Part:BBa_K1648002

Construct Map for K1648002

Recombination Template for mamAB Operon with Promotor and Terminator

Consist of a template for recombination of mamAB operon(K1648000), a promotor & RBS(J13002) in front of each template and a double terminator(B0015) at the back of each template.

This part is designed for homologous recombination to introduce the large magnetosome forming operon into Azotobactor vinelandii.

This part is introduced into A. vinelandii genome via random integration before full operon sequences to be transformed into A. vinelandii.

This Part were verified by double digestion (Fig. 1) and sequencing.
Fig 1. Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: Recombination Template for mamAB Operon with Promotor and Terminator (BBa_K1648002) without digestion, with single digestion at XbaI site, with double digestion cut at XbaI and PstI site.

This part was introduced into Azotobacter vinelandii by stable genomic integration. Every coding parts of it were successfully expressed. The expression of proteins was shown in SDS-PAGE (Fig .2).

Fig 2. SDS-PAGE showing expression of Recombination Template for mamAB Operon with Promotor and Terminator (BBa_K1648002) in Azotobacter vinelandii. L: Protein ladder. Lane 1: Wild-type Azotobacter vinelandii. Lane 2: transformed Azotobacter vinelandii. Lane 2 shows 3 more bands compared to lane 1, in which the ~25 kDa band is the protein coded by chloramphenicol resistant gene in BBa_K1648002, the ~15 kDa and ~ 13kDa bands are the protein coded by Recombination Template for mamAB Operon.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 855
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 371
  Illegal AgeI site found at 890
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 616