Part:BBa_K539691

promoter(lacI regulated)+Alss+ilvC+ilvD(each preceded by own RBS)and RNA thermometer+terminator

Please refer to the wiki for our overall concept:[http://2011.igem.org/Team:NCTU_Formosa/BP_design 2011 NCTU_FORMOSA]

In traditional genetic engineering method, 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.

Figure 1.Traditional genetic engineering method.

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).

Figure.2 Overall conception of isobutanol synthesis pathway

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.)

We cotransformed Part:BBa_K539691 and Part:BBa_K539742 into DH5α, so the expression of Alss, ilvC, ilvD and kivd are under the control of temperature.

And to realize whether Alss,ilvC and ilvD have any impact to the production of isobutanol, we compare the circuit which contain Alss,ilvC and ilvD genes with the one with only a single kivd gene in it.

Figure.3 Additionally insert the following genes, alss, ilvC, and ilvD(three precursory genes), the production of isobutanol will extremely increase. (About 80 times)

By viewing our two circuits we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet. In this way, Part:BBa_K539742 will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli.

Figure 4. When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet. In this way, Part:BBa_K539742 will not be expressed.

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 5. 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℃

Circuit 1

Built in Plac, two strong and one weak expressing RBS, Alss, ilvC, ilvD, 37’celcius regulator RBS, tetR and terminator (Part:BBa_K539651 with Part:BBa_K539653).

It catalyze pyruvate into 2-Ketoisovalerate in isobutanol biosynthesis pathway and inhibit Ptet contained circuit when the temperature hit 37’C or higher.

In the new method we design, we control the pathway by stopping the mechanism when it reach the non-toxic intermediate production step which we want to accumulate, then under specific thermal control, the mechanism would continue to express under specific thermal control.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 5198
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6087
  Illegal AgeI site found at 2564
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2150
  Illegal BsaI site found at 4984
  Illegal BsaI site found at 5241
  Illegal BsaI.rc site found at 535
  Illegal BsaI.rc site found at 1129
  Illegal BsaI.rc site found at 2969