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| - | == <font size=5>'''Our project''' </font>== | + | == <font size=5>'''Basic project''' </font>== |
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| - | == <font size=5>'''Aplication project''' </font>==
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| - | About one paper, 94 times activity was observed at 30MPa for the lac
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| - | promoter compared with the level expressed at atmospheric pressure.</div>
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| - | (cf.Takako Sato, Chiaki Kato,and Koki Horikoshi(1994) Effect of high
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| - | pressure on gene expression by lac and tac promoters in Escherichia
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| - | coli.Marine Biotechnology 3:89-92)</div>
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| - | === <font size=5>'''Development of low pressure inducible promoter'''</font> ===
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| - | It is known that lac promoter is induced under 30 MPa (Kato et al. 1994).However, 30 MPa is too high to use as input.
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| - | Therefore, we are trying to develop low pressure inducible promoter.
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| - | ==== Method ====
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| - | ===== Principle =====
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| - | LacI binds to lacI binding site and repress lac promoter.
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| - | In addition, pressure of 30 MPa activated the lac promoter.
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| - | We propose a hypothesis that 30 MPa induce a conformational change in lacI reprssure and change the affinity for lacI binding site weaker. Therefore, lacI can't repress lac promoter under 30 MPa.
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| - | ===== Strategy =====
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| - | <gallery widths="360px" heights="190px" >
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| - | Image:Tech dev 1.JPG|Figure 1 - RCP random mutagenesis
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| - | Image:Tech dev 2.JPG|Figure 2 - Strategy of sorting with FACS
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| - | </gallery>
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| - | We are trying to develop low pressure inducible promoter by PCR random mutagenesis to lac promoter in order to reduce affitity to lacI.
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| - | And we are screening an E. coli library for promoters that are induced under low pressure using a fluorescence activated cell sorter (FACS).
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| - | This scheme is based on the ability to separate bacteria with a FACS in response to expression, or lack of expression, of a fluorescent marker.
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| - | *'''step 1''' - Fluorescent bacteria without repressor protein were collected by FACS. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.
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| - | *'''step 2''' - Constitutive promoter are removed with repressor protein and sorting all non-fluorescent bacteria.
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| - | *'''step 3''' - A final passage through under pressure with repressor protain and sorting for fluorescent bacteria removes false negatives and enriches for bacteria bearing promoter that are low pressure inducible.
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| - | ==== Results ====
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| - | We finished step 1.
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| - | Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.
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| - | ===== Sequence and Characterization =====
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| - | <gallery widths="430px" heights="220px" >
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| - | Image:Dev 3.JPG|Figure 3 - FACS
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| - | Image:Tech dev 4.JPG|Figure 4 - Sequensing
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| - | </gallery>
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| - | We sorted fluorescent (A) and non-fluorescent bacteria (B) with a FACS.
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| - | Then, we analyze these base sequences.
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| - | *A have mutations in lacI binding site or non-functional DNA.
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| - | *B have mutations in CAP binding site, -35 or non-functional DNA.
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| - | ==== Conclusion ====
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| - | We have successfully demonstrated that it is possible to collect objective promoter by PCR random mutagenesis and screening with a FACS.
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| - | So we clearly believe that we can screen low pressure inducible lac promoter mutant with this strategy.
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| - | === <font size=5>'''Genetic toggle switch to implement rewritable function'''</font> ===
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| - | In order to implement rewritable function, we tried to construct Pressure Inducible Genetic Toggle Switch (in short PIGTS).
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| - | We thought two types of model - genetic circuit model and mathematical model - so as to construct PIGTS.
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| - | ==== Genetic circuit model ====
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| - | [[Image:Toggle_genetic_circuit_model.png|thumb|400px|right|figure 5-1 genetic circuit model]]
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| - | '''1. Writable function'''
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| - | * '''Plac''' is induced under 30MPa pressure
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| - | * Express CI and GFP
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| - | * CI represses PL and causes low LacI concentration
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| - | * Express increasingly CI and GFP('''inserted destruction tags''') ⇒ '''Bright!!'''
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| - | '''2. Erasable function'''
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| - | * '''PL''' is induced by heat
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| - | * Express LacI and LacI represses Plac
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| - | * Therefore, GFP expression decreases
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| - |
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| - | ==== Mathematical model ====
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| - | <gallery widths="200px" heights="150px" perrow="1" align="right" caption="Differential equation models">
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| - | Image:Room_pressure_model.png|figure 5-2 Atmospheric pressure model. It is known that n<sub>P<sub>L</sub></sub> = 3.0 from earlier studies
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| - | Image:30MPa_pressure_model2.png|figure 5-3 30MPa pressure model
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| - | </gallery>
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| - | We used two differential equation models and we call them 'atmospheric pressure model' and '30MPa pressure model'.
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| - | In order to implement rewritable function, we decided to use the difference of nullclines of each system so as to satisfy the following condition
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| - | * Converge to high CI concentration state under 30MPa pressure (writable)
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| - | * Bistable under atmospheric pressure (erasable)
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| - | [[Image:Desirable_condition.png|700px|thumb|left|figure 5-4 Desirable condition<!--. left:under atmospheric pressure (nullclines of figure 5-2), right:under 30MPa pressure (nullclines of figure 5-3).-->]]
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| - |
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| - | {{clear}}
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| - | ===== IPTG assay =====
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| - | [[Image:Transfer_Function.png|thumb|300px|left|figure 1 Hill function]]
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| - | '''Objective'''
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| - | By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.
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| - | '''Result & Conclusion'''
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| - | GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in figure 1 and the formulation of Hill function fitting described above, the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained '''n<sub>P<sub>lac</sub></sub> = 2.15'''.
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| - | {{clear}}
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| - | ==== Calculate the domain of appropriate parameters ====
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| - | [[Image:Desirable_condition_94.png|400px|thumb|right|figure 5-5 The domain of appropriate parameters if α<sub>Plac</sub> = 3.0]]
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| - | It is known that Plac activity under 30MPa pressure is '''94.0''' times stronger than Plac activity under atmospheric pressure, so, '''β<sub>30P<sub>lac</sub></sub>''' = 94.0.
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| - | If 30MPa additional activity rate of PL ('''β<sub>30P<sub>L</sub></sub>''') or the ratio of Plac - RBS strength ('''α<sub>P<sub>lac</sub></sub>''') and PL-RBS strength('''α<sub>P<sub>L</sub></sub>''') are not appropriate value, we can't construct PIGTS.
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| - | So we calculated the domain of appropriate β<sub>30P<sub>L</sub></sub> and the ratio of α<sub>P<sub>lac</sub></sub> and α<sub>P<sub>L</sub></sub>.
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| - | [[Image:PL_pressure_response.png|200px|none|thumb|left|figure 5-6 Pressure-response ability of PL]]
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| - | We identified '''β<sub>30P<sub>L</sub></sub> = 1.42''' by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose '''P<sub>L</sub> - RBS strength range from 2.4 to 7.4'''.
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| - | == <font size=5>'''Our Team'''</font> ==
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| - | <div align='right'>
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| - | [[Team:Tokyo_Tech/Home|see more]]
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| - | </div>
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| - | == <font size=5>'''Acknowledgements'''</font> ==
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| - | <div align='right'>
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| - | [[Team:Tokyo_Tech/Acknowledgements|see more]]
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| - | </div>
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For confirming pressure-response ability of pressure-inducible promoter, we experimented under 0.1MPa and 30MPa pressure. We chose TetR promoter(PtetR) as pressure-inducible promoter and we constructed two plasmids - one is PtetR-GFP on pSB6(BBa_K121010), the other is promoter less-GFP on pSB6(BBa_K121013) as a negative control.
Dilute this culture medium by 1% by adding fresh medium and suitable antibiotic (ampicilin; 50㎍/ml). Next, seal this culture medium with oxygen-saturated fluorinert (25% volume of medium) in polypropylene tubes with parafilm (figure3). Put this tube into pressure vessel filled with water (figure4). Next cup pressure vessel.
Finally, pressurize pressure vessel by pressure device(figure5). and then incubation was started at 37℃ immediately at each pressure vessel(original designed) for 16h at 37℃.After cultivation(figure6), the cells were examined by fluorescence microscope.
The result was that PtetR activity under 30MPa pressure is about 2.5 times stronger than PtetR activity under 0.1MPa pressure. Therefore, we confirmed that PtetR was induced under 30MPa pressure.
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