Part:BBa_K2384011

mf-Lon

mf-Lon is endogenous Lon protease based on the Gram-positive M.florum tmRNA system. And it is combined with lac operator*2+trc promoter+lac operator+RBS+CDS+rrnB T1 terminator.

Usage and Biology

Exogenous control of protein biosynthesis through transcriptional and translational regulation has been well established, but robust and tunable control of protein degradation in bacteria remains elusive.

Here we present a synthetic degradation system based on the Gram-positive M.florum tmRNA system that does not rely on host degradation systems and function in a wide range of bacteria.

Figure 1:Schematic of the tunable protein degradation system in which ATc-induced mf-Lon expression allows the protease to degrade constitutively expressed GFP in pdt-dependent manner.

Protein degradation in bacteria occurs in part through the transfer-messenger RNA (tmRNA) system, which uses C-terminal fusion of the ssrA peptide to direct proteins to the endogenous ClpXP and ClpAP proteases for rapid degradation in E. coli. Variants of the E. coli ssrA tag (ec-ssrA) are commonly used to modify the degradation rate of attached proteins in both bacteria and eukaryotes, but these tags do not provide inducible control of degradation. Recently developed inducible eukaryotic systems rely on degradation machinery not present in bacteria and bacterial systems such as the one developed by Davis et al. require disruption of the endogenous tmRNA system and are therefore not easily transferred to other organisms.

Toggle switch control of targeted essential protein degradation. Inclusion of the mf-Lon–specific pdt#1 tag on the specified essential gene causes mf-Lon–mediated degradation of the essential protein upon circuit activation.

Figure 2:Toggle switch.

This part is the combination of other two parts(BBa_K2384009) and (BBa_K2384010) , which could work together as a kill switch that uses a transcription-based monostable toggle design to provide rapid and robust target cell killing, dubbed ‘Toggle switch’

This part has made a condon optimization.

Sequence optimization information:

Expression System: Bacillus megaterium

Gene Length:2364 (bp)

Show the relative codon used distribution

Figure 4 :Codon Used Distribution after optimization.

Figure 5:GC Content after optimization.

Figure 3:Digested with EcoRI/PstI.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 25
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2498
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]