Team:WITS-South Africa/The Machine

From 2010.igem.org

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== '''Cassette 1 in population 1''' ==
 
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=== Lac/AraC Promoter ===
 
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The Lac/Ara-1 promoter will be a synthetic fusion promoter comprised of the operator from the arabinose operon and the Lac promoter from the lac operon. The AraC protein is constitutively expressed and binds to the arabinose operon in the absence of the arabinose sugar. When arabinose is present, or an isomer thereof, it induces a conformational change in AraC thus preventing it from binding the operon and thus allowing transcription. Since IPTG is an isomer of β-galactosidase, it will induce the same conformational effect on AraC thus inducing transcription. In this way, the exogenous addition of IPTG will serve as a proxy for the HPV virus and induce the transcription cassette 1. Furthermore, as illustrated by Lutz and Bujard, the degree of induction is influenced by both IPTG and arabinose. Hence, promoter activity can be regulated and finely tuned by the addition of varying concentrations of IPTG and arabinose. The Lac/Ara-1 promoter will be synthesized by primer-primer annealing and subsequent PCR elongation.
 
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=== PlcR-PapR Quorum Molecule ===
 
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The PlcR regulon in ''Bacillus thuringiensis'' and ''Bacillus cereus'' houses approximately 15 genes required for the production of many virulence factors. The activation of this regulon is under the control of the PlcR peptide which binds a region within the promoter known as the PlcR box. The ''papR'' gene - which is found within the PlcR regulon - encodes a 48 amino acid peptide that is crucial for the binding to and subsequent activation of the PlcR box. The PapR pro-peptide is secreted from the cell (via the SecA pathway) following translation, cleaved extra-cellularly and the resulting pentapeptide is re-imported into the cell via an oligopeptide permease (a protease ubiquitous to the extra-cellular matrix of Gram-positive bacteria). Once inside the cell, PapR allows PlcR to bind to the PlcR box thus activating the regulon. Due to the presence of the plcR gene within the regulon, PlcR positively regulates its own transcription.  The PlcR regulon within ''Bacillus anthracis'' houses a gene that codes for a PlcR-PapR fusion protein which was found to strongly induce transcription of the native plcR gene. It has been shown, by Pomerantsev and colleagues, that binding to the PlcR box and activation of the PlcR regulon is achieved by the expression of a hetrologous PlcR-PapR fusion protein.  So as to ectopically express both PlcR and PapR in ''Lactobacillus gasseri'', a sequence coding for the PlcR-PapR fusion protein will be derived from bioinformatic analysis of the native sequence in ''Bacillus anthracis''. This will be implemented due to the inherent compactness of the fusion protein as well as ease of transportability when inserting into a foreign bacteria. The ''plcR-papR'' sequence will be synthesized by Gene Art and furthermore, the sequence will be optimized by an online tool.
 
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=== Venus ===
 
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So as to assess the degree of activation of cassette 1, a variant of the Yellow Fluorescent Protein, Venus, has been included in the construct. So as to obtain quantitative data as to the efficacy of IPTG in inducing the activation of cassette 1, a host of fluorometric analysis will be performed on a cassette-containing colony. Venus, which possesses a known excitation:emission characteristic, will be used as a reporter protein to quantify the degree of transcription (by proxy of fluorescence) with use of a fluorometer. Although venus is a ubiquitous fluorescent protein found in the laboratory, specialised sequences will be ordered from Gene Art. Furthermore, the sequences will be codon optimized so as to produce the greatest protein yield and fluorometric activity in ''Lactobacillus gasseri''. 
 
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=== Terminator ===
 
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So as to compartmentalize the cassette and explicitly terminate transcription, the inclusion of a double terminator from the Registry of Standard Parts is included in the design. The terminator used to ‘cap’ cassette 1 is a rho-independent terminator and will inhibit polymerase functioning by the formation of a stem-loop structure. The terminator DNA will be sourced from the Standard Registry of Parts.       
 
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== '''Cassette 2 in population 2''' ==
 
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=== PlcR Promoter ===
 
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Through the identification of the PlcR box within the promoter of the PlcR regulon, a promoter that contains the PlcR box as well the requisite transcription-initiating landmarks within the promoter will be synthesized. Hence, any construct that is placed downstream of the promoter will be activated by the addition of exogenous PlcR and the facilitating peptide PapR. The PlcR-PapR promoter will be synthesized through primer-primer annealing and subsequent PCR elongation.
 
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===PlcR-PapR Fusion Gene===
 
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The PlcR-PapR fusion gene that codes for the activating peptides PlcR and PapR has been included in construct so as to amplify the initial signal presented by population 1. Since the fusion protein of PlcR and PapR is translated into its inactive form, there will be no direct self-stimulatory effects within the same bacterium. Once secreted and processed, the peptides will serve to activate neighboring bacteria thus ensuring the propagation of the signal throughout the entire population. 
 
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===Phage Activator===
 
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Developed by the winners of 2009 iGEM competition, Team Cambridge developed an array of activator and promoter combinations (derived from bacteriophages) that have varying degrees of binding and activating strengths. The intention was to create a library of activators and promoters than can be combined in such a way so as to vary the degree of negative-feedback SpoOA exerts on the PlcR-PapR promoter. As such, several alternative endings of cassette 2 will be created, and based on experimental procedure a combination will be chosen so as to introduce an effective delay in repression. This delay is crucial so as to allow sufficient transcription of both the PlcR-PapR fusion peptide as well as the E. chromi pigment as this ensures adequate propagation of the signal throughout the colony as well as sufficient E. chromi being produced to ensure a distinctly visible report. The phage activator will be sourced from the Registry of Standard Parts.
 
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===mCherry===
 
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Akin to the function of Venus in cassette 1, mCherry - a monomeric fluorescent protein - will be used in assaying the degree of transcription induced in colony 2 by incident PlcR-PapR quorum molecules.  In addition to the visual report that will be produced as the response signal that propagates throughout the colony, mCherry will aid in the quantification of the mean fluorescent response of the colony. mCherry will be sourced from the Registry of Standard Parts. 
 
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===Terminator===
 
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As described above, the terminator ends transcription. By placing a terminator in the middle of cassette 2, it introduces a time delay between the activation of the initial region of cassette 2 and the feedback component, SpoOA. The terminator DNA will be sourced from the Registry of Standard Parts.
 
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===PO Phage Promoter===
 
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Forming the receptor of the cognate activator-promoter pair of phage sensitivity tuners, the PO promoter will initiate transcription when the phage activator binds the promoter. Since the genes downstream of the promoter will only be transcribed after the phage activator is transcribed, translated and folded, a time delay is inserted due to this unavoidable metabolic process. The PO promoter will be sourced from the Registry of Standard Parts.
 
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===E. chromi===
 
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So as to produce a chromogenic indication of the presence of HPV (or a proxy thereof), the E. chromi pigment will be used. The E. chromi biobrick is a composite biobrick comprised of sequential enzymes that process a substrate until a visible dye is produced. The combination of four sequential enzymes converts the substrate farnesyl pyrophosphate (which is colourless) to the pigment beta-carotine via several intermediates.  The E. chromi biobrick will be sourced from the Registry of Standard Parts.
 
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===SpooA===
 
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So as to provide a suitable negative-feedback loop to the ‘machine’, SpoOA is a protein that binds to the regions adjacent to the PlcR box in the PlcR-PapR promoter. Once produced, the repression of SpoOA will negate the production of both mCherry as well as E. chromi. Furthermore, SpoOA will inhbit it’s own production this derepressing cassette 2 in such a way that the system, if left to its own devices in the presence of a suitable concentration of PlcR-PapR, will exhibit oscillatory behavior. SpoOA will be synthesized by Gene Art and will be codon optimized for ''Lactobacillus gasseri'' so as to ensure optimal production and expression.
 
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===Terminator===
 
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So as to provide a final cap to cassette 2 and the machine in whole, a terminator will be placed to end transcription. The terminator will be sourced from the Registry of Standard Parts. 
 
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Revision as of 14:21, 26 October 2010



Project overview

Our machine was designed as a proof of concept that commensal bacteria, which live normally and harmlessly on the human body, can be engineered to function as diagnostic and prophylactic devices against pathogens which are otherwise difficult to detect or control. Basically we wish to explore the possibility that commensal bacteria can be used as a biological “suit of armour” against disease.


We decided to focus on Human Papillomavirus, which is a sexually transmitted infection which causes cervical cancer. HPV invades the vaginal mucosa, which is where commensal Lactobacillus gasseri and various other Lactic Acid Bacteria can be found in high numbers in healthy females, making L. gasseri a good candidate as a chassis for our protective machine.


When designing the machine we considered the following:


The ability of the bacteria to detect and respond to a virus/viral element

Engineering a bacterium to alter its gene expression based on recognition of a specific virus is a major difficulty. For the purposes of this project we chose to focus, instead, on building and tweaking the ability of the machine to respond in certain ways to a specific (but non-viral) stimulus as a “proxy” for infection. Once this was achieved, it would be possible to investigate how to make the machine specific for HPV.


The nature of the response – How will the machine alert an individual that she has been exposed to the virus?

Traditional whole cell biosensors have been engineered to respond to one or more stimuli with the expression of fluorescent proteins such as firefly luciferase or GFP. When these biosensors are added to an environmental sample, such as potentially contaminated water, and then the sample is viewed under a fluorescent microscope, a positive response can easily be detected. However, if the biosensor is part of a population of commensal bacteria in the vaginal mucosa, this would not be appropriate or useful! Thus, we wanted our machine to respond by producing an output easily visible to the naked eye. Preferably, an individual could monitor the response by herself, with no need to go to a doctor for examination or undergo a complicated procedure. Thus, we decided to experiment with Cambridge’s winning iGEM 09 project – E.chromi biobricks which cause bacteria to produce easily visible chromogenic reporter molecules. Simultaneous production of a reporter molecule and one which blocks, contains or neutralises the viral particle would be ideal, but for the purposes of this project, we decided to focus on the reporting mechanism, with the option to add in a neutralising molecule at a later stage.


The strength of the response needed for detection

If a viral particle is present in the vaginal mucosa, and one, or even a few local bacterial biosensors detects it, the colour change in those cells will not be sufficient for detection by the host. In order for our machine to be of practical use, infection needs to trigger a coordinated response from the entire population, switching every biosensor on. For this we explored the use of quorum sensing molecules, which are a method of bacterial communication which can directly activate gene expression. Gram-positive bacteria, such as L. gasseri, produce species specific quorum peptides which can be used to create a positive feedback loop to propagate an initial infection signal amongst an entire bacterial population.


How to switch the machine off again after infection has been reported

A continuous “on” state is obviously not desirable, given that the eventual effect of machine activation would be an entire bacterial population in the vaginal mucosa that is producing a bright, visible colour. Once infection has been noted by the host, allowing them to seek medical treatment, the population needs to return to an off, non visible state – Firstly to avoid discomfort or embarrassment for the host and secondly to ensure that the machine can be used to further detect any subsequent infections. A repressor protein, which can switch off expression of the quorum molecule and shut down the positive-feedback loop, creating a negative-feedback loop, was selected. This would stop expression of the chromogenic reporter, and eventually, the bacterial cells would return to their normal colour.


How to avoid false positives and false negatives


Machine Schematic

Schematic wiki.JPG

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