Part:BBa_K2144100
Coding sequence for Nuclease with His6 and LPXTG-tag regulated by T7-promoter
Coding sequence for Nuclease from Staphylococcus Aureus, regulated by T7-promoter. This enzyme has the ability to cleave DNA.
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]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage:
Nuclease is an enzyme with the capability of cleaving phosphodiester bonds between nucleotides in DNA. This enzyme can be used in applications concerning degradation of DNA. In our project the aim was to evaluate and use Nuclease degrading abilities to inhibit the biofilm formation produced by Staphyloccus Auerus.
The encoding part of the BioBrick is derived from BBa_K729004, made by iGEM London 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:
Nuclease is an extracellular enzyme found in Staphyloccus Aureus. S.Aureus encodes for two Nucleases, Nuc1 and Nuc2, [1] whereof the last-mentioned is a part of this BioBrick. Nuc2 is connected to the membrane with a N-terminal anchor. [1]
There are several bacterial pathogens using Nuclease to combat the immune response in the host by for instance avoiding neutrophil extracellular traps. (NETs). [2] Furthermore, since eDNA is an important component of biofilm, Nuc is able to work as a regulator of biofilm formation.[3] Studies have suggested that the expression of Nuc contributes to the inability to produce biofilm [3] and during biofilm formation-conditions the expression of Nuc is repressed.[1]
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