Part:BBa_K1053209

Pconst.- RBS-YF1/FixJ-DT- Pfixk2- taRNA- DT- Pconst.- HHR*- GFP- DT

This part is used negative control against BBa_K1053208. HHR is expressed constantly and YF1 and FixJ is expressed too. In dark YF1 phospholylates FixJ response regulator and FixJ response regulator activates Pfixk2, so taRNA(taR12) is expressed. taRNA binds expressed HHR and inactivates HHR’s self cleavage. In blue light YF1 doesn’t phospholylate FixJ response regulator, so P_fixk2 isn't activated. taRNA doesn’t bind HHR but this HHR is mutated at active site, so HHR doesn’t do self cleavage. GFP’s RBS isn’t exposed, so GFP isn’t expressed.

Evaluation
The GFP fluorescence intensity was taken after main culture for 12 h by Plate Reader in this evaluation method.

Result Evaluation of transformed E. coli under the light or dark condition.
If HHR*’s active site is mutated difference between in light or dark isn't observed.The cause of this result is that excitation light of GFP and YF is almost the same.

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 585
  Illegal NgoMIV site found at 657
  Illegal NgoMIV site found at 747
  Illegal NgoMIV site found at 765
  Illegal NgoMIV site found at 1251
  Illegal NgoMIV site found at 1544
  Illegal NgoMIV site found at 1638
  Illegal AgeI site found at 299
  Illegal AgeI site found at 1419
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1308