Part:BBa_K4417019

CuO-RBS-ureABC-rrnB T1 Terminator-CuO-RBS-ureEFG-rrnB T1 Terminator MTU

Description

This part was created by cloning the master plasmid (BBa_K4417017) with the coding sequence of ureABC (BBa_K4417012) and ureEFG (BBa_K4417013). Both ureABC and ureEFG genes have their own cumate inducible promoter, RBS, and terminator.

Figure 1:CuO-RBS-ureABC-rrnB T1 Terminator-CuO-RBS-ureEFG-rrnB T1 Terminator MTU.

Usage and Biology

• This part can be transformed in both B. subtilis and E. coli.
• Inducer: p-isopropyl benzoate (cumate).
• Cumate is non-toxic to the host. In our experiment, we induced the urease expression with 50 μM cumate.
• E. coli ori is a pMB1 derivative
• B. subtilis ori is unknown
• The copy number of this plasmid in B. subtilis and E. coli is unknown.
• NdeI restriction can be used to change promoter. In this way, the performance of ureABC and ureEFG could be compared, each from a different promoter.

Method

Golden Gate Assembly was used to assemble the master plasmid with TU1 and TU2.

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 3844
  Illegal BamHI site found at 1
  Illegal BamHI site found at 2779
  Illegal XhoI site found at 4598
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]