded (ds) DNA was immobilized to the SPR chip via a streptavidin-biotin linkage.
Synthetic zippers are used for scaffolding proteins to increase product production by concentrating enzymes and substrates. A collection of KD, kon, and koff values was achieved for combinations of synthetic zipper pairs when SPR was used to study their binding interactions (Figure 2). This created a rich resource for finding synthetic zipper pairs with desired KD, kon, and koff values, which would be useful for protein scaffolding applications [3].
Figure 2. A general setup for SPR assay to study binding interactions between different pairs of synthetic zipper. Synthetic zipper B-coupled single domain antibody D (ricin binding) was immobilized on ricin-covered surfaces. Synthetic zipper A and B represent zippers of different amino acid sequences.
A glucose biosensor was designed by using SPR as a tool to assess the binding affinity of a protein to glucose for potential glucose monitoring in human blood samples (Figure 3). The glucose/galactose-binding protein (GGBP) was mutated to change its binding affinity towards glucose. A triply mutated GGBP resulted in a KD that matched closely to the lower glucose concentration range found in human blood. Thus, SPR was able to evaluate the binding affinity of glucose to GGBP, and GGBP can be further mutated to exhibit different binding affinities towards a full physiological range of glucose concentrations [4].
Figure 3. SPR scheme for testing various GGBP mutants towards the binding of glucose. GGBP was immobilized via EDC/NHS or thiol coupling.
Affinité's P4SPR™ in Synthetic Biology Applications
Affinité's P4SPR is a revolutionary SPR instrument that provides real-time kinetic and affinity measurements in a compact and portable design. It is very user-friendly; even individuals without extensive lab skills can learn how to use it in a short amount of time. There are two kinds of microfluidic cell (4- vs. 2-channel) to choose from, and it comes with its own software with fitting capability. Most importantly, complex biological samples such as human serum (see second example below) can be used. Please read further to view examples of synthetic biology applications that have used the P4SPR to investigate binding interactions.
The P4SPR was used to study the interaction of lacl repressor with the lac operon (Figure 4). The double-stranded DNA of the lac operon was immobilized onto a SPR chip via a streptavidin-biotin linkage. The KD value was determined by exposing the DNA-immobilized surface to increasing concentrations of the lacl repressor. A KD value of 6.4 ± 1.2 nM was obtained, which agreed well with literature values. Moreover, sensorgrams could be collected in real-time within 10 min.
Figure 4. a) SPR setup of the lacO and lacl binding assay. lacO was immobilized via a streptavidin-biotin linkage; b) sensorgrams obtained from different concentrations of lacl repressors.
A SPR platform was designed to detect antibodies specific for the SARS-CoV-2 nucleocapsid protein [5]. The nucleocapsid protein was first immobilized onto the SPR chip via EDC-NHS chemistry. The sensor surfaces were then exposed to increasing antibody concentrations in human serum and the P4SPR collected the data in real-time. The SPR signal correlated with the increase in antibody concentration. Furthermore, after surface immobilization, each sensorgram was collected within 15 min. Therefore, this SPR platform sets precedence for a potential sensor to detect immunity in individuals that have been naturally infected by SARS-CoV-2 or vaccinated with a COVID vaccine.
Figure 5. SPR biosensor to detect antibodies specific for the SARS-CoV-2 nucleocapsid protein. The nucleocapsid protein was immobilized using EDC/NHS chemistry.
Conclusions
SPR is a rapid, real-time, label-free method to obtain affinity and kinetic data for protein-DNA, protein-protein, and protein-small molecule interactions applicable to the synthetic biology field. In addition, Affinité's P4SPR is a portable, compact, and user-friendly device. The gold sensor chip is customizable for different types of immobilization strategies and can be used for complex biological samples.
The Affinité Advantage
Affinité Instruments’ P4SPR™ is a very user-friendly instrument that is equipped with a gold sensing chip can be tailored to accommodate all types of surface chemistries. In addition, samples do not need much sample preparation and can be directly injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.
Simplicity - Fast training, fast results
Versatility - Pharmaceutical, biosensing, assay development applications
Economy - Affordable, accessible
We help life science labs and biotech companies to do rapid assay development and characterization. Feel free to reach out to us about the expertise we offer at info@affiniteinstruments.com
References
[1] Marc-Sven Roell and Matias D. Zurbriggen. The impact of synthetic biology for future agriculture and nutrition. Curr. Opin. Biotechnol. 2020, 61, 102-109.
[2] Edwige Moyroud, , Mathieu C. A. Reymond, Cecile Hames, Francois Parcy, and Charles P. Scutt. The analysis of entire gene promoters by surface plasmon resonance. Plant J. 2009, 59, 851-858.
[3] George P. Anderson, Lisa C. Shriver-Lake, Jinny L. Liu, and Ellen R. Goldman. Orthogonal Synthetic Zippers as Protein Scaffolds. ACS Omega 2018, 3, 4810-4815.
[4] Helen V. Hsieh, Zachary A. Pfeiffer, Terry J. Amiss, Douglas B. Sherman, J. Bruce Pitner. Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein. Biosens. Bioelectron. 2004, 19, 653–660.
[5] Abdelhadi Djaileb, Benjamin Charron, Maryam Hojjat Jodaylami, Vincent Thibault, Julien Coutu, Keisean Stevenson, Simon Forest, Ludovic S. Live, Denis Boudreau, Joelle N. Pelletier, Jean-Francois Masson. A Rapid and Quantitative Serum Test for SARS-CoV-2 Antibodies with Portable Surface Plasmon Resonance Sensing ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12118914.v1