Team:Caltech/Project/Phage Pathogen Defense

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==Introduction==
==Introduction==
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[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic E. coli.]]
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[[Image:PathogenicEcoli.jpg|thumb|left|Pathogenic ''E. coli''.]]
There are <math> 10^{14} </math> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1].  However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3]  
There are <math> 10^{14} </math> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1].  However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3]  
There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this fact, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria.  
There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this fact, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria.  
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Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to create a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within E. coli..  
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Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to create a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within ''E. coli''..  
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===System Design===
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The engineered probiotic acts as a delivery vehicle for phage into the large intestine.  There are four important design goals for such as system: (1) the system releases phage into the large intestine, (2) the production of phage is regulated with a high dynamic range between a distinct on and off state, (3) the system can target a wide range of pathogenic bacteria, and (4) the system integrates well within the existing iGEM probiotic.
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==Part I: Lambda Phage==
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An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA with the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, ''E. coli''. We can avoid this limitation by incorporating as a lysogen a phage which is not normally infectious to ''E. coli''. This phage could potentially target pathogenic bacteria besides  ''E. coli''. To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of ''E. coli''  immune to λ infection due to ''lamB'' deletion[5]. In this model, the host is immune to infection unless lamB is expressed on a plasmid. After infection and selection for lysogens, the ''lamB'' plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage which targets a pathogenic bacterial species, constitutively expressing the phage receptor within ''E. coli'' to create lysogens, and inducing the lysogens to infect non-''E. coli'' pathogenic bacteria.
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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.
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===System Design===
 
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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. In the final system, the lysogens will have to be immune to infection, thus, the lamB+ plasmid will have to be cured from the JW3996-1 strain. This is proposed to be done via fusaric acid tetracycline counter-selection [4], a procedure which will allow for selective pressure against tetracycline resistance. To apply this to the lamB+ plasmid, a tetracycline resistant cassette (P1005) with a terminator (B0015) has been cloned downstream of the lamB gene.
 
[[Image:SystemDesign 1.jpg |frame|center|50px|Design for the lamB+tetA constructs used for lambda infection and tetracycline counterselection.]]
[[Image:SystemDesign 1.jpg |frame|center|50px|Design for the lamB+tetA constructs used for lambda infection and tetracycline counterselection.]]
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The second plasmid which must be designed controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. In general, λ lysogens stay in lysogeny until some trigger, usually cellular stress. However, we want to be able to induce the lysogens into the lytic cycle, this is done through the over expression of the E. Coli gene rscA, which has been shown to bring lysogens into the lytic phase. Currently, the rscA gene has been placed behind the luxR repressor/activator. This repressor prevents expression of rscA until activation via acyl-homo-serine lactone (AHL). However, within the final project, rscA will most likely be placed in the control of Allen's random differentiation generator. Furthermore, the final system will be similar in nature to Doug's, with a inverter after the activation of rscA, this is shown below.  
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The second plasmid which must be designed controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950 [6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the ''recA'' pathway was not readily feasible, so an alternative effector was used to elicit induction.  A secondary plasmid expressing the ''E. coli'' regulatory gene, ''rcsA'', is used as the trigger for λ phage induction. ''rcsA'' is one of several regulatory genes which provide a ''recA'' independent pathway for λ induction[7].  Overexpression of ''rcsA'' leads to λ induction, and, furthermore, ''rcsA'' appears to directly compete with the λ suppression gene ''cI''. [6] In this project, ''rcsA'' is used to trigger phage production. The effectiveness and dynamic range of ''rcsA'' induction is explored.  
[[Image:SystemDesign 2.jpg |thumb|center|650px|Design for the control system for control of lambda phage lysogenic/lytic life cycles.]]
[[Image:SystemDesign 2.jpg |thumb|center|650px|Design for the control system for control of lambda phage lysogenic/lytic life cycles.]]
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==Part II: B. Subtilis Lysogens==
==Part II: B. Subtilis Lysogens==

Revision as of 05:07, 23 October 2008


iGEM 2008



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Phage Pathogen Defense


Introduction

Pathogenic E. coli.

There are <math> 10^{14} </math> bacterial cells that naturally reside in the human gut, an order of magnitude greater than all the cells in the body. Humans enjoy a mutualistic relationship with intestinal microbiota, wherein the microorganisms perform a host of useful functions, such as processing unused energy substrates, training the immune system, and inhibiting growth of harmful bacterial species [1]. However, not all bacteria populations within the human intestine are benign. There are many pathogenic bacteria which cause a wide variety of diseases when present in the human gut. As one example, cholera is one of the most widespread and damaging of such infectious microorganisms, with the World Health Organization reporting 132,000 cases in 2006 [2]. But cholera is just one of many examples. In the United States alone, there were more than 325,000 hospitalizations from food-borne illnesses in the year of 2006 [3]

There are many medical treatments for food-borne illnesses, the most commonly prescribed being antibiotics. Unfortunately, antibiotics are indiscriminant and lead to rapid depletion of benign bacterial populations within the intestine. Due to this fact, dietary supplements have been popularized which aim to introduce beneficial bacteria back into the gut after antibiotic treatment; these substances have been termed ‘probiotics’. Natural probiotics have two main advantages: they provide useful functions for the host and they competitively inhibit growth of pathogenic bacteria. Natural probiotics do not convey any more advantages than bacteria in a healthy human intestine. Modern synthetic biology techniques should allow us to create an engineered probiotic that goes beyond the limitations of natural probiotics. The viability of such engineered probiotics within humans has already been established [4]. As part of a collaborative effort by the Caltech iGEM team to create a novel probiotic with improved medical applications, this project focuses on engineering a pathogen defense system within E. coli..

System Design

The engineered probiotic acts as a delivery vehicle for phage into the large intestine. There are four important design goals for such as system: (1) the system releases phage into the large intestine, (2) the production of phage is regulated with a high dynamic range between a distinct on and off state, (3) the system can target a wide range of pathogenic bacteria, and (4) the system integrates well within the existing iGEM probiotic.

An obvious delivery mechanism would be the adaptation of natural lysogens, a phase of the phage life cycle in which the phage integrates its DNA with the host organism. Lysogens lie dormant until disturbed by some external stimuli, but can be stimulated to enter the lytic cycle and release phage. There is one major limitation to this method: phage released will be specific to the lysogenic hosts, E. coli. We can avoid this limitation by incorporating as a lysogen a phage which is not normally infectious to E. coli. This phage could potentially target pathogenic bacteria besides E. coli. To demonstrate the feasibility of this approach, a simple model system was developed using λ phage and JW3996-1, a strain of E. coli immune to λ infection due to lamB deletion[5]. In this model, the host is immune to infection unless lamB is expressed on a plasmid. After infection and selection for lysogens, the lamB plasmid is counterselected against to regain immunity. Extending this model to a more realistic situation, we envision using a phage which targets a pathogenic bacterial species, constitutively expressing the phage receptor within E. coli to create lysogens, and inducing the lysogens to infect non-E. coli pathogenic bacteria.


Design for the lamB+tetA constructs used for lambda infection and tetracycline counterselection.

The second plasmid which must be designed controls the induction of the lysogens to release phage into the environment. Control of this aspect of the system is vital for integration with the overall iGEM project. The induction of λ phage lysogens has been explored since 1950 [6], and serves as a model system for the study of other temperate phages. The most famous induction mechanism is ultraviolet radiation (UV). In UV induction of λ lysogens, UV induced DNA damage activates the cellular SOS mechanism, which activates the expression of RecA, a protein involved in SOS response. RecA, in turn, cleaves the λ repressor and leads to phage activation. In this study, activation of the recA pathway was not readily feasible, so an alternative effector was used to elicit induction. A secondary plasmid expressing the E. coli regulatory gene, rcsA, is used as the trigger for λ phage induction. rcsA is one of several regulatory genes which provide a recA independent pathway for λ induction[7]. Overexpression of rcsA leads to λ induction, and, furthermore, rcsA appears to directly compete with the λ suppression gene cI. [6] In this project, rcsA is used to trigger phage production. The effectiveness and dynamic range of rcsA induction is explored.

Design for the control system for control of lambda phage lysogenic/lytic life cycles.


Part II: B. Subtilis Lysogens

Basic Idea

Phasmid Assembly

We wish to create a phasmid, basically the lysogen genome with an E. Coli plasmid origin of replication, using B. Subtilis lysogens. This allows the phage genome to pass on as a plasmid within our engineered E. Coli, but when the plasmid is conjugated to B. Subtilis, the virus is induced and destroys the pathogens. Phasmid construction will an E Coli origin of replication with a Subtilis specific promoter in front of RecA or another inducer.

Current Progress

Three different bacteriophage lysogens and three wildtype strains of B. Subtilis has been ordered from the Bacillus Stock Center. However, efforts to induce the bacteriophage from the lysogens with UV exposure has proved to be futile thus far, however, this was possibly due to a contamination of the lysogen stocks. The strains have been reordered and B. Subtilis lysogen induction with UV occurring soon, most likely the week of 8/4.

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