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Team:NCTU Formosa/Project-sub4
From 2012.igem.org
Optimization
Protocol of the M9 medium and trace metal solution.
To maximize the isobutanol production, we optimize E.coli strains, culture medium, time, temperature and carbon source. Amazingly, our production surpasses the published reference whose production is 6.8g/L for 24 hr by using modified JCL16 strain（Smith KM, Liao JC, An evolutionary strategy for isobutanol production strain development in Escherichia coli,2011.）.
Medium optimization
First, we tried to find the most suitable medium for DH5α to produce isobutanol.
Figure 17.Medium test: Incubated host cell (DH5α)in M9,M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract)medium in 37℃.Until OD600 up to 0.5, we transfer to 27℃ environment(blue) to see which medium is the best to produce isobutanol.
We cultured the DH5α strain in the common medium, M9,M9T(M9+ trace metal mix) and M9TY(M9+ trace metal mix+ yeast extract) in the low temperature release system. Data shows that when we changed M9 into M9T medium, the yield increased 10 times from 0.05% to 0.5%. Furthermore, when changed M9T into M9TY, the yield increased more than 50% from 0.5% to 0.8%! Consequently, M9TY is the most appropriate medium. And, this medium conforms to the journal we refers to（Smith KM, Liao JC, An evolutionary strategy for isobutanol production strain development in Escherichia coli,2011.）, so we decided to use M9TY as our medium in our upcoming experiments.
E.coli strain optimization
Next, we wanted to know that if other strains could have great productivity of isobutanol. So, we tested the following five strains: DH5α,DH10b,JM109,MG1655,EPI300.
Figure 18.Comparison of isobutanol production obtained with DH5α, DH10b,JM109,MG1655,EPI300, incubating in 37℃,3.6% glucose, M9TY medium. Until OD600 reached 0.2(set as 0 hr), strains were cultured for 24 hours then measure the production by GC.
According to the result (Figure 18.), we knew that DH5α produced the most isobutanol. So, we choose DH5α to produce isobutanol on our upcoming experiments.
How do we change our culture conditions?
After activating overnight in 37℃,we transferred 1/100 of volume to the new medium then cultured in 37℃ until OD600 reached 0.2, we set this point as 0 hour and transferred each tube to different condition at specific time. (Figure 19.)
Figure 19.This diagram explains our main idea in designing the experiment of the temperature control system. Green line means: 37℃(24hr); red line means: 37℃(12hr)27℃(12hr); blue line means: 27℃(24h). So, we can adjust different temperature and culture time likewise.
Culture time and temperature optimization
After knowing the most appropriate medium and strain, we tried to find out the best condition, including temperature and culture time, for our host cell to produce isobutanol.
Figure 20.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.
The report indicates that changing from 37℃ environment into lower temperature environment did produce more isobutanol. (Figure 20.) We could see that E.coli being in 37℃ environment for 20 hours and in 32℃ environment for 4 hours have the highest production quantity of isobutanol.
Figure 21.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 21.), 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.
Because of these factors affect the final isobutanol concentration,many versions of synthetic circuits which have different protein expression levels are needed to created to test performance in reliability and consistency, but this process is both tedious and time consuming. To overcome this problem, we develop a temperature control method to construct a isobutanol production circuit that can use culture temperature shifts to control the expression levels of a series of metabolic proteins at the precise times. The experimental data in Fig. 7 reveal the design method works successfully, and E.coli under 42℃ environment for 24 hours have higher production of isobutanol (~0.75%).
After we found the 42oC temperature phenomena, we then decided to tested our system on temperatures around 42 oC, in this case we tested on 40 oC and 44 oC. The figure below shows the E. coli production under 40 oC, 42 oC, and 44 oC. The graph indicated that the isobutanol production rate under 40 oC appears to be lower than that of 42 oC and the isobutanol production rate under 44oC wasn’t better than that of 42oC.
Figure 22.
The graph above shows that 42 oC still appeared to be the best temperature for our isobutanol production system. Furthermore, the graph below shows the overall production rate under various temperatures ranging from 25 oC to 44 oC after 16 hours incubation. This graph concludes that 42 oC is the best temperature for our isobutanol production system.
Figure 23.
qPCR
In order to figure out what happened behind the high temperature phenomena, we then do quantitative real-time polymerase chain reaction (qPCR) experiment on our system to find out the mRNA expression of our second circuit (kivD).
First, we incubated our bacteria till the OD600 reached 0.2. Afterwards, we changed the environment into various different temperatures, ranging from (25oC, 30 oC, 37 oC, 40 oC, 42 oC, 44 oC) for 16 hours. Next, we extracted mRNA and converted it into cDNA. Finally, by using our designed primer, we conducted qPCR. The result was as follows.
Figure 24.
Our result above shows that the 25 oC, 30 oC, 37 oC and 40 oC bars are in corresponding with our low temperature control concept. However, 42 oC and 44 oC are out of our expectation. It is most likely that in higher room temperature, tetR was unfavorable to bind the Ptet promoter for inhibition of the second circuit.
Carbon source optimization
Knowing that glycerol is the redundant product of the petroleum pyrolysis, we wanted to reduce the useless byproduct of petroleum Industry on earth. We fed our host with glycerol to see whether it is possible to be our E.coli‘s carbon source or not.
Figure 25.Culture our DH5α host cell in the medium of 3.6% glucose or 5% glycerol for three days in 30℃,37℃,and42℃. Test the sample by GC every 24 hours.
We discovered that using glycerol as carbon source could get the 1/3 of the yield of isobutanol produced by using glucose as carbon source. In this result (Figure 25.), the industrial byproduct, glycerol, can also be digested by our host and turned into the promising bio-fuel.
Furthermore, we also compared the productivity of different temperature.
Figure 26.After activating our host in the medium overnight, we transferred it to new medium, and continued culturing in 37℃. Until OD600 reached 0.2, we set this point as 0 hour, and cultured tubes in three different temperature. To analyze the production rate, we collected 1ml of the broth every 24 hours and measured the yield by GC.
By comparing 37℃ & 30℃ to 42℃, we found out that the best temperature to produce isobutanol is 42℃. From this figure, the productivity of all temperature decreased obviously after 24 hours. Thus, we conjectured that the isobutanol effused out to the air or the intermediate products of isobutanol pathway were converted into other byproducts.
