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 highly expressing promoter to initiate our gene, so E.coli will over express the protein we need in synthetic pathway.
However, this overexpression will let E.coli waste its limited growth resources and too much intermediates will be accumulated.
These exceeded intermediates are poisonous to organisms, which then slow down the growing of E.coli and cannot achieve optimum productivity.

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.
The advantage of our new method is that the precursor is much less toxic for E.coli than our target product be.
Applying this new method to our project, we first accumulate lots of the non-toxic intermediate as the precursor, which is 2-Ketoisovalerate, to a certain amount, and then convert all the non-toxic precursor into the product, isobutanol, all at once.

To optimize the performance of the butanol pathway, we use the intermediates of E. coli’s amino acid metabolic system as raw material to produce butanol through the butanol biosynthesis pathway by adjusting the performace of gene we put into to the host cell.

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. Those enzymes are crucial for producing butanol.

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