Part:BBa_K1362093

pSB1K30: High copy BioBrick cloning/expression backbone carrying Kan resitance

Standard BioBrick cloning is a universal way of putting two BioBrick parts together to build a new BioBrick part. Despite several alternative cloning methods allow the assembly of multiple parts at one its simplicity and the broad availability of compatible parts keep it the 'de facto' standard of the iGEM-community.

Using standard BioBrick cloning, the generation of translationally active parts requires often more than one round of cloning. The ability to easily test the functionality of a protein before cloning them into complicated circuits has the potential to prevent many unsuccessful experiments of iGEM teams and may improve the characterization of the parts in the parts registry. However the extra amount of work required to clone such an additional construct may inhibit this behavior. We therefore improved the standard plasmids pSB1X3 and pSB4X5 by inserting a lacI repressible T7 promoter directly upstream to the BioBrick prefix of those plasmids. This promoter is completely inactive in 'E. coli' strains lacking a T7 RNA polymerase such as TOP10 or DH10beta bute inducible in strains carrying the T7 RNA polymerase under a lacI repressible promoter such as DE3 strains. This enables the use of the same backbone for cloning and over expression. Using 3A assembly a translational active part can be cloned from an RBS and a coding part in one step while maintaining the full flexibility of standard BioBrick assembly. These new RFC 10 conform backbones eliminate one cloning step needed for the expression and thus the characterization of a newly BioBricked protein. Version number 30 was claimed for the high copy variants and version number 50 for the low copy variants.

High copy BioBrick expression backbone:

pSB1A30(Part:BBa_K1362091): High copy BioBrick cloning/expression backbone carrying Amp resistance pSB1C30(Part:BBa_K1362092): High copy BioBrick cloning/expression backbone carrying Cm resistance pSB1CK30(Part:BBa_K1362093): High copy BioBrick cloning/expression backbone carrying Kan resistance pSB1CT30(Part:BBa_K1362094): High copy BioBrick cloning/expression backbone carrying Tet resistance Low copy BioBrick expression backbone:

pSB4A50(Part:BBa_K1362095): High copy BioBrick cloning/expression backbone carrying Amp resistance pSB4C50(Part:BBa_K1362096): High copy BioBrick cloning/expression backbone carrying Cm resistance pSB4K50(Part:BBa_K1362097): High copy BioBrick cloning/expression backbone carrying Kan resistance

Sequence and Features

Assembly Compatibility:

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

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2234
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2240
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2234
  Illegal XhoI site found at 1033
  Illegal XhoI site found at 2059
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2234
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2234
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2249
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.