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Team:NCTU Formosa/BP data
From 2011.igem.org
Butanol pathway
Data
Our butanol circuit device(Figure 8) indicate that the amount of Kivd protein will be regulated by different incubation temperature, because when the temperature is below 37℃, tetR will not be expressed and ptet will not be inhibited by TetR. In this way, part BBa_K539742 will express. Therefore, the production of isobutanol is adjustable. With the help of low-temperature released devices, we can improve the production of butanol.
Figure 8. BBa_K539691 and BBa_K539742 indicate that the amount of Kivd protein will be regulated by incubation temperature, because when the temperature doesn’t reach 37℃, tetR will not be expressed and ptet will not be inhibited by TetR. In this way, part BBa_K539742 will express. Therefore, the production of isobutanol is adjustable.
Built isobutanol concentration standard curve by Gas chromatography(GC) (Figure 9).
We dilute the pure butanol sample to get different concentration of butanol and make a standard curve for later comparison with our sample. The experimental data means that different butanol concentration will lead to different GC value(the area of the peak). Thus, GC value of our unknown sample can be applied into the standard line to get the unknown isobutanol concentration.
Figure 9. Isobutanol concentration standard curve: The concentrations of diluted samples are 800ppm, 400ppm, 200ppm, 100ppm, 50ppm, 25ppm and 12.5ppm.
Expression analysis in different host cell (Figure 10).
We transform the circuit 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.
Figure 10. This diagram indicates that DH5α is very suitable for this circuit BBa_K539742 , because it can provide higher productive rate of isobutanol.
Compare the production of isobutanol with precursory enzyme Alss, IlvC and IlvD or not(Figure 11).
In order to know the other three enzymes, Alss, IlvC and IlvD, whether impact the production of isobutanol, we compare the circuit with alss, ilvC and ilvD genes and the circuit with kivd gene only.
Figure 11.: Additionally insert the following genes, alss, ilvC, and ilvD(three precursory genes), the production of isobutanol will extremely increase. (About 80 times)
Analysis of the isobutanol production in different temperature(Figure 12).
Figure 12A is the curve of utilizing the temperature controlled simulation from 2010 iGEM team NCTU Formosa(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.
In figure 12B, after we make sure our low-temperature released system is available in 30℃,37℃,and42℃(by figure 12A), we cotransform BBa_K539691 and 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. (See more details in GC graph below)
Figure 12.
(A.) Mean fluorescence intensity (MEFL) at 30℃,37℃,42℃ The vertical axis is mean fluorescence(MEFL), and the horizontal axis is time. 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.
(B.) Production of isobutanol at 30℃,37℃,42℃ Cotransform BBa_K539691 and 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.
GC (Gas Chromatography) graph
Figure 13 and 14 represent the GC analysis of our bacterial culture with circuits BBa_K539691 and BBa_K539742. The peaks represent different kinds of alcohol respectively. The time axis is above the peaks. The peak of isobutanol appears at 4.55 to 4.62 minute. First , we incubate the bacteria at 37℃ and until O.D. reaches 0.5, then analyze the concentration of isobutanol via GC(Figure 13). The area of the peak in the red square of Figure 13 represents the related concentration of isobutanol in the beginning. After the bacteria is cultured at 30℃ for 24hr, we measure the medium composition again. The result is shown in Figure 14, and we calculate the area of same peak and find out that the related concentration of isobutanol increase approximately 20 times. It indicates our circuits can make bacteria produces isobutanol.
Figure 13. Cotransform two circuits, BBa_K539691 and BBa_K539742, into DH5α. Incubate DH5α at 37℃ until OD reaches 0.5. Before we switch the temperature to 30℃, we analyze the chemical composition of medium by GC.
Figure 14. Cotransform two circuits, BBa_K539691 and BBa_K539742 , into DH5α. Incubate DH5α at 37℃ until OD reaches 0.5, then we switch the temperature to 30℃ incubating for 24hr and analyze the chemical composition of medium by GC.
Comparison of isobutanol production under low-temperature released device or not(Figure 15).
We construct two devices. The first one is normal butanol synthetic device that includes alss, ilvC, ilvD and kivd only.(BBa_K539671 and BBa_K539742 ) The second one includes alss, ilvC, ilvD and kivd with low-temperature released device.( BBa_K539691 and 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.
Figure 15. Control group (non-temperature controlled device): BBa_K539671 and BBa_K539742 Experimental group(low-temperature released device): BBa_K539691 and BBa_K539742 GC graph in 30℃, 37℃, 42℃
Figure 16, 17 and 18 represent the GC analysis of our bacterial culture. The peaks represent different kinds of alcohol respectively. The time axis is above the peaks. The peak of isobutanol appears at 4.55 to 4.62 minute.
First ,we incubate the bacteria at 37℃ and until O.D. reaches 0.5, then culture it at 30℃, 37℃ and 42℃ for 24hr respectively. We measure the medium composition by GC. The results are shown in Figure 16, 17 and 18, and we calculate the area of the peak of isobutanol. As the result, we find out that the related concentration of isobutanol decreases when the temperature rises. Combine all the experiments did, our butanol pathway works perfectly with our low-temperature device.
Figure 16. Cotransform two circuits, BBa_K539691 and BBa_K539742 , into DH5α. Incubate DH5αat 37℃ until OD reaches 0.5, then transfer it into 30℃ and incubate for 24hr and we analyze the chemical composition of medium by GC.
Figure 17. Cotransform two circuits, BBa_K539691 and BBa_K539742, into DH5α. Incubate DH5αat 37℃ until OD reaches 0.5, then transfer it into 37℃ and incubate for 24hr and we analyze the chemical composition of medium by GC.
Figure 18. Cotransform two circuits, BBa_K539691 and BBa_K539742 , into DH5α. Incubate DH5α at 37℃ until OD reaches 0.5, then transfer it into 42℃ and incubate for 24hr and we analyze the chemical composition of medium by GC.
Comment
In our experiment, we do test by using two kinds of competent cell, DH5α and EPI300 culture to express kivd in a media with glucose. Comparing these two different host cells, we find an obviously different outcome of isobutanol production. DH5α is more efficient than EPI300, so we chose DH5α as our host cell.
To produce isobutanol, the alss, ilvC, ilvD genes under the control of the Plac promoter were over expressed to enhance 2-ketoisovalerate biosynthesis. Combine three genes, alss, ilvC, ilvD ,with kivd, we can increase the production of isobutanol.
We control the temperature when cultivate E.coli, as what we expected that the production of isobutanol will increase in lower incubation temperature.
Concluding above, we make the gene expression under low-temperature released decice. High temperature contributes to low production of cytotoxic isobutanol but high accumulation of the non-toxic intermediate, 2-ketoisovalerate. When we control the temperature to lower degree, the production of isobutanol will increase. In this way, we successfully gain our target product with the most efficient method.
