Part:BBa_K887002:Experience

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Applications of BBa_K887002

Our circuit was equipped on backbone PSB3K3.

1.Isobutanol Tolerance

We did an experiment to prove the isobutanol is truly toxic to the E.coli. The data shows that the higher concentration of the isobutanol was in the medium, the lower OD600 value could be obtained.

Figure 1.Activate our E.coli overnight. Then, transfer it into the new medium with the microaerobic environment until OD600 reached 0.2. After that, measure the OD600 every 4 hours.

Figure 2. When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet.KivD can't be translated.

2.Our system Do Work

Figure 3.Mark the second circuit with the fluorescent protein to test the expression of kivD enzyme.

According to Figure 3, our low temperature release system do truly work !We used the fluorescent protein to mark the second circuit of our biobrick. The data tells us that kivD enzyme under 37℃ environment had the lower expression than under 30℃ and the 25℃ environment.

3.The production quantity of isobutanol.

27°C culture condition

Figure 4.Transfer to different temperature at different timing. We inoculated our E.coli in the 37℃ environment until OD600 up to 0.2 , which we set as 0 hour. Then, E.coli was transferred into 27℃ after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

32°C culture condition

Figure 5.Transfer to different temperature at different timing. We inoculated our E.coli in the 37℃ environment until OD600 up to 0.2 , which we set as 0 hour. Then, E.coli was transferred into 32℃ after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

42°C culture condition

Figure 6.Transfer to different temperature at different timing. We inoculated our E.coli in the 37℃ environment until OD600 up to 0.2 , which we set as 0 hour. Then, E.coli was transferred into 42℃ after being 0 hour, 12 hours , 16 hours, 20 hours and 24 hours.

According to the data (Figure 6.), we discovered that E.coli under 42℃ environment for 24 hours would have higher production of isobutanol than under 37℃ environment at the beginning. This result is totally out of our expectation, and the high production really surprised us! There are several factors that influence the production rate of isobutanol as follows:

(1) Concentration of enzymes

A higher concentration of enzymes leads to more effective collisions per unit time, which leads to an increasing reaction rate. However, the higher protein expression level is a metabolic load of host cells and decrease the growth rate.

(2) Temperature

Usually, an increase in temperature is accompanied by an increase in the reaction rate. Temperature is a measure of the kinetic energy of a system, so higher temperature implies higher average kinetic energy of molecules and more collisions per unit time. But the enzyme has its suitable reaction temperature, higher temperature may decrease the enzyme activity.

(3) Presence of the byproducts

The α-ketoisovalerate decarboxylase (Kivd) is a unique lactococcal key enzyme in the decarboxylation of branched-chain α-keto acids derived from branched-chain amino acids transamination into aldehydes. The promiscuous nature of kivd decarboxylase does not allow good selectivity in the decarboxylation step. Intermediate byproducts such as isobutyrate were present in the fermentation broth. The byproducts decreas the production rate of isobutanol

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