Part:BBa_K887000

Plac+B0034+zif268+alsS+B0034+PBSII+ilvC+B0034+HIVC+ilvD+37℃ induced RBS+tetR+double terminator

In traditional genetic engineering method, we use strong promoter to initiate our gene expression, so that 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, so the activity and performance of the enzymes may be too low. In this situation, the synthetic pathway is unbalanced, 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.
We make a big change to improve the traditional gene.
To solve the problem, we need to adjust the expression of the genes. We use the cellulose to produce glucose. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step.But isobutanol and isobutyraldehyde have biological toxicity and also can denature proteins,so we design a temperature control system to stop at the step that produce 2-Ketoisovalerate. We 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. The toxic isobutanol and isobutyraldehyde will produce at the last minute and cause less harm.

In our design, we put E.coli in 37°C and control the pathway by stopping the pathway when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate. Then under specific thermal control that moves E.coli to 30°C, the mechanism would continue to express isobutyraldehyde at once. Then isobutyraldehyde will been produced into isobutanol with the enzyme of the widetype E.coli.
But we are not satisfied.
We encode four zinc fingers in front of each enzyme.Zinc fingers could tightly bind to specific DNA or RNA sequence. We replace the zinc fingers' functional domains with our enzymes to create fusion proteins to put the enzymes in order. When the intermediates are produced, it could have the next reaction as quickly as possible.

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 6049
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6938
  Illegal AgeI site found at 3158
  Illegal AgeI site found at 4143
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2744
  Illegal BsaI site found at 5835
  Illegal BsaI site found at 6092
  Illegal BsaI.rc site found at 836
  Illegal BsaI.rc site found at 1430
  Illegal BsaI.rc site found at 3563