Part:BBa_K1392991

with constitutive promoter (J23108), XylE(K316003) coding sequence

Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2.Catechol or catechol 2,3-dioxygenases (XylE) (C2,3O) + O(2) is converted by a ring cleavage into 2-hydroxymuconate semialdehyde which is the cytotoxic and bright yellow-coloured product[1]. This is a key enzyme in many (soil) bacterial species used for the degradation of aromatic compounds. Catechol 2,3-dioxygenase[2] was originally isolated from Pseudomonas putida and is active only as a homotetramer. With araC-Pbad promoter,the production of XylE start and bacteria degrade catechol to 2-hydroxymuconate semialdehyde.

For this part characterization first we have dissolved 10 grams of catechol in 250 mL. distilled water.

Then we added culture media which has contained our part of K1392991 (J23108 + K316003) to two tubes for 5 mL. and only we have added 1000 μL. of catechol at one tube. After 1 hour later we have observed the color change from non catechol added to catechol added tube.

You can see the left tube is catechol added and right tube is non catechol added tube. After 1 hour later left tube’s colr changes to yellow with the presence of catechol induced indicator.

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This graph shows us that E.coli during adding catechol to media, diffuses quickly because of the high diffusion rate. Therefore you can see catechol concentration inintiates with a high value. Then our bacteria degrade catechol to 2-hydoxymuconate semialdehyde then it degrades to 2-oxopent 4-enoate then continues to 4-hydroxy 2-oxopentoonate, finally we get our last degraded product of pyruvate. Our paramaeters of this cycle shows us that. While our bacteria diffuses catechol in its cytosol, its enzymes starts to degrade catechol fast to obtain pyruvate.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 380
  Illegal NgoMIV site found at 552
  Illegal AgeI site found at 903
  
• 1000
  COMPATIBLE WITH RFC[1000]