Team:NCTU Formosa/BP data

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.

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.

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.

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 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 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.

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 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.

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.