Part:BBa_K887002

Plac+B0034+alsS+B0034+ilvC+B0034+ilvD+37℃RBS+tetR+double terminator+Ptet+B0032+kivD+B0015

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.
We use the cellulose to produce glucose. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. And we stop at the step to accumulate the intermediates we want (see picture Overall Reaction).

Figure 2. Overall conception of isobutanol synthesis pathway But isobutanol and isobutyraldehyde have biological toxicity and also can denature proteins,so we design a low temperature release system to stop at the step that produse 2-Ketoisovalerate. Figure 3. When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet.KivD can't be translated.

When being in 37°C environment, the first part will be translated and produced tetR protein to inhibit Ptet promoter and the first part will be translated to double terminator. The second part will not be translated because Ptet has been inhibited. Then we can accumulate non-toxic intermediate , 2-Ketoisovalerate,and it won't be toxic to E.coli. After getting enough 2-Ketoisovalerate ,E.coli will be moved to 30°C environment. The ribosome will not bind on the 37°C ribosome binding site and tetR genes will not be translated. Then promotor, Ptet, will not be inhibited. So the second part can be translated successfully. At the end , we can get the isobutanol .
Built in Plac, two strong and one weak expressing RBS, Alss, ilvC, ilvD, 37’celcius regulator RBS, tetR and terminator.

And in the end, terminatorPart:BBa_B0015 is included.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 6860
  Illegal XhoI site found at 5191
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6080
  Illegal AgeI site found at 2560
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2146
  Illegal BsaI site found at 4977
  Illegal BsaI site found at 5234
  Illegal BsaI.rc site found at 534
  Illegal BsaI.rc site found at 1128
  Illegal BsaI.rc site found at 2965