The experimental workflow can be divided into two distinct concurrent projects. The first involves the engineering of the encapsulin-MBP system which will be responsible for the bioaccumulation of arsenic. This system is designed to be modular, with all components contained on one operon meaning Golden Gate cloning can easily be applied to substitute in various encapsulins and MBPs to optimise the system for different organisms and environments. The second involves designing a phage delivery system. This allows for targeted expression of our construct within a chosen environment, which can easily be modified to target different species without the need to genetically modify the species directly.
To increase our chance of successful gene expression we have chosen four species to apply our platform to, with up to four phages for each species. We have selected two model species, B. subtilis (gram positive) and P. putida (gram negative); and two species more representative of species found in pollution sites, C. glutamicium (gram positive) and V. natriegens (gram negative). E. coli will also be used to produce proteins for purification and testing of protein properties such as binding and localisation to encapsulin.
Before translating this system to specialist bacteria commonly found in heavy-metal polluted sites, we will use recombinant expression in E. coli as a proof of concept. The formation of nanocompartments and their capacity to internalise metal binding proteins (MBPs) will be assessed using native PAGE, negative stain transmission electron microscopy (TEM), and isothermal titration calorimetry (ITC). Using our control constructs, ITC and TEM in particular will provide information regarding the degree to which these nanocompartments can internalise heavy metals, where we will use antimony as an arsenic analogue for these experiments. After this characterization, the most promising encapsulins and MBPs will be transformed into specialist alkaliphilic bacteria using a phage-based delivery platform, and expressed under species-specific regulatory elements.
To get stable expression of our constructs within bacteria we have decided to use lytic phages, preventing the virus from going into a lysogenic phase and becoming dormant. We have identified multiple candidate phages for each bacterial species of interest, and will test which phages are best at expressing foreign proteins by using GFP as a marker gene for successful expression. This will allow for the phage expression system to be developed before our encapsulin-MBP operons are ready for expression. Our gene constructs will be inserted into the structure and assembly portion of the viral genome through homologous recombination, and successful clones selected using Cas13 counterselection. This will allow us to select against phages without successful insertion of our gene. Further modification of phages such as removal of lysis genes, or modification of structural proteins may be pursued to optimise properties such as viral specificity and bioaccumulation capacity.
1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5mL centrifuge tube.
2. dd 10 μL Membrane Binding Solution per 10mg of gel slice. Mix and incubate at 50-65°C until gel slice is completely dissolved.
3. Insert SV Minicolumn into Collection Tube.
4. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
5. Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
6. Add 700μL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
7. Repeat Step 4 with 500μL Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.
8. Empty the Collection Tube and centrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
9. Carefully transfer Minicolumn to a clean 1.5mL microcentrifuge tube.
10. Add 50μL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 1 minute twice
11. Discard Minicolumn and store DNA at 4°C