Part:BBa_K2762016

P_gadA-RiboJ-B0034-GFP

Background

This is a composite part that including P_gadA, a pH sensitive promoter, a ribozyme sequence, RiboJ, a RBS and a GFP sequence. Promoter gadA is a pH sensitive promoter from E. coli, described by Dundee iGEM 2016. For the part of RiboJ, it was first described as an insulator in genetic construct but there has no any data shown this insulation affected the downstream genetic parts. The mechanism of RiboJ is that the upstream sequence as will as RiboJ sequence will cleave after the transcription. The cleavage of promoter and RiboJ sequence aids the design of predictable genetic constructs and the iGEM team William and Mary 2016 has reported that RiboJ could increase the expression of the downstream gene.

Function of gadA promoter

Our team, NCKU_Tainan 2018 has decided to use gadA promoter as one of the promoter in our pH sensing system but there has a problem, the fluorescent intensity is too low to be observed. To solve this problem, we have added RiboJ at the downstream of gadA promoter. In result, the fluorescence intensity of the strain that contains RiboJ is more than the strain that without RiboJ. In conclusion, we have successfully strengthen the signal and increase the specificity of gadA promoter. Thus, we could determine the pH condition of the medium when the color of the medium turns from turbid yellow to green.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1020
  

References

Kalen P Clifton, Ethan M Jones, Sudip Paudel, John P Marken, Callan E Monette, Andrew D Halleran, Lidia Epp, Margaret S Saha. ( 2018.May.9). The Genetic Insulator RiboJ Increases Expression of Insulated Genes. bioRxiv. doi: https://doi.org/10.1101/317875