Part:BBa_K1392991

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

Part J23108 is a constituve promoter and its backbone is pSB1C3.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 J23108 constitutive promoter,the production of XylE start and bacteria degrade catechol to 2-hydroxymuconate semialdehyde.

We did gel electrophoresis of the part K1392991 and we observed that; First two wells ares approximately 2100 but it is circular. Other one is linearized and it is approximately 3000bp

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 colour changes to yellow with the presence of catechol induced indicator.

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.

Our parameter values are below in the chart:

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]