Part:BBa_K3697002

manP expression cassette

This part is the expression cassette for expressing manP in B. subtilis. It includes the natural RBS, Promoter, and coding sequence for the manP gene found in Bacillus Subtilis 168. Because of this, this cassette can be used in B. subtilis to produce this transporter protein at normal levels.

The manP gene in B. Subtilis is a part of the mannose phosphotransferase system (PTS). The mannose PTS is primarily encoded by two genes manA and manP. ManP encodes for the specific transporter used by B. subtilis to uptake mannose into the cell and phosphorylate it, whereas manA encodes for mannose-6-isomerase which converts mannose into fructose [1]. These two genes work in tandem to take mannose up into the cell and convert it into a form that is usable in the cell.

When manP is expressed in alone in a B. subtilis cell, it acts as a negative selection marker in the presence of mannose because it leads to an over accumulation of mannose with in the cell [1]. Mannose is continuously brought into the cell and phosphorylated but never is metabolized as mannose-6-isomerase is not there to convert it into fructose. This over accumulation of phosphorylated mannose eventually inhibits cell growth [1].

The easiest way to use manP as a negative selection marker is into to transform a plasmid containing it into a strain of B. subtilis that does not contain the mannose PTS. One example of a strain that is good for this is the strain 1A1276 from the Bacillus Genetic Stock Center (http://www.bgsc.org/catalogs.php). This strain is a good candidate for this kind of work because it not only has the mannose PTS system knocked out, but super-competence can be induced in this strain within 1.5 hours by adding mannitol to a liquid culture of cells and allowing them to grow in a shaker at 37C and 200rpm.

This manP can be easily put into a integration vector for B. Subtilis by adding the BioBricks suffix and prefix to the ends of this cassette when ordering the sequence and then assembling it into PBS1C (a common integration backbone for B. Subtilis).

[1] Wenzel, M. and Altenbuchner, J. (2015) Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate‐dependent phosphotransferase system. Microbiology (United Kingdom), 161(10), 1942–1949.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 129
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 722
  Illegal AgeI site found at 372
  Illegal AgeI site found at 466
  Illegal AgeI site found at 2132
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 272
  Illegal SapI site found at 1294