Part:BBa_K539691:Experience

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Experience by 2011 iGEM NCTU_FORMOSA

Figure.1 Firstly , we ligase B0030 with ilvC.

Figure.2 At the sametime , we ligase B0030 with ilvD.

Figure.3 Meanwhile , we ligase B0032 with Alss.

Figure.4 To make sure we obtain the correct part , colony PCR is needed.

Figure.5 Secondly , we ligase Plac with B0032+Alss

Figure.6 Thirdly , we ligase Plac+B0032+Alss with B0030+ilvC

Figure.7 At the same time , we ligase B0030+ilvD with RNA thermometer+tetR+double terminator

Figure.8 Finally , we ligase the whole circuit:promoter(lacI regulated)+Alss+ilvC+ilvD(each preceded by own RBS)and RNA thermometer+terminator

Analysis of the isobutanol production in different temperature

Figure 9.

(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 10. Cotransform two circuits, Part:BBa_K539691 and Part: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 11. 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 12. 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℃

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