Team:WITS-South Africa/Machine Design

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===Lacto-detect===
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<div style="padding:40px;">
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[[Image:Wits_Machine_1_wiki.jpg‎]]
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==The Machines==
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===Lacto-report===
 
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===Intermediate Machines===
 
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===Machine Testing===
 
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===Design Rationale===
 
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So how did we end up selecting the final parts of our Machines? Each one was selected after much consideration and scouring of the literature to select the most suitable biological system.
 
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'''Cassette 1 in population 1'''
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===Lacto-detect===
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</div>
 
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[[Image:Cassette_1.JPG]]
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This genetic machine within a population of bacteria will prime these bacteria to act as ‘Detectors’. It causes the production of a quorum signalling peptide in response to an input signal, which activates another machine, Lacto-report.  
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<div class ="heading">
 
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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/AraC promoter will be synthesized by primer-primer annealing and subsequent PCR elongation.
 
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<div class="heading"> 
 
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'''PlcR-PapR Quorum Molecule'''
 
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</div>
 
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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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<div class="heading">
 
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'''Venus'''
 
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</div>
 
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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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<div class ="heading">
 
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'''Terminator'''
 
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</div>
 
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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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<div class="heading">
 
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== '''Cassette 2 in population 2''' ==
 
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</div>
 
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[[Image:Cassette_2_wiki.JPG]]
 
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<div class="heading">
 
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=== PlcR Promoter ===
 
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</div>
 
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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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[[Image:Wits_Machine_1_wiki.jpg‎]]
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'''Figure 1. The Lacto-sense construct'''
   
   
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<div class="heading">
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So how did we end up selecting the final parts of our Machines? Each one was chosen after much consideration and scouring of the literature to select the most suitable biological system. For a design rationale of why we selected the parts that we did to create this machine, click [https://2010.igem.org/Team:WITS-South_Africa/machine1_design here]
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===PlcR-PapR Fusion Gene===
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</div>
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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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All machines were constructed via sequential cloning steps in ''E.coli'' using the Standard Assembly method.
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===Lacto-report===
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<div class="heading">
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This genetic machine is present in a second bacterial population, mixed in with the first. The quorum peptide produced when Lacto-sense is activated will be imported into the bacterial cells containing this machine and induce expression of this construct. This will have several effects:
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===Phage Activator===
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</div>
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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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* Production of more quorum peptide, to activate neighbouring cells and produce a positive feedback loop
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* Production of a chromogenic reporter compound which is visible to the naked eye
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* Production of a negative regulator which will block the binding of the quorum peptide and create a negative feedback loop, resetting the system to a negative state
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[[Image:Wits_Machine_2.jpg]]
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<div class="heading">
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'''Figure 2. The Lacto-report construct'''
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===mCherry===
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</div>
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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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For a design rationale of why we selected the parts that we did to create this machine, click [https://2010.igem.org/Team:WITS-South_Africa/machine2_design here]
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We were not able to finish constructing Lacto-report in its entirety, due to time constraints and issues with the E.chromi Biobrick parts. (For more details, click [https://2010.igem.org/Team:WITS-South_Africa/e-chromi here].)
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<div class="heading">
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For testing and characterising the behaviour of the quorum sensing mechanism in a model Gram-positive bacillus, we used an intermediate machine construct we dubbed Lacto-test.
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===Terminator===
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</div>
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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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===Lacto-test===
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<div class="heading">
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This construct is a simplified of version of Lacto-report. When transformed into a Gram-positive bacteria, the presence of the PlcR promoter will allow us to determine if the PlcR-PapR quorum peptide can be correctly processed, imported into the cell and whether or not it activates the promoter. This can be determined by visualising expression of mCherry in a mixed culture of Lacto-detect and Lacto-report, after the addition of an inducer for Lacto-detect.  
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===PO Phage Promoter===
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</div>
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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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[[Image:Wits_Lacto-test.jpg‎]]
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<div class="heading">
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'''Figure 3. The Lacto-test construct'''
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===E. chromi===
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</div>
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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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Lacto-test was cloned into the Gram-positive/E.coli shuttle vector pNDW5 and electroporated into a model Gram-positive bacillus for testing via fluorescent microscopy.
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<div class="heading">
 
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===SpoOA===
 
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</div>
 
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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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==Machine Testing==
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<br />
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All of our constructs contained different fluorescent protein markers. We added these in order to gain some preliminary data on the behaviour of our machines using fluorescent microscopy. Images were obtained by laser excitation of the fluorescent proteins at their excitation maximum wavelengths and filtered through a yellow/green channel to observe Venus or a red channel to observe mCherry.
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<div class="heading">
 
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===Terminator===
 
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</div>
 
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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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A culture of 2ml of ''E. coli'' containing Lacto-detect was grown overnight at 37°C and then transferred to 25 ml of ampicillin LB broth. This culture was then grown at 37°C in a shaking incubator for 2.5 hours so as to ensure that the bacteria were in their exponential growth phase. A baseline reading (Fig 4) was taken at this point (Tile A), to determine the presence of non-specific promoter activation. 10% 1mM IPTG was then added and the culture returned to the shaking incubator. An aliquot was then imaged after 30min (Tile B), and again after 1 hour (Tile C). All aliquots used in imaging were 100 ul.
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As is seen in the baseline image (Tile A), the degree of fluorescent activation is minimal before the addition of IPTG. Tile B shows the fluorescent activation after 30min incubation with IPTG - there is very little visible fluorescent activation. After an hour of incubation (Tile C) there is a marked increase in the degree of fluorescence, thus providing an indication that IPTG has a positive effect on the Lac/Ara-1 promoter, activating gene expression.  <br /> <br />
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[[Image:Montage_of_M1I_d.jpg|850px]] <br />
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'''Figure 4. Time course fluorescent microscopy of Lacto-detect before and after induction with IPTG''' <br /> <br /> <br />
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Once the induction of Lacto-detect had reached an appreciable level, it was added to an aliquot of ''B. subtilis'' containing the Lacto-test construct and imaged(Fig 5). Baseline images of the combined Lacto-detect and Lacto-test populations are shown. Tile A shows the Lacto-detect construct which has been activated (Imaged on a GFP filter which will only detect yellow/green fluorescence) and Tile B visualised red fluorescence which is indicative of activated mCherry in Lacto-report. Tile C is the same images overlaid to show a combined fluorescence profile of the sample. The degree of activation (Tile A) of Lacto-detect is appreciable due to the previous activation with IPTG, while the observed activation of Lacto-report (Tile B) shows no significant observable induction. <br /> <br />
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[[Image:Montage_of_Mixed.jpg|850px]] <br />
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'''Figure 5. Imaging of a mixed population of induced Lacto-detect with Lacto-test, immediately after the cultures were combined''' <br /> <br /> <br />
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Once the induced aliquot of Lacto-detect was added to Lacto-test, the combined aliquot was imaged at 30min intervals for 6 hours so as to observe the possible activation of Lacto-test by Lacto-detect (Figure 6). Figure 7 shows the imaged aliquot from Figure 6 with the Venus and mCherry fluorescent channels separated and presented at each epoch as alphabetical sets - A&B, C&D etc. This is done as to fully appreciate the direct induction of Lacto-test by Lacto-detect. Taking particular note of tile G of Figure 6 and the corresponding tiles M&N of Figure 7 (after 3.5h), there is  marked increase in both Venus and mCherry fluorescence. This is indicative of the active communication between Lacto-detect and Lacto-test, thus illustrating the efficacy of the PlcR-PapR quorum network. <br /> <br />
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[[Image:Montage_of_SuperMontage.jpg|850px]] <br /> '''Figure 6. Combined Lacto-test and Lacto-report populations imaged over time to demonstrate induction''' <br /> <br />
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[[Image:Montage_of_Combo.jpg|850px]] <br /> '''Figure 7. Combined Lacto-test and Lacto-report populations imaged over time to demonstrate induction, showing the seperate fluorescent channels'''
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==Conclusions==
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</div>
 
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== Machine Construction ==
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This data is promising, indicating that a quorum sensing mechanism from one Gram-positive organism such as ''Bacillus thuriengiensis'' can potentially be imported into another, such as ''Bacillus subtilis'', and successfully allows for co-ordinated control of gene expression.
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== Machine Testing  ==
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It also indicates that it is possible to obtain functional quorum peptides from a Gram-negative such as ''E.coli'', which would ordinarily not produce these molecules. 
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Final Machine 1 - LactoDetect
 
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Purpose:
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The synthetic PlcR-PapR fusion peptide we created appears to be fully functional in it's ability to bind to the PlcR promoter and activate gene expression.
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a) To act as the ‘Detector’ Machine within the population and produce the quorum signalling peptide in response to an input signal
 
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Construction:
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Both the Lac1/AraC promoter and the PlcR promoter appear to be fairly tightly regulated, with very little baseline expression, making them promising candidates as inducible promoters.  
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LactoDetect was constructed via a series of cloning steps using the Standard Assembly method. These are outlined in Figure 1 below.
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[[Image: machine_1_small.jpg|650px]]
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[[Image:Venus-tt_screen.jpg|350px]]
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Intermediate Machine 2 (Figure 1)
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Purpose:
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a) To characterise the strength of the induction of the Lac/Ara-1 promoter by measuring fluorescence after IPTG is added
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b) To show that the PlcR promoter is activated in ''L. gasseri'' by measuring fluorescence after the addition of exogenous PlcR and PapR proteins
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Fig1. The proposed intermediate Machine 2
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Fig 2. The proposed Final Machine 1
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Final Machine 2 (Figure 3)
 
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Purpose
 
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a) To act as the ‘Reporter’ Machine within the population and respond to infection as previously described
 
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Fig 3. The proposed Final Machine 2
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'''The PlcR promoter and PlcR-PapR protein system is thus a useful inducible gene expression system for Gram-positive bacteria and can be used in the most commonly used Gram-positive chassis, ''Bacillus subtilis''.'''

Latest revision as of 21:01, 27 October 2010


Contents

The Machines

Lacto-detect

This genetic machine within a population of bacteria will prime these bacteria to act as ‘Detectors’. It causes the production of a quorum signalling peptide in response to an input signal, which activates another machine, Lacto-report.


Wits Machine 1 wiki.jpg

Figure 1. The Lacto-sense construct


So how did we end up selecting the final parts of our Machines? Each one was chosen after much consideration and scouring of the literature to select the most suitable biological system. For a design rationale of why we selected the parts that we did to create this machine, click here

All machines were constructed via sequential cloning steps in E.coli using the Standard Assembly method.


Lacto-report

This genetic machine is present in a second bacterial population, mixed in with the first. The quorum peptide produced when Lacto-sense is activated will be imported into the bacterial cells containing this machine and induce expression of this construct. This will have several effects:

  • Production of more quorum peptide, to activate neighbouring cells and produce a positive feedback loop
  • Production of a chromogenic reporter compound which is visible to the naked eye
  • Production of a negative regulator which will block the binding of the quorum peptide and create a negative feedback loop, resetting the system to a negative state


Wits Machine 2.jpg

Figure 2. The Lacto-report construct


For a design rationale of why we selected the parts that we did to create this machine, click here

We were not able to finish constructing Lacto-report in its entirety, due to time constraints and issues with the E.chromi Biobrick parts. (For more details, click here.)

For testing and characterising the behaviour of the quorum sensing mechanism in a model Gram-positive bacillus, we used an intermediate machine construct we dubbed Lacto-test.


Lacto-test

This construct is a simplified of version of Lacto-report. When transformed into a Gram-positive bacteria, the presence of the PlcR promoter will allow us to determine if the PlcR-PapR quorum peptide can be correctly processed, imported into the cell and whether or not it activates the promoter. This can be determined by visualising expression of mCherry in a mixed culture of Lacto-detect and Lacto-report, after the addition of an inducer for Lacto-detect.


Wits Lacto-test.jpg

Figure 3. The Lacto-test construct


Lacto-test was cloned into the Gram-positive/E.coli shuttle vector pNDW5 and electroporated into a model Gram-positive bacillus for testing via fluorescent microscopy.


Machine Testing


All of our constructs contained different fluorescent protein markers. We added these in order to gain some preliminary data on the behaviour of our machines using fluorescent microscopy. Images were obtained by laser excitation of the fluorescent proteins at their excitation maximum wavelengths and filtered through a yellow/green channel to observe Venus or a red channel to observe mCherry.


A culture of 2ml of E. coli containing Lacto-detect was grown overnight at 37°C and then transferred to 25 ml of ampicillin LB broth. This culture was then grown at 37°C in a shaking incubator for 2.5 hours so as to ensure that the bacteria were in their exponential growth phase. A baseline reading (Fig 4) was taken at this point (Tile A), to determine the presence of non-specific promoter activation. 10% 1mM IPTG was then added and the culture returned to the shaking incubator. An aliquot was then imaged after 30min (Tile B), and again after 1 hour (Tile C). All aliquots used in imaging were 100 ul.


As is seen in the baseline image (Tile A), the degree of fluorescent activation is minimal before the addition of IPTG. Tile B shows the fluorescent activation after 30min incubation with IPTG - there is very little visible fluorescent activation. After an hour of incubation (Tile C) there is a marked increase in the degree of fluorescence, thus providing an indication that IPTG has a positive effect on the Lac/Ara-1 promoter, activating gene expression.

Montage of M1I d.jpg
Figure 4. Time course fluorescent microscopy of Lacto-detect before and after induction with IPTG


Once the induction of Lacto-detect had reached an appreciable level, it was added to an aliquot of B. subtilis containing the Lacto-test construct and imaged(Fig 5). Baseline images of the combined Lacto-detect and Lacto-test populations are shown. Tile A shows the Lacto-detect construct which has been activated (Imaged on a GFP filter which will only detect yellow/green fluorescence) and Tile B visualised red fluorescence which is indicative of activated mCherry in Lacto-report. Tile C is the same images overlaid to show a combined fluorescence profile of the sample. The degree of activation (Tile A) of Lacto-detect is appreciable due to the previous activation with IPTG, while the observed activation of Lacto-report (Tile B) shows no significant observable induction.

Montage of Mixed.jpg
Figure 5. Imaging of a mixed population of induced Lacto-detect with Lacto-test, immediately after the cultures were combined


Once the induced aliquot of Lacto-detect was added to Lacto-test, the combined aliquot was imaged at 30min intervals for 6 hours so as to observe the possible activation of Lacto-test by Lacto-detect (Figure 6). Figure 7 shows the imaged aliquot from Figure 6 with the Venus and mCherry fluorescent channels separated and presented at each epoch as alphabetical sets - A&B, C&D etc. This is done as to fully appreciate the direct induction of Lacto-test by Lacto-detect. Taking particular note of tile G of Figure 6 and the corresponding tiles M&N of Figure 7 (after 3.5h), there is marked increase in both Venus and mCherry fluorescence. This is indicative of the active communication between Lacto-detect and Lacto-test, thus illustrating the efficacy of the PlcR-PapR quorum network.

Montage of SuperMontage.jpg
Figure 6. Combined Lacto-test and Lacto-report populations imaged over time to demonstrate induction


Montage of Combo.jpg
Figure 7. Combined Lacto-test and Lacto-report populations imaged over time to demonstrate induction, showing the seperate fluorescent channels


Conclusions

This data is promising, indicating that a quorum sensing mechanism from one Gram-positive organism such as Bacillus thuriengiensis can potentially be imported into another, such as Bacillus subtilis, and successfully allows for co-ordinated control of gene expression.


It also indicates that it is possible to obtain functional quorum peptides from a Gram-negative such as E.coli, which would ordinarily not produce these molecules.


The synthetic PlcR-PapR fusion peptide we created appears to be fully functional in it's ability to bind to the PlcR promoter and activate gene expression.


Both the Lac1/AraC promoter and the PlcR promoter appear to be fairly tightly regulated, with very little baseline expression, making them promising candidates as inducible promoters.


The PlcR promoter and PlcR-PapR protein system is thus a useful inducible gene expression system for Gram-positive bacteria and can be used in the most commonly used Gram-positive chassis, Bacillus subtilis.