Part:BBa_K2144005
Coding sequence for trunctated Lysostaphin with His6 and LPXTG-tag regulated by T7-promoter
Coding sequence for truncated Lysostaphin regulated by T7-promoter. This enzyme has the ability to cleave cross-linking pentaglycine bridges in the cell wall peptidoglycan found in certain Staphylococci.
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 540
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage:
Lysostaphin is an enzyme with the ability to cleave cross-linking pentaglycine bridges in the cell wall peptidoglycan found in certain Staphylococci.[1] Furthermore, studies have shown that Lysostaphin can degrade S.Aureus biofilm.[2] Thus, Lysostaphins antimicrobial functions can be used in applications to combat certain Staphylocci. In our project the focus was to evaulate and use Lysostaphins lytic properties to combat S.Aureus and its biofilm formation.
The encoding part of the BioBrick is derived from BBa_K748002, made by iGEM Harbin 2012. The T7 promoter (BBa_k525998) 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:
Lysostaphin is an extracellular enzyme produced by Staphylococcus simulans. The gene encoding for Lysostaphin is found on a large penicilinase plasmid. However, the maturement occurs extracellularly in S.simulans cultures and involves removal of the NH2-terminal containing seven tandem repeats of a 13 amino acids sequence. [3]
The enzyme has a glycylglycine endopeptidase acitivity lysing practically all known Staphylococci strains. The target, the interpeptide bridge of peptidoglycan, consists of five glycine residues found in many Staphylococci strains. However, if one glycine or more are substituted for serine residues , the lytic property of Lysostaphin will not be as effecient. The cell wall of Staphylococcus simulans has these replacements protecting it from lysing itself. [4]
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