Team:UM Macau/Design
Experiment Design
Design Overview
Our project, Biofilm Removing E. coli for Aquarium Cleaning (BREAC) aims to engineer the E. coli strain of bacteria to be capable of attaching to biofilms. Along with that, expressing the enzymes that are able to biodegrade our biofilm targets. Our project demonstrates the concept of biofilm degradation in an aquarium system. In the future, teams can use our result as a reference for the design and method on how to engineer microorganisms for cleaning purposes in aquariums. The application of our genetically modified organism would be safe, cheaper and have very high efficiency.
The project demonstrates a biofilm cleaning system for aquariums that utilizes our BREAC. Biofilms have and secrete a molecule called Acyl homoserine lactone (AHL), which can be detected and combined with LuxR(BBa_K3630003) in our E. coli, and promote the degradation of the biofilm via the expression of enzymes. Initially, we only had this detection and degradation system. However, after interviewing the ocean park staff, they raised several concerns with us. First, the aquarium ecosystem requires bacteria in the biological filter to remove the toxic ammonia and nitrogen compounds from the waste. These bacteria usually are in the dark environment. Through the addition of a light inducible system, we would prevent our BREAC from degrading useful bacteria in the aquarium ecology. The second problem is about the removal and clearance of our BREAC. To address this, we decided to make our bacteria magnetizable. We chose to over express ferritin to accumulate Fe3+ion to let our biofilm become positive. In addition to that, we will also make a magnetic field near the filter side to attract and extract our bacteria. The design of our bacteria has evolved to have three features, a light inducible system, a biofilm detection and degradation system, and a magnetization system.
Our bacteria construction will be divided into 3 parts. Originally, we were only constructing AHL-LuxR detection system. However, after getting insights of the real conditions in the aquarium and concerns on our BREAC’s application; we have optimized and evolved our design further. The optimization includes constructing a plasmid containing a light switchable mechanism and magnetization system. Both system would be mainly using the BL21 strain of E. coli , which is suitable for protein expression as it lacks the proteases Lon and OmpT that may affect the integrity of protein. However, we were not able to start the testing for the two systems due to time constraints. With all these additions, we hope that our BREAC would be able to perform better its purpose.
The core part of our project construction is related to the mechanism of biofilms. AHL are the primary quorum sensing molecules utilized by Gram-negative bacteria. Available molecules of AHL increases along with the bacteria population number and they are secreted into the extracellular environment. It is expected that the biofilm density in aquariums is going to be high, therefore we expect that there are enough AHL molecules in the extracellular environment that would help our BREAC detect and degrade biofilms.
Our engineered bacteria contain the LuxR promoter (PLuxR (BBa_K3630005)), an adhesion protein is located downstream of it. The adhesion protein, known as antigen 43 (Ag43) (BBa_K3630010), a self-recognizing outer membrane protein, will stay on the surface of our engineered bacteria. Ag43 is an autotransporter protein which has an adhesion site that is exposed to the extracellular environment. As a result, our bacteria will be able to bind on the biofilm.
After the bacteria binds on the biofilm, more AHL will combine with LuxR (BBa_K3630003), which is under the T7 promoter (BBa_K3630000). This event activates the pLuxR (BBa_K3630005) even more, resulting in the expression of other constructs as well. Others constructs mainly focus on expressing different types of enzyme. The enzymes that we are using are Alkaline serine protein (BBa_K3630009), Dispersin B (DspB)(BBa_K3630007), DNaseTA(BBa_K3630008). All of which are under the pLuxR (BBa_K3630005). We have tried to construct the plasmids. However, due to the Covid-19 pandemic, we did not have sufficient time to perform the practical experiment to examine the expression levels and degradation efficiency of each of the enzyme both in the lab and in the aquarium system.
Overall design
In order to prevent our bacteria from degrading useful biofilm which normally exists behind the filter of the aquarium system where no light could reach. We need to a conditional expression system. Hence, we designed a light sensor system to prevent our BREAC from activating in a dark area. Our design has two parts: The light sensor system synthesis and the light sensitive LuxR expression and repression system.
Figure 7 below, shows our Light sensor system synthesis design. Our light sensor system utilizes a light sensitive complex Cph8 (BBa_I15010) made by the 2004 UTAustin iGEM team. This chimeric complex has three parts: The phytochrome Cph1, a transferase EnvZ histidine kinase domain, and finally a chromophore called Phycocyanobilin. This complex translocates onto the membrane of our bacteria. However, Phycocyanobilin (PCB) is not naturally produced in E. coli. Therefore, we have to express heme oxygenase 1(Ho1) (BBa_K3630013) and phycocyanobilin reductase (PcyA) (BBa_K3630014), both of which would be converting Haem (which naturally exist in E. coli) to PCB.
Ho1 (BBa_K3630013) and PcyA (BBa_K3630014) are required for chromophore synthesis in photosynthetic light-harvesting complexes. In PCB pathways, Haem first undergoes cleavage towards biliverdin. Ho1 (BBa_K3630013) induces the formation of a stable Haem. HO1 (BBA_K3630013) and PcyA (BBa_K3630014) induces protein-protein interactions.
After PCB has been synthesized, it responds to light intensity and binds to the Cph1 Phytochrome form a complex. When Cph1 absorbs light, it induces a conformational change in the complex which inhibits the autophosphorylation capability of the EnvZ histidine kinase domain. In the natural EnvZ/OmpR regulatory system, EnvZ histidine transfers phosphate to OmpR after which the OmpR will binding onto the OmpC (BBa_K3630015) promoter region to regulate the expression of downstream genes. Our OmpC promoter in the context of our system is a dark dependent promoter and will only express genes downstream of it when our BREAC is in the dark. We utilize this mechanism in our second part of the light sensitive expression system.
In Figure 3 above, shows our design for the Light sensitive LuxR expression and repression system. We use the constitutive promoter (BBa_J23106) to constitutively express LuxR so that under illuminated conditions or red light conditions our LuxR detection system would still be active. Since we want our bacteria to not produce the digestive enzymes when it’s in the dark: We have incorporated a Lac operon (BBa_K3630002) just downstream of the Constitutive promoter (BBa_J23106) for the LuxR expression system. We then put the Lacl (BBa_K3630020) gene under the OmpC (BBa_K3630015) promoter where OmpR would bind. In a dark environment or far-red illumination, our BREAC would express the Lacl (BBa_K3630020) gene and LacI (BBa_K3630020) would bind to the Lac operon (BBa_K3630002) to repress transcription of our LuxR protein. Summarized in Figure 4 below, through our light conditional expression system, we would be able to detect AHL and produce adhesive proteins and digestive enzymes to help degrade biofilms, when our BREAC has been exposed to an environment filled with light. But, under dark or Far-red illumination conditions, such as in the biofilters of the aquarium, our BREAC would not be able to detect AHL and thus prevent the degradation of important aquarium biofilms.
As our BREAC is a microorganism that could tip the balance of the aquarium ecosystem, its removal is another necessary problem that we have to address. Aquariums usually have a filter to clean the water. However, it cannot guarantee the removal of our bacteria. Hence, our design has evolved to include to have the third feature of the project, that is, the magnetization system. When a magnetic field is applied, E. coli can be magnetized and stick on the magnet and it could be cleared.
Our team learned the mechanism from the former UM iGEM team that by overexpressing FtnA which can encode for a protein complex called ferritin (BBa_K3033014). Consisting of 24 protein subunits which forms a shell to store and crystalize iron ions, these crystallized forms of irons have paramagnetic properties. Thus, they found that the overexpression of FtnA (BBa_K3033014) proteins and help E. coli to store more crystallized Fe3+ ions and give render E. coli magnetizable.
As a contribution to their idea, we have modified FtnA (BBa_K3033014) and increase the iron storage capacity for magnetic manipulation. We have found a protein called Inkabox-PAK4cat which can spontaneously form crystals when expressed in mammalian cells. In order to increase the amount of iron that can be stored. In our design, FtnA (BBa_K3033014) is linked to iBOX (BBa_K3630017) and PAK4 (BBa_K3630018). We expect the resulting complex Ftn-PAK4 would be crystallize accumulate more Fe ion to enhance the sensitivity of our BREAC to magnetic force. With this improvement, we can make an external magnetic field which can attract and remove our engineered bacteria from the aquarium to collect our BREAC and prevent it from being released to the environment.
Optimized construction:
Reference
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