Team:British Columbia/Project Phage

Introduction

The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.

The phage standard presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.

The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.

The Details

The phage standard describes the process of adding a given Biobrick part, which we will call source DNA into the genome of a lysogenic phage, referred to as host DNA. This will require secondary DNA sequences including the phage genome integration site, some garbage DNA (flanked by essential restriction enzyme cut sites) and the low-copy number BioBrick plasmid.

The first step is to choose restriction enzymes by using webcutter 2.0 to select restriction sites that appear only once, closest to the region of the phage genome that is going to be modified. See Figure 1 below.

Next, 2 cute sites are found which are absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the 2 absent cut sites. Please see Figure 2 for more details, note that A1 refers to the first absent restriction enzyme site and A2 refers to the other absent restriction enzyme site.

Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid. Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid. The state of the plasmid is shown below in Figure 3.

Lysogenic phages have evolved 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 at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as insertion vectors. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.

Results & Discussion

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.

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.