manP expression cassette
This part is the expression cassette for expressing manP in B. subtilis. It includes the natural RBS and promoter for the manP gene found in Bacillus Subtilis 168 and the coding sequence is also the same with the exception of two sites: the starting at position 348 and ending at 350 and the codon starting at 2012 and ending at 2014 where a silent mutation for a CTG codon was turned into a TTA codon in accordance with TTA being B. Subtilis' favored codon for leucine in B. subtilis . 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 . 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 . 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 .
Notes On Using ManP as a Negative Selection Marker
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).
Unfortunately, due to limitations in lab space due to the pandemic the team was not able to independently verify manP as a negative selection marker, but its use as a negative selection marker is well documented in the 2015 paper by Marian Wenzel and Josef Altenbuchner which can be found at this link: https://www-microbiologyresearch-org.stanford.idm.oclc.org/content/journal/micro/10.1099/mic.0.000150a)
 Hershberg R, Petrov DA. General rules for optimal codon choice. Plos Genetics. 2009 Jul;5(7):e1000556. DOI: 10.1371/journal.pgen.1000556.
 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
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 129
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 722
Illegal AgeI site found at 372
Illegal AgeI site found at 466
Illegal AgeI site found at 2132
- 1000Illegal BsaI.rc site found at 272
Illegal SapI site found at 1294