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| | ===Applications of BBa_K539742=== | | ===Applications of BBa_K539742=== |
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| − | == '''Experience by 2011 iGEM NCTU_FORMOSA''' ==
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| − | '''Expression analysis in different host cell'''
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| − | We transform the circuit [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] ,which is inserted in pSB4A5, into DH5α and EPI300 and collect the bacteria incubated for 0HR, 24HR and 40 HR to find out the most appropriate host cell. After the analysis of GC we find out that DH5α is much better than EPI300 for this circuit, because it can provide higher productive rate of isobutanol. Therefore, we chose DH5α as the host in the following experiments.
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| − | https://static.igem.org/mediawiki/2011/9/9d/Butanol-10.1.png
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| − | '''Figure 1. This diagram indicates that DH5α is very suitable for this circuit [https://parts.igem.org/Part:BBa_K539742 K539742] , because it can provide higher productive rate of isobutanol.'''
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| − | '''Analysis of the isobutanol production in different temperature'''
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| − | Figure 2A is the curve of utilizing the temperature controlled simulation from 2010 iGEM team NCTU Formosa [https://parts.igem.org/Part:BBa_K332032 BBa_K332032]. We use green fluorescence protein(GFP) as reporter protein and detect mean fluorescence intensity after incubated under 30℃,37℃,and42℃ . Green fluorescence intensity was measured by flow cytometer. The result shows that our low-temperature released device can work at these three temperatures, so we can do the following experiment.
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| − | In figure 2B, after we make sure our low-temperature released system is available in 30℃,37℃,and42℃(by figure 2A), we cotransform [https://parts.igem.org/Part:BBa_K539691 BBa_K539691] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] into DH5α.Now the expression of alss, ilvC, ilvD and kivd are under the control of temperature. After we incubate three tubes of the E.coli at 37℃ until O.D.(optical density)reaches 0.5, we transfer two of them into different incubation temperature, 30℃ and 42 ℃, then detect the production of isobutanol at 0 hours and after 24 hours. In figure 12B suggests that it tends to produce isobutanol increasingly in lower incubation temperature, and the expression of Kivd will affect the production of isobutanol in different temperature.
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| − | https://static.igem.org/mediawiki/2011/6/66/Butanol-12.png
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| − | <br><b> Figure 2.</b><br>
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| − | (A.)<b> Mean fluorescence intensity (MEFL) at 30℃,37℃,42℃</b>
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| − | The vertical axis is mean fluorescence(MEFL), and the horizontal axis is time.
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| − | We can regard GFP as our target protein,Kivd. Under different temperature, a tendency shows that the production of GFP proteins will increase in lower incubation temperature. The result shows that our low-temperature released device can work at these three temperatures. In this way, we can do the experiment as the same, but changing the genes of our butanol circuit.<br>
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| − | (B.)<b> Production of isobutanol at 30℃,37℃,42℃</b>
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| − | Cotransform [https://parts.igem.org/Part:BBa_K539691 BBa_K539691] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] into DH5α, so the expression of alss, ilvC, ilvD and kivd are under the control of temperature. Coupling A. and B. diagram, the Kivd protein expression and the production of isobutanol are under temperature control, and both of their production will increase in lower incubation temperature. Therefore, we can conclude that the Kivd protein expression is related to the production of isobutanol.
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| − | '''Comparison of isobutanol production under low-temperature released device or not'''
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| − | We construct two devices. The first one is normal butanol synthetic device that includes alss, ilvC, ilvD and kivd only.([https://parts.igem.org/Part:BBa_K539671 BBa_K539671] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] ) The second one includes alss, ilvC, ilvD and kivd with low-temperature released device.( [https://parts.igem.org/Part:BBa_K539691 BBa_K539691] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] )As the result, in both devices, they tend to produce isobutanol increasingly in lower incubation temperature. However, the tendency is much more significant in low-temperature released device. We successfully improve the production of isobutanol by low-temperature released device.
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| − | https://static.igem.org/mediawiki/2011/3/3f/Butanol-15.png
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| − | <br><b> Figure 3.</b> Control group (non-temperature controlled device):[https://parts.igem.org/Part:BBa_K539671 BBa_K539671] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742]
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| − | Experimental group(low-temperature released device):[https://parts.igem.org/Part:BBa_K539691 BBa_K539691] and [https://parts.igem.org/Part:BBa_K539742 BBa_K539742] GC graph in 30℃, 37℃, 42℃
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| − | <br><b>In order to verify the cI repressed promoter is working, we made the modeling of BBa_K098988.</b>
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| − | The fluorescence intensity ofBBa_K098988 is measured by the flow cytometry (Figure. 1).
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| − | <br>https://static.igem.org/mediawiki/parts/d/d3/Modeling-1.JPG
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| − | <br>Figure1. Part BBa_K098988 Design. The heat induced device BBa_K098995uses gene BBa_K098997 coding for cI repressor to inhibit the cI promoter BBa_R0051. The activity of cI repressor is decreased by elevating temperature from 30 ℃ to 42 ℃. A differential equation is used to calculate protein expression activity of BBa_K098995 as follows.
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| − | <br>https://static.igem.org/mediawiki/parts/9/95/Modeling-2.JPG
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| − | <br>This equation describes the concentration of GFP in BBa_K098988change with time (Figure. 1). Alpha-Temp is the protein expression rates corresponding to BBa_K098995which is a temperature sensitive expression device. To describe transition during log phase and stationary phase, the alpha-Temp is assumed to zero in stationary phase. Gamma-GFP are decay rates of the GFP proteins. When bacteria divide, the molecular in a bacterium will be dilute. Because bacteria grow faster, the dilution rate d(t) is included in this model and can be calculated from OD ratio of medium (Figure. 2). The values of the kinetic parameters used in the simulation were initially obtained from the literature and experimental data. Data computations were performed with Matlab software. A program was written and used as a subroutine in Matlab for parameter optimization using nonlinear regression (Figure. 3).
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| − | <br>https://static.igem.org/mediawiki/parts/2/20/Modeling-3.jpg
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| − | Figure 2. The OD ratio is increased faster in log phase than it in stationary phase. The dilution rate d(t) can be calculated from OD ratio and used in out model.
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| − | <br>https://static.igem.org/mediawiki/parts/1/1a/Modeling-4.jpg
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| − | Figure 3. The behavior of high temperature induced device BBa_K098988 at 25°C, 37 °C and 42°C. Experimental data (dot) and simulated results (line) of the model suggest this temperature-dependent device can control the expression level of the target protein by the host cell’s incubation. The fitting results indicate our dynamic model can quantitatively assess the protein expression activity of BBa_K098988during log phase and stationary phase.
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| − | <br>Using least squares estimation from experimental data, the relative the protein expression activity of BBa_K098988 at 25°C, 37 °C and 42°C were estimated (Figure. 4).
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| − | <br>https://static.igem.org/mediawiki/parts/7/78/Modeling-5.JPG
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| − | Figure 4. The relative the protein expression activity of BBa_K098988at 25°C, 37 °C and 42°C estimated using least squares estimation from experimental data. The protein expression activity at 42°C is higher than 25°C, 37 °C
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| − | <br>According to the fitting results (Figure. 3), the dynamic model successfully approximated the behavior of our high-temperature induced system. The model equation presents interesting mathematical properties that can be used to explore how qualitative features of the genetic circuit depend on reaction parameters. This method of dynamic modeling can be used to guide the choice of genetic ‘parts’ for implementation in circuit design in the future.
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