"
Team:NCTU Formosa/CI data
From 2011.igem.org
High Temperature Induced System – cI Promoter & cI repressor
Data
To verify that this circuit can work as expectation, we use GFP (Green Fluorescent Protein, BBa_E0040) as reporter protein. Part BBa_K098988 is also designed by Team Harvard 2008. In this circuit BBa_K098988, we can regard GFP (Green Fluorescent Protein, BBa_E0040) as reporter protein to tell us that the gene after previous circuit BBa_K098995 can be induced or not(Figure 3).
Figure 3. Part BBa_K098988 Design, with Part BBa_K098995 followed by Part BBa_E0240 (include BBa_B0032+BBa_E0040+BBa_B0010+BBa_B0012). Among this circuit, GFP (Green Fluorescent Protein, BBa_E0040) is reporter protein to tell us that the gene after the part BBa_K098995 will be induced or not.
First day, we incubated E.coli which contains this circuit (BBa_K098988) with GFP (Green Fluorescent Protein, 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, 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).
| 25℃ | 37℃ | 42℃ | |
| 0 | 9.02 | 8.76 | 9.14 |
| 1 | 4.66 | 8.36 | 17.54 |
| 2 | 2.82 | 6.46 | 29.24 |
| 3 | 2.37 | 5.00 | 26.44 |
| 4 | 1.70 | 4.69 | 22.27 |
| 5 | 1.80 | 4.40 | 21.94 |
| 6 | 1.52 | 4.72 | 21.57 |
| 7 | 1.58 | 4.78 | 21.01 |
| 9 | 1.22 | 3.9 | 22.21 |
Table 1. Related fluorescent unit at 25℃,37℃,42℃ in 9 hours
| 25℃ | 37℃ | 42℃ | |
| 0 | 2400.9500 | 2326.2350 | 2435.4930 |
| 1 | 1175.9040 | 2211.6410 | 4926.7290 |
| 2 | 683.2785 | 1673.7370 | 8559.5680 |
| 3 | 566.2294 | 1268.9040 | 7677.1730 |
| 4 | 395.3901 | 1184.0880 | 6377.2450 |
| 5 | 420.5876 | 1105.1540 | 6160.8630 |
| 6 | 350.3407 | 1192.2770 | 6160.8630 |
| 7 | 365.3116 | 1208.6670 | 5988.1660 |
| 9 | 276.2400 | 970.0604 | 6358.6760 |
Table 2. Molecules of equivalent fluorescence at 25℃,37℃,42℃ in 9 hours
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℃.
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 peak of green fluorescent intensity of E.coli incubated at 37℃ for 9 hours moves right a little bit than incubated at 25℃ for 9 hours. 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.
Figure5-1. Fluorescence of sample at 25℃, 0hr &9 hour. X-axis is related green fluorescent intensity, and Y-axis is cells number.
Figure5-2. Fluorescence of sample at 37℃, 0hr &9 hour. X-axis is related green fluorescent intensity, and Y-axis is cells number.
Figure5-3. Fluorescence of sample at 42℃, 0hr & 9 hour. X-axis is related green fluorescent intensity, and Y-axis is cells number.
Discussion
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 (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.
