IGEM:Caltech/2008/Project/Phage Pathogen Defense: Difference between revisions
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=Phage Pathogen Defense= | =Phage Pathogen Defense= | ||
==General Idea== | |||
Bacterial food poisoning is a prevalent problem around the world; in the year of 2006, the United States had more than 325,000 hospitalizations from food borne illnesses. The iGEM 2008 team is designing a probiotic for medical applications, and given the prevalence of foodborne illnesses, pathogen defense is high on the list of priorities. Bacteriophages were chosen because they have evolved to be highly effective at infecting and killing their bacterial hosts, and their highly infectious and replicative nature means just one phage can lead to decimation of the pathogen population. | Bacterial food poisoning is a prevalent problem around the world; in the year of 2006, the United States had more than 325,000 hospitalizations from food borne illnesses. The iGEM 2008 team is designing a probiotic for medical applications, and given the prevalence of foodborne illnesses, pathogen defense is high on the list of priorities. Bacteriophages were chosen because they have evolved to be highly effective at infecting and killing their bacterial hosts, and their highly infectious and replicative nature means just one phage can lead to decimation of the pathogen population. | ||
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Bacteriophage λ is a temperate phage with an E. Coli. host, λ infects E. Coli through the lamB receptor, and absence of this receptor prevents λ infection. Our project takes advantage of this aspect of bacteriophage λ to create E. Coli which are resistant to the phage, but release the phage to destroy susceptible pathogenic E. Coli. To achieve this, lamB deficient E. Coli must first express the surface protein through a constitutively active version of the gene on a plasmid. This allows the lamB deficient E. Coli to be infected by λ phage. Lysogens are selected for using antibiotic resistance, and then the plasmid possessing the lamB receptor gene is counter-selected against, producing a strain lysogenic for λ, but is immune to infection. | Bacteriophage λ is a temperate phage with an E. Coli. host, λ infects E. Coli through the lamB receptor, and absence of this receptor prevents λ infection. Our project takes advantage of this aspect of bacteriophage λ to create E. Coli which are resistant to the phage, but release the phage to destroy susceptible pathogenic E. Coli. To achieve this, lamB deficient E. Coli must first express the surface protein through a constitutively active version of the gene on a plasmid. This allows the lamB deficient E. Coli to be infected by λ phage. Lysogens are selected for using antibiotic resistance, and then the plasmid possessing the lamB receptor gene is counter-selected against, producing a strain lysogenic for λ, but is immune to infection. | ||
The system design revolves around the construction of two plasmids, both of which to be placed within E. Coli Strain JW3996-1, a strain deficient in the maltose outer membrane porin lamB, a surface protein integral to λ phage infection. One of these two plasmids is first responsible for creating JW3996-1 λ lysogens. However, creating lamB- lysogens is complicated by the fact that the JW3996-1 strain is immune to λ phage infection. Thus, to allow for λ infection, lamB must be expressed on a plasmid. | ===System Design=== | ||
The system design revolves around the construction of two plasmids, both of which to be placed within E. Coli Strain JW3996-1, a strain deficient in the maltose outer membrane porin lamB, a surface protein integral to λ phage infection. One of these two plasmids is first responsible for creating JW3996-1 λ lysogens. However, creating lamB- lysogens is complicated by the fact that the JW3996-1 strain is immune to λ phage infection. Thus, to allow for λ infection, lamB must be expressed on a plasmid. The gene coding for lamB was obtained from E. Coli genomic DNA using PCR. For regulation of lamB expression, a weak constitutive promoter, J23113, and 2 weak ribosomal binding sites, B0032 and B0033, were placed upstream of the lamB gene. | |||
[[Image:systemdesign 1.jpg |frame|center|50px|Design for the lamB+tetA constructs used for lambda infection and tetracycline counterselection.]] | |||
Revision as of 05:15, 1 August 2008
Phage Pathogen DefenseGeneral IdeaBacterial food poisoning is a prevalent problem around the world; in the year of 2006, the United States had more than 325,000 hospitalizations from food borne illnesses. The iGEM 2008 team is designing a probiotic for medical applications, and given the prevalence of foodborne illnesses, pathogen defense is high on the list of priorities. Bacteriophages were chosen because they have evolved to be highly effective at infecting and killing their bacterial hosts, and their highly infectious and replicative nature means just one phage can lead to decimation of the pathogen population. Two methods have been used to approach the goal of ‘manufacturing’ phage. The first utilizes the Escherichia coli bacteriophage λ as a defense against pathogenic E. coli. A strain of E. coli has been developed which is immune to λ phage infection, but is lysogenic for λ. Furthermore, a plasmid has been developed to control the induction of λ using the E. Coli gene rscA, thus, with some application of signal, the lysogens can be triggered to enter the lytic cycle and release phage into the environment to infect other E. Coli. Due to the fact that the probiotic is immune to λ phage infection, the λ phage will only target pathogenic E. coli. The second method developed can be adapted to any temperate bacteriophage to target many species of bacteria. By circularizing phage DNA with a E. Coli plasmid origin of replication, the phage resides within the probiotic as a plasmid, however, upon conjugation with a bacteria of the host species, the ‘phasmid’ will produce virulent phage to destroy the pathogenic host population. Part I: Lambda PhageBacteriophage λ is a temperate phage with an E. Coli. host, λ infects E. Coli through the lamB receptor, and absence of this receptor prevents λ infection. Our project takes advantage of this aspect of bacteriophage λ to create E. Coli which are resistant to the phage, but release the phage to destroy susceptible pathogenic E. Coli. To achieve this, lamB deficient E. Coli must first express the surface protein through a constitutively active version of the gene on a plasmid. This allows the lamB deficient E. Coli to be infected by λ phage. Lysogens are selected for using antibiotic resistance, and then the plasmid possessing the lamB receptor gene is counter-selected against, producing a strain lysogenic for λ, but is immune to infection. System DesignThe system design revolves around the construction of two plasmids, both of which to be placed within E. Coli Strain JW3996-1, a strain deficient in the maltose outer membrane porin lamB, a surface protein integral to λ phage infection. One of these two plasmids is first responsible for creating JW3996-1 λ lysogens. However, creating lamB- lysogens is complicated by the fact that the JW3996-1 strain is immune to λ phage infection. Thus, to allow for λ infection, lamB must be expressed on a plasmid. The gene coding for lamB was obtained from E. Coli genomic DNA using PCR. For regulation of lamB expression, a weak constitutive promoter, J23113, and 2 weak ribosomal binding sites, B0032 and B0033, were placed upstream of the lamB gene.
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