Rice/Project A: Phage Project
From 2007.igem.org
Contents |
Phage Project
Background
Motivation
Due to widespread usage of antibiotics, bacteria are developing multiple resistances to overcome the selective pressure put on them by our many “magic bullets.” In bacterial hotspots such as hospitals, these resistances are growing at an alarming and life-threatening rate. Some conventional strategies have been developed to counter this problem including use of large amounts of antibiotics, development of new antibiotics, and utilization of mutually antagonistic antibiotics. These methods tend to be expensive, harmful to the patient, and require a long time to implement. None of these methods remove the selective pressure to develop multiple antibiotic resistances.
Goals
By conferring selective advantage on bacterial population, we intend to artificially create changes in evolutionary fitness landscape such that bacteria harboring antibiotic resistance are competed out.
System and Circuit Design
Bacteriophage Selection - When selecting the bacteriophage to be used in this circuit, we desired a phage whose life cycle and genome had been well characterized. Bacteriophage lambda has all of these characteristics as well as the benefit of being commonly used as a cloning vector. Specifically, the lambda strain we selected can be induced to undergo lytic growth by growing lysogenic cells at 42°C. Also, due to the infectious nature of bacteriophage lambda, we selected a strain that is unable to lyse cells (amber mutation preventing the expression of lytic enzyme, protecting other bacteria within the lab from phage infection). Though the phage will not lyse the cells, it will produce fully functional phage particles that can be isolated via chloroform extraction.
Antibiotic Selection - The antibiotic resistance we will be selecting against in this circuit will be tetracycline. Tetracycline was selected because Tn10 tetracycline resistance transposon has been extremely well characterized and can be activated by a non antibiotic molecule anhydrotetracycline that binds to the TetR transcription factor, allowing us to use a variety of approaches when characterizing the genetic circuit. Also, the human body is extremely tolerant of tetracycline, making it a good target for resistance reduction.
Basic Circuit Design - The general strategy for linking lsyis to tetracycline resistance, involves putting a lambda transcription factor (CI) under control of the tet promoter. This circuit makes the amount of CI in a cell proportional to the amount of tetracycline in the cell. Cells with higher concentrations of tetracycline have higher levels of CI thus preventing lysis of the cell via phage. Cells with lower levels of tetracycline (cells that are resistant to tetracycline) will have less CI present and thus be more likely to be lysed by phage.
Strain Selection – To compare the relative fitness of tetracycline resistant vs. tetracycline sensitive phage infected cells, we selected two strains of E.coli genotypically identical with the exception that one has the Tn10 tetracyline resistance transposon. These nearly identical strains will allow us to asses the impact of tetracycline resistance on relative fitness.
Phage Engineering – We will construct a recombination plasmid designed to integrate our circuit into lambda DNA located chromosomally in lysogen E.coli. The plasmid will not contain an origin of replication (to limit recombination sites), so we will have to amplify it using high fidelity PCR. The plasmid will contain our circuit flanked by homologous recombination sites that will combine at non-critical location of the phage chromosome (yet to be determined) .
Circuit Characterization – To assay relative fitness of the cells, we will measure doubling times of the strains at sub mic (minimum inhibitory concentration) tetracycline by measuring optical density (OD600nm) of cells.
