Part:BBa_K4435305

level 2 JUMP plasmid, pBBR1 ori, AmpR

BBa_J428367 derived plasmid with the replacement of SpecR for AmpR

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 3060
  Illegal suffix found at 16
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3060
  Illegal SpeI site found at 17
  Illegal PstI site found at 31
  Illegal NotI site found at 24
  Illegal NotI site found at 3066
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3060
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3060
  Illegal suffix found at 17
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3060
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3075
  Illegal SpeI site found at 17
  Illegal PstI site found at 31
  Illegal NgoMIV site found at 749
  Illegal NgoMIV site found at 878
  Illegal NgoMIV site found at 1373
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3082
  Illegal BsaI.rc site found at 10
  

Construction and description

According to multiple scientific papers, standard E. coli compatible replication origins are not functional in Komagataeibacter. Unusual chassis frequently require plasmids with broad species origins such as RK2, pBBR1, or RSF1010. Plasmids BBa_J428346, BBa_J428347, BBa_J428349 also encode the KanR marker and whereas BBa_J428366, BBa_J428367 and BBa_J428369 encode SpecR (the latter of which has not been proven effective in Komagataeibacter). Hence, the level 2 vectors supplied with the distribution kit were not compatible with our chassis. To overcome this, we designed primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers. In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. PCR products were purified and the backbone and the new antibiotic resistance marker with combined by GoldenGate assembly with SapI and T4 DNA ligase. Competent DH5alpha were transformed with the ligation products and transformants selected on LBamp supplemented with either ampicillin or chloramphenicol. Successful assemblies yielded plasmids encoding green fluorescence and either AmpR (BBa_K4435301 to BBa_K4435305) or CmR (BBa_K4435308 to BBa_K4435312).  We confirmed the functionality of the novel backbones by transformation into E. coli followed by restreaking in different media. We also successfully transformed Komagataeibacter rhaeticus AF1 with