Part:BBa_K590017

mamHIEJKL in vector pGA3K3

This part is made by [http://2011.igem.org/Team:Washington UW iGEM Team 2011]. It includes 6 genes mamHI, mamE, mamJ, mamKL as well as the native mamAB promoter, all in a pGA3k3 plasmid backbone. All the genes were found essential for magnetosome formation in Magnetospirillum magneticum strain AMB-1.

Usage and Biology

We made this construct from 2 kilobase gene extractions from AMB-1. These fragments were assembled using the [http://2010.igem.org/Team:Cambridge/Gibson/RFC Gibson cloning] method instead of standard assembly. This super-assembly is known as "Part one" of the full 16kb mamAB reassembly project. This part is in a medium copy backbone and it belongs to the [http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit].

The gel image on the left is HIEJKL(~9000bp), we extracted this band and "gibsoned" it with pGA3k3 plasmid backbone(~3000bp), the right image.

1kb Ladder (left), mamHIEJKL (right)

1kb Ladder (left), 3k3 backbone (right)

After Gibson cloning and transformation, cell chain formation was observed in microscopy(below) when this plasmid was transformed in E.coli.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 5175
  Illegal PstI site found at 8836
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 5175
  Illegal PstI site found at 8836
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 8540
  Illegal XhoI site found at 89
  Illegal XhoI site found at 6375
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 5175
  Illegal PstI site found at 8836
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 5175
  Illegal PstI site found at 8836
  Illegal NgoMIV site found at 238
  Illegal NgoMIV site found at 1254
  Illegal NgoMIV site found at 2589
  Illegal NgoMIV site found at 3920
  Illegal NgoMIV site found at 6754
  Illegal NgoMIV site found at 8562
  Illegal NgoMIV site found at 8929
  Illegal AgeI site found at 5849
  Illegal AgeI site found at 6264
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 651
  Illegal BsaI site found at 1986
  Illegal BsaI site found at 3317
  Illegal BsaI.rc site found at 6947
  Illegal SapI site found at 825
  Illegal SapI site found at 2160
  Illegal SapI site found at 3491
  Illegal SapI.rc site found at 9208
  Illegal SapI.rc site found at 9529