Team:British Columbia/Project Phage

Introduction

The goal of the Phage and Phage standard sub-team was to develop the new phage standard for submission to the BioBrick registry and to test the characteristics of the phage that would be used for our wet lab experiments.

The Phage Standard

The phage standard presented itself as a necessity once we made the decision to use a phage as a vector to complete our project goals. As we explored it further it became obvious that the phage standard is a useful addition to the BioBricks registry as a whole.

The necessity of the phage standard stems from some key factors. First, lysogenic phages are natural vectors. They have evolved to insert and propagate their DNA through specific bacterial strains. Second, it is impossible to work with phages using the existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Thirdly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids.

Our project goal was to negate the issues from genome size, exploit phage characteristics for use as a vector and develop a BioBrick compatible standard applicable to all lysogenic phages.

The Details

The phage standard is the process of adding some source DNA into the genome of a lysogenic phages, referred to as host DNA. This will require secondary DNA sequences including the integration site DNA, the low-copy number BioBrick plasmid

The first step of the phage standard is to decide on which restriction enzymes to use. Using webcutter 2.0 (Link) it is easy to see all restriction enzyme sites that are present multiple times, present once or not present in the genome. For this standard you need to identify the cut site that appears only once, closest to the region of the phage that you are intending to modify. Next you need to find 2 cut sites that do not appear anywhere in a) The phage genome, b) The BioBrick plasmids and c) The DNA you are inserting into the phage.

First, lysogenic phages are a natural vector - they are designed to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only in a very specific location in the genome (INSERT REFERENCE HERE). This specificity of integration allows the integration sites of different lysogenic phages to be used as insertion vectors. These insertion vectors (example shown below in Figure 1) will be a low-copy BioBrick plasmid containing the integration site of a given phage, flanked by chosen restriction sites.

• Every phage genome contains multiple instances of illegal cut sites
• Phage genomes are too large to be manipulated using normal BioBrick plasmids.
• Phages are inherently useful due to their specific nature and ability to insert DNA into bacterial genomes

Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.

Approach

Results & Discussion

The Wet-Lab Phage

Since our wet lab experiments were focusing on S. Aureus biofilms we had a finite list of phages to choose from. We originally chose to work with phage φMR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose φ11, a prophage found in S. Aureus strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage φMR11 and S. Aureus strain φ11.