One of the main points of discussion that we have encountered is the bacteria of the biosensor. The main issues are related to the presence of our biosensor bacteria in the human body. 

The bacteria need to be contained in some way that allows analytes to reach it while preventing the bacteria from dispersing in the body. One option we explored was encapsulating or immobilizing the cells in a porous polymer with pores large enough to allow bacteriophages and signaling molecules to go through, but not the E. coli.

At the same time, the biosensor needs to be placed in such a way that the light sensor is able to detect the signal it emits. There should be enough bacteria present such that a strong enough signal is produced. For this, some modeling could be done where the sensitivity threshold of the device is linked to the amount of bacteria. Further experiments should be carried out to determine what the function of light produced by a certain amount of bacteria is. Then, the total population of our biosensor required to pass the detection threshold of the device can be calculated.  

To overcome potential problems with passing the detection threshold, the cells could be immobilized at the end of the optic fiber cable used to detect the signal. Additionally a shorter cable length should also decrease the detection threshold, requiring less bacteria to be present.

Another important concern is whether the bacteria can actually survive the human body. Depending on the position of the biosensor, the bacteria will either be exposed to the blood, the interstitial fluid, or the synovial fluid around the joint. More research needs to be done to compare the different amounts of nutrients, metabolites and oxygen present in these fluids compared to the required amounts for cell survival. 

Perhaps the most important point regarding the bacterial cells is their biocontainment. A detailed biocontainment approach can be found in the [Safety page]. The method we thought of was physical containment, but leakage can occur. In the case of leakage, a different killswitch should be implemented. One idea would be adding specific antigens on the surface of the cell that could be recognized by the human antibodies and subsequently eliminated. CRISPR-based killswitches have also been recently reported for applications in gut bacterial treatments, using chemical or temperature induction [1]. 

Bacteria also tend to be present in two forms, either as planktonic or in a biofilm, which means that the biosensor bacteria is also capable of forming a biofilm. It is still unclear what induces bacteria to go into biofilm mode, but it involves several environmental and transcriptional factors [2-3]. Bacteria actually are mostly present in nature in biofilm form, as it increases the colony's chances of survival. Through differentiation, bacterial cells in a biofilm can assume different roles in order to divide labor amongst themselves [4]. 

If we want our biosensor bacteria to survive long-term, perhaps having them in biofilm form might actually be more beneficial, providing there is a way to avoid them escaping the physical containment. The downside would be reduced metabolism and dispersion of inducers amongst the population. Lab experiments can be conducted where different sizes of biosensor biofilm are exposed to expected concentrations of inducer and measuring their fluorescent output. A balance can be found so that the population produces a signal that is above the detection threshold.

However, we want to avoid pathogens forming biofilms on the outside of the enclosure. This makes the choice of material more difficult, as it should promote biofilm formation inside the enclosure but not outside. 

One idea we had that would solve most of these issues is having the bacteria physically contained outside of the body and administering purified phage+Dispersin B constructs upon the signal of an infection. Subsequently, a non-living biosensor strategy could be implemented that senses biofilm formation and induces phage production in the bacteria. Researchers have found non-invasive as well as constant monitoring systems for biofilm sensing that do not include live bacteria [12-13].

One thing that needs to be accounted for is the amount of cyclic-di-GMP that is expected to be present in the area around the biofilm. In order to model the behavior of our system inside the body, we would need information regarding the diffusion coefficient of cyclic-di-GMP in blood or the fluid where the device would be present, as well as the expected concentrations in and around the biofilm. However, we had a very difficult time retrieving such information, as it is not easy to quantify these parameters. There is not a lot of literature available about cyclic-di-GMP levels in or around cells, as this molecule is quite difficult to detect with conventional lab methods.

One major factor we did not take into account when designing the project is that the biosensor cells also produce cyclic-di-GMP endogenously. The promoter we decided to work with was designed by the CUG China 2022 iGEM team and is able to measure different concentrations of cyclic-di-GMP [10]. What we did not realize, however, was that in their measurements they detected endogenous levels of cyclic-di-GMP and then used a hydrolase to reduce the amount for the subsequent measurements. In this case, any other amounts of cyclic-di-GMP that we would like to measure would be considerably lower than the endogenous amounts already present in the cells, not making a difference in the expression. We had this realization while designing the constructs and experiments, which was too late for us to change the promoter. What we decided to do is to also introduce the hydrolase into our design so that we could reduce endogenous levels of cyclic-di-GMP as much as possible. We also explored the option of knocking out genes responsible for cyclic-di-GMP production in our cells, but we soon realized that the expression system for this signaling molecule is more complex and cannot be easily suppressed [9].

We also struggled to express and test the promoter in our lab, which further made us wonder if it would perhaps be a better idea to switch to a different promoter that is activated by a different signaling molecule. We also toyed with the idea of making the promoter modular by changing the repressor protein that cyclic-di-GMP binds, also referred to as the sensor module, in order to promote expression. In theory, modularity could be introduced by devising a well-working method for obtaining proteins or protein complexes that bind specific analytes, or by providing a library of several repressor proteins/protein systems for specific targets.

Some questions we ran into while researching phage therapy and phage applications in biofilm treatment were also related to the specificity of the phages. We initially decided to use specific phages for E. coli, since that would be easiest to test in the lab. However, we received feedback that it might actually be a better idea to use non-specific phages since oftentimes biofilms are not composed of solely one species of bacteria. A cocktail of different phages can also be released as a temporary treatment, however, that would require intensive cloning efforts. 

Phages can be administered via various routes [6]. Besides our biosensor designing, two routes that we thought also had potential in the context of orthopedic infections are intramuscular injections. Perhaps oral administration upon biofilm detection would reduce the complexity of the design and be less invasive. However, the phage construct would need to be able to effectively pass through the digestive tract and an M13 phage in combination with Dispersin B could negatively impact the biofilms in the microbiome. Therefore intramuscular injections may be the most realistic alternative.

Producing a product for medical use requires stringent testing procedures and safety guarantees, for good reason. In the European Union, for a medicinal product to be available for market use, the European Medicine Agency (EMA) has to approve the application [8]. Especially with our set-up that requires a live and metabolizing bacterial host, one can assume the difficulty of getting such a system approved for medical use. Additionally, there are many more advanced options that are less risky and less invasive. However, with the problem of antibiotic resistance of biofilms in particular, we believe that the system of Dispersin B phage display has potential when it comes to medical implementation.