Proof of Concept
Our product QBlock would serve as a treatment for chronic wounds, particularly in their initial phases. Commonly, they manifest as a subsequent complication of longer-lasting health issues. Most chronic wounds have biofilms, which are mediated by quorum sensing. Our goal with the QBLOCK project is to reduce biofilm development by preventing bacterial autoinducing peptides (AIPs) from inducing other bacteria or the bacteria themselves through the use of DARPins (Designed Ankyrin Repeat Proteins). We were needed to answer a few crucial questions in order to demonstrate our capacity to accomplish these actions necessary for the final product and its intended effect.
- Is the DARPin production and the process of their affinity selection feasible?
- Can our bioreporter gauge the extent of quorum sensing and can the pathway be inhibited?
- Can QBlock be used in a practical setting?
Several experiments were undertaken to address these questions. Figure 1 shows the workflow of the project.
The Process of Affinity Selection and DARPin Production
DARPin library
In order to find a DARPin that would effectively bind to our peptide of interest AIP, we designed and optimised a DNA library of different DARPin molecules by modifying and randomising specific amino acid positions so that the biofilm formation in Staphylococcus epidermidis (S. epidermidis) would arrest. For wet-lab experiments, we synthetically designed and ordered 42 DARPin molecules due to the lack of finance support and time. Our experiments would have been undertaken with the initial library of 1023 unique DARPin molecules if sufficient time and resources had been available, thereby increasing the success of DARPin selection via ribosome display. For additional information about our DARPin library, please refer to Model page.
Affinity selection via ribosome display
To determine the optimal DARPin, we conducted a ribosome display assay which is an affinity selection method.In light of this, we intended to determine if our pool of sequences possessed an AIP-binding DARPin effect. After successfully completing the ribosome display, we forwarded the reverse transcribed products to the sequencing unit. In addition to the 42 sequences, we performed in silico computational simulations in order to validate the binding characteristics and probabilities of our DARPins.Through the NGS (next-gen sequencing) data we received, we can conclude that the ribosome display assay worked as an affinity selection method for our DARPins, and that a DARPin would bind our target molecule AIP. To learn more about our ribosome display results, please refer to the Results page.
Expressing and purifying a DARPin
To effectively suppress quorum sensing with a DARPin, the AIP-binding DARPin must be produced and purified independently. As a control for our ability to complete these processes, we employed a GFP-binding DARPin with a known sequence. We were able to produce DARPin in E. coli BL21 cells in large quantities and purify some of it. Optimising the expression environment to increase the yield of the purified product would have been the next step. Refer to the results to understand more about our accomplishments in DARPin expression and purification.
Measurement and inhibition
Measuring quorum sensing levels with a bioreporter
Our bioreporter was engineered to recognize the AIP molecules and trigger the expression of GFP upon receptor binding. Using the bioreporter, we demonstrated that AIP can successfully bind to its receptor and induce GFP expression. Our DARPin's ability to block AIP binding could be demonstrated using a bioreporter. Previous attempts to block this pathway have been effective, so we can confidently say that the discovery of an AIP-binding DARPin will likewise block this pathway.(Rémy et al., 2018).
Modelling
For the modelling, the protein structures of our DARPin molecules were predicted to further explore their molecular characteristics and possible binding sites. Second, we projected protein-peptide docking in silico making use of a variety of softwares to find out which of the chosen and optimised DARPin sequences would have the best likelihood of binding to the AIP peptide. After analysing the data, we saw that our molecules exhibited a certain pattern and degree of binding phenomena. Read more here.
Inhibition of the pathway
Attempts to inhibit the quorum sensing pathway, particularly those based on AHL and AI-2 (Rémy et al., 2018), have been made on many stages. These pathways are known to be inhibited by quorum quenching enzymes in nature and can also be synthetically produced. This can be accomplished, for instance, by using tiny compounds to interfere with the quorum sensing pathway, such as a DARPin in our case. Quorum quenching, as proposed by Rémy and coworkers, has great promise in reducing pathogenicity and biofilm formation.
QBlock in the real world
We have interviewed numerous medical experts and gathered first-hand accounts of living with chronic wounds. According to the comments we've received so far, both wound care professionals and their patients have a pressing need for a unique treatment that can be easily coupled with other treatments and has the potential to prevent the wound from chronifying in the first place. Several testing would need to be done to make sure QBlock is safe and effective before it could be released to the public. Refer to implementation if you want to know what to do next with bringing QBlock into the real world..