Part:BBa_K2273115

YhcR cell wall anchor derived from Bacillus subtilis

This membrane anchor is a part used in the Beta-Lactam Biosensor project of [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)]. The sequence encodes the C-terminal end of the yhcR gene found in the Bacillus subtilis W168 genome. YhcR is an extracellular endonuclease that processes RNA. Due to its C-terminal sequence it is anchored into the inner cell membrane by so-called sortases. The sortase YhcS is able to recognize the sorting motif in the YhcR protein (that is also localized at the C-terminus) and subsequently facilitates the attachment to the cell membrane. We chose this genetic sequence of the yhcR gene to create a cell wall anchor that enables the easy attachment of the enzyme BlaZ (a beta-lactamase found in Staphylococcus aureus) to the outer cell wall. To enable translational fusions, the RFC25 prefix and suffix have been added to the anchor sequence:

Prefix with	EcoRI, NotI, XbaI, RBS, spacer sequence, Start Codon and NgoMIV	GAATTCGCGGCCGCTTCTAGAAGGAGGTGTCAAAATGGCCGGC
Suffix with	AgeI, Stop Codon, SpeI, NotI and PstI	ACCGGTTAAACTAGTAGCGGCCGCTGCAGA

Sites of restriction enzymes generating compatible overhangs are indicated by sharing one color. (EcoRI and PstI are marked in blue, NotI in green, XbaI and SpeI in red, AgeI and NgoMIV in orange)

Beta-Lactam Biosensor

In this subproject, we developed a functional and complete heterologous beta-lactam biosensor in Bacillus subtilis. By the time these specified cells sense a compound of the beta-lactam family, they will respond by producing a measurable luminescence signal. We further investigated the detection spectrum of the biosensor by testing different beta-lactam antibiotics from various subclasses. For increased control and easy handling of the biosensor strain during a potential field application, we demonstrate that the encapsulation of the cells into Peptidosomes is quite advantageous.

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 153
  Illegal BamHI site found at 168
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 124