Part:BBa_K887004

Plac+alsS+ilvC+ilvD(each preceded by own zinc-finger and RBS)+Ptet+B0032+kivD+B0015

The main goal of our project this year is to improve the production of isobutanol in E.coli. In addition to the temperature control system, we also find the zinc fingers to be a perfect solution for our project. According to 2010 Slovenia team, the yield of violacein was at least 2-fold higher with the application of the zinc finger. Therefore, We expect higher concentration of isobutanol.

• More details and results about our temperature control system, please refer to Part:BBa_K539691 from 2011 NCTU_formosa iGEM team.
  

Figure 1.The Whole circuit of BBa_87004.

Figure 2.The source of our enzymes(names, strains, length and point mutation site).

This part（Part:BBa_87004) ,which is ligated from Biobrick(Part:BBa_K887000) with another one(Part:BBa_K539741)contain four enzymes. Each of the former three enzymes(alsS, ilvC& ilvD) linked with a zinc finger. The three enzymes could convert pyruvate into 2-ketoisoalerate(the Intermediate of isobutanol). The final enzyme(kivD)could change 2-ketoisoalerate into isobutyraldehyde, which would be converted into isobutanol by ADH in E.coli. This is a complete circuit to produce isobutanol.(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.)

Figure 3.The isobutanol biosynthesis pathway.

However, the isobutyraldehyde and isobutanol are toxic to E.coli. Why don't we stop producing toxic substance at first, and accumalate a large quantity of the non-toxic intermediate? Therefore, We accumulate lots of the non-toxic intermediate , 2-Ketoisovalerate, as the precursor to a certain amount, and then convert the entire non-toxic precursor into the final product, isobutanol. The advantage of our new method is that we could store large amount of the non-toxic 2-Ketoisovalerate, and convert all the precursor into the final product, all at once. It also cause less harm to the bacteria.

We also encoded three zinc fingers zif268(Part:BBa_K323105), PBS II(Part:BBa_K323107) and HIVC(Part:BBa_K165006)in our host (DH5a). Our current isobutanol yield by using temperature control system is about 0.7%, thus our conservative estimate of the isobutanol production is about 1.4%v/v(11.2g/L) which is better than recent paper(2011 Metabolic Engineering 13, James C. Liao) ,8.0g/L .

Figure 4.The modification of DNA program.

Furthermore, We inserted a lost base into DNA program(Part:BBa_K887011)from 2010 Slovenia(Part:BBa_K323066). We use DNA program to make a scaffold, which the enzymes fused with the Zinc finger can bind on it. The faster transport of intermediates could from the enzyme to the next one. It is just like a production line in a factory. With this strategy, E.coli could produce higher amounts of the final product efficiently with lower metabolic burden.

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 7718
  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