OVERVIEW

To ensure the ability of our system, it is composed of four main biological components. The parts we are using are bacteriophage, a host bacteria, an elastin-like-polymer ( ELP), and a metal binding protein.

Together, the four components will work together to sequester and remove metal ions from a solution.

BACTERIAL HOST

When considering a potential bacteria host that would be compatible with our system, the following criteria must be met.

I) The host can be parasitized by a bacteria-phage amenable to engineering.
II) The host can survive in high metal concentrations.
III) The host can resist temperature fluctuations and aggregation.

Chosen accordingly to the criteria, we have decided to explore the host bacteria: Escherichia coli as our proof of concept as it is a well-studied model organism with defined methods of genetic manipulation and is easily accessible in our lab.

In real-life applications of our system, we will be using methanotrophic bacteria as it is one of the few natural organisms that can survive in the detrimental environment of tailings ponds. By using the methanotrophic bacteria it also ensures a cost-efficient and sustainable system as the bacteria already naturally exists in the tailings pond. The additional benefit of working with E. coli is that results obtained in the system will inform in silico modelling, allowing us to design other phage systems for different organisms.

In summary, the choice of host defines numerous properties of the system in terms of energy input, phage production, and efficiency of removing the metal ions from the oil and mining tailings ponds. Our initial efforts with E. coli will inform our design of other host-phage systems, and alter the way in which our device works to capture and sequester ions in mining and oil tailings ponds.

BACTERIOPHAGE CASPID PROTEIN

The T4 Bacteriophage capsid is an icosahedron with proteins Gp23, 24, and 31 contributing to its assembly. The Highly antigenic and small outer capsid proteins serve as accessory proteins on the outer shell. Capsid proteins from this bacteriophage will be used as a proof of concept for further usage in the finalized construct.

PHAGE SELECTION

In future design application, we have looked at phages that have a host range corresponding to our potential hosts: E. coli as the proof of concept and the methanotrophic bacteria as the real-life bacteria application.

Through the utilization of phage display technology, we will attempt to express ELPs fused to metal binding proteins on the surfaces of the T4 phage capsid for E. coli and in later experimentation, the M4 phage for the methanotrophic bacteria.

The T4 bacteriophage belongs to the phage family Myoviridae and interacts with E. coli. The capsid of this phage has been successfully modified to display peptides and protein domains on the surface. In past experiments, biologically active, full-length foreign proteins were displayed by fusion to SOC (small outer capsid protein) and HOC (highly antigenic outer capsid protein) accessory protein genes found on the T4 capsid surface. In fact, a bipartite T4 SOC–HOC protein display system allowed two different proteins to be displayed on one T4 particle simultaneously. The high abundance of these proteins (960 SOC and 160 HOC molecules per phage capsid) increases the efficiency with which potential metal-binding proteins may function, once attached to this phage.

The M4 bacteriophage will be used in the real-life application of our system in the mining and oil tailing ponds as it is a natural occurring phage that can survive in the environment of the tailings pond.

As a proof of principle, we will be using the capsid protein taken from the T4 phage: gp23, 24 and 31. The proteins gp23 and gp24 are coat proteins making up the phage capsid, and gp31 acts as a chaperone in the gp23 folding process. These proteins will produce an empty viral capsid to test the parts individually with the bacteria. This will allow for further experimentation with the full bacteriophage construct including the other parts.

ELASTIN LIKE POLYMERS