Part:BBa_K539742
TetR repressible promoter (BBa_R0040)+ kivd (alpha-ketoisovalerate decarboxylase)+Ter (BBa_B0015)
Please refer to the wiki for our overall concept:[http://2011.igem.org/Team:NCTU_Formosa/BP_design 2011 NCTU_FORMOSA]
The part is built in kivd and TetR repressible promoter that constitutively ON and repressed by tetR. And in the end, terminatorPart:BBa_B0015 is included. The coding sequence of kivd is from Lactococcus lactis subsp. It encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. This is part of the isobutanol biosynthesis pathway. We want to make the isobutanol biosynthetic pathway under low-temperature induced system that has been constructed by 2010 NCTU Formosa. Combining this circuit with BBa_K539691 which is included gene of alss, ilvC, ilvD achieve the high production in isobutanol.
In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, but this way E.coli will overexpress the proteins we need in synthetic pathway. However, this overexpression of target proteins will cause E.coli wastes its limited growth resources, or the activity and performance of the enzymes may be too low. In this situation, it unbalances the synthetic pathway, and the production of isobutanol will not be optimum. This is also a problem in the production of isobutanol which is poisonous to E.coli.
To solve the problem, we need to adjust the expression of the genes, and make sure every intermediate can be catalyzed by the very next enzyme. The intermediates of isobutanol can be catalyzed step by step till they become the target products we want. In the new method we design, we control the pathway by stopping the mechanism when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then Apply this new method to our project. We first accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once.
In order to achieve the goal of our experimental design, we apply carbohydrate fermentation to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. We can also stop any step to accumulate the intermediates we want (see picture Overall Reaction).
We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)
Comparison of isobutanol production under low-temperature released device or not
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 2. 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℃
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 658
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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