Part:BBa_K2144006
Coding sequence for ESP with His6 and LPXTG-tag regulated by T7-promoter
Esp, extracellular serine protease, is capable of inhibit the biofilm formation and nasal colonization of S. aureus. This by protelysing receptors essential for biofilm formation and prevention of eDNA exudation.
The inserted LT-sequence contain the gene encoding for His-tag which enables the use of IMAC to purify the combat protein. Furthermore it contains the LXPTG-tag which enables the use of the "match making" enzyme Sortase. The key feature of sortase is the specific conjugation reaction it carries out, where the enzyme recognizes a specific amino acid sequence, a so called sorting motif (LPXTG motif in the case of S.aureus) and conjugate this sequence with another unit carrying an oligo glycine motif. A new peptide bond is formed. The Linker in the LT-sequence enables proper folding of the protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 431
Illegal XhoI site found at 180
Illegal XhoI site found at 1001 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1254
Illegal AgeI site found at 1377 - 1000COMPATIBLE WITH RFC[1000]
Usage:
Esp, extracellular serine protease, is capable of inhibit the biofilm formation and nasal colonization of S. aureus. This by protelysing receptors essential for biofilm formation and prevention of eDNA exudation. Thus, the biofilm degrading properties of ESP can be used in applications concerning biofilm degradation and infectious diseases caused by S.Aureus. In our project the aim was to evaulate and use ESP’s abilities to disrupt and eridicate the biofilm formation S.Aureus.
The encoding part of the BioBrick is derived from BBaK531005 , made by iGEM Grinnell 2011. The T7 promoter (BBak525998) has been inserted to control protein expession under IPTG induction. To regulate the protein expression under T7 promoter control, E.coli containing a T7 polymerase must be used, for instance BL21(DE3).
Biology:
Extracellular serine protease (Esp) is secreted by a subset of Staphylococcus epidermidis, which is a non-pathogenic bacterium in human nasal cavity and skin. Staphylococcus epidermis prevent colonization and biofilm formation of S.Aureus.One underlying mechanism for this is ESP’s ability to degrade proteins critical for the attachment of bacteria to the host; thus, changing S. aureus sessile form to planktonic form [10].
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