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| - | Data
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| - | To verify that this circuit can work as expectation, we use GFP (Green Fluorescent Protein, <a href=””>BBa_E0040) as reporter protein. Part <a href=””>BBa_K098988 is also designed by Team Harvard 2008. In this circuit <a href=””>BBa_K098988, we can regard GFP (Green Fluorescent Protein, <a href=””>BBa_E0040) as reporter protein to tell us that the gene after previous circuit <a href=””>BBa_K098995 can be induced or not(Figure 3).
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| - | Figure 3. Part <a href=””>BBa_K098988 Design, with Part <a href=””>BBa_K098995 followed by Part <a href=””>BBa_E0240 (include <a href=””>BBa_B0032+<a href=””>BBa_E0040+<a href=””>BBa_B0010+<a href=””>BBa_B0012). Among this circuit, GFP (Green Fluorescent Protein, <a href=””>BBa_E0040) is reporter protein to tell us that the gene after the part <a href=””>BBa_K098995 will be induced or not.
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| - | First day, we incubated E.coli which contains this circuit (<a href=””>BBa_K098988) with GFP (Green Fluorescent Protein, <a href=””>BBa_E0040) at 37℃ overnight. The next day, we transferred them to new three tubes with M9 medium. When the O.D. reached 0.08, we incubated them to 25℃, 37℃, and 42℃, respectively. We collected the samples once an hour, diluting them 200x for accurate GFP (Green Fluorescent Protein, <a href=””>BBa_E0040) measurements. Here is our experiment raw data (Table 1). The mature data will be obtained by unit-conversion according to beads control data (Table 2). We can use Table2 data to draw line chart data, which is easy for us to compare the mean fluorescence at different temperature, 25℃, 37℃, and 42℃. (Figure4) The vertical axis is mean fluorescence, and the horizontal axis is time (unit: an hour).
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| - | Original Data (Related fluorescent unit)
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| - | 25 37 42
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| - | 0 9.02 8.76 9.14
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| - | 1 4.66 8.36 17.54
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| - | 2 2.82 6.46 29.24
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| - | 3 2.37 5 26.44
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| - | 4 1.7 4.69 22.27
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| - | 5 1.8 4.4 21.94
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| - | 6 1.52 4.72 21.57
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| - | 7 1.58 4.78 21.01
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| - | 9 1.22 3.9 22.21
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| - | Table 1. Related fluorescent unit at 25℃,37℃,42℃ in 9 hours
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| - | Data after unit conversion
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| - | 25℃ 37℃ 42℃
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| - | 0 2400.95 2326.235 2435.493
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| - | 1 1175.904 2211.641 4926.729
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| - | 2 683.2785 1673.737 8559.568
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| - | 3 566.2294 1268.904 7677.173
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| - | 4 395.3901 1184.088 6377.245
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| - | 5 420.5876 1105.154 6275.167
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| - | 6 350.3407 1192.277 6160.863
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| - | 7 365.3116 1208.667 5988.166
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| - | 9 276.24 970.0604 6358.676
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| - | Table 2. Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours
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| - | Figure 4. Line chart of molecules of equivalent fluorescence performance at 25℃,37℃,42℃ in 9 hours. The vertical axis is mean fluorescence, and the horizontal axis is time. Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples at 25℃,37℃.
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| - | The following diagrams, Figure5-1.~Figure5-3., are from the Flow cytometer experiment we did. The green fluorescent intensity of E.coli incubated at 25℃ (Figure5-1) and 37℃(Figure5-2) for 9 hours is lower than incubated at 42℃(Figure5-3). Also can refer to the data from Table 1. In Figure5-2., the green fluorescent intensity of E.coli incubated at 37℃ moves right a little bit. Last, in Figure5-3., incubated at 42℃ for 9 hours, the peak of green fluorescent intensity is significantly stronger than incubated at 25℃ and 37℃ for 9 hours.
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| - | Figure5-1. Fluorescence of sample at 25℃, 0hr &9 hour.
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| - | Figure5-2. Fluorescence of sample at 37℃, 0hr &9 hour.
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| - | Figure5-3. Fluorescence of sample at 42℃, 0hr & 9 hour.
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| - | Discussion
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| - | Form the experimental data, we find that MEFL increases steeply at 42℃ in first two hours, which means the expression of GFP is high. Although after the first two hours its performance decreases and maintains at certain volume, it still expresses a lot more than the other two samples in different temperatures. We can regard GFP as our target protein. This means that our hypothesis has been confirmed that any protein placed after this circuit (<a href=””>BBa_K098995) can be regulated by elevating or lowering temperature. At higher temperature(42℃), our target protein will perform significantly. On the contrary, at lower temperature(25℃), it will express slightly.
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Design
In order to make our target protein has a large amount of performance at high temperature, we use the circuit BBa_K098995, which is designed by Team Harvard 2008. (Figure1)
Figure 1.
Part
BBa_K098995 Design, with constitutive strong promoter
BBa_J23114 , followed by RBS(Ribosome Binding Site)
BBa_B0034, cI repressor
BBa_K098997, terminator
BBa_B0014, and high temperature-sensitive cI promoter
BBa_R0051. Among this circuit, cI repressor(
BBa_K098997) will be produced constantly to inhibit the cI promoter(
BBa_R0051) at low temperature. At high temperature, cI repressor(
BBa_K098997) is still produced, however; cI repressor(
BBa_K098997) will degrade soon. In this way, cI promoter(
BBa_R0051) will be activated. This part is designed by 2008 Harvard on backbone pSB1A2, and we transform this part to host strain DH5α for further use.
This circuit uses gene BBa_K098997coding for cI repressor to inhibit the cI promoter BBa_R0051. The activity of cI repressor is decreased by elevating temperature from 30 ℃ to 42 ℃. We just need to place our target gene after this circuit BBa_K098995, and then regulate the performance of our target protein by incubating E.coli at different temperature between 30℃ to 42℃. At higher temperature, our target protein will produce abundantly. On the contrary, at lower temperature, it will not express well. The function of this circuit is illustrated as followed. (Figure 2.) At lower temperature, cI dimer (cI repressor, BBa_K098997) will block the RNA polymerase from further transcription, which leads to the gene after this circuit BBa_K098995 can’t be expressed. Although, at higher temperature, cI dimer (cI repressor, BBa_K098997) will degrade and RNA polymerase can transcribe the DNA sequence again, which means that the expression of the gene after this circuit can be recovered.
Figure 2.
First,
BBa_K098995 on backbone PSB1A2 is transformed to host strain DH5α. Then we can find out that, at lower temperature, cI dimer (cI repressor,
BBa_K098997) will block the RNA polymerase from further transcription, which leads to the gene after this circuit
BBa_K098995 can’t be expressed. Although, at higher temperature, cI dimer (cI repressor,
BBa_K098997) will degrade and RNA polymerase can transcribe the DNA sequence again, which means that the expression of the gene after this circuit can be recovered.
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