Parts
BioBricks made by iGEM Stockholm 2016:
BBaK2144000 T7-Nuclease
BBaK2144001 T7-Lysostaphin
BBaK2144002 T7-ESP
BBaK2144003 T7-Defensin
BBaK2144004 T7-Nuclease-LT
BBaK2144005 T7-Lysostaphin-LT
BBaK2144006 T7-ESP-LT
BBaK2144007 T7-Defensin-LT
BBa_K2144008 Sortase
Basic Parts
Composite Parts
T7-Nuclease (BBa_K2144000)
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 BBaK729004, made by iGEM London 2012. 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: 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 [3] 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]
T7-Lysostaphin (BBa_K2144001)
Usage:
Lysostaphin is an enzyme with the ability to cleave cross-linking pentaglycine bridges in the cell wall peptidoglycan found in certain Staphylococci.[4] Furthermore, studies have shown that Lysostaphin can degrade S.Aureus biofilm. Thus, Lysostaphins antimicrobial functions can be used in applications to combat certain Staphylocci. [5]
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 BBaK748002, made by iGEM Harbin 2012.
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: 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. [6]
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. [7]
T7-ESP (BBa_K2144002)
Usage: Esp, extracellular serine protease, is capable of inhibit the biofilm formation and nasal colonization of S. aureus [8]. 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. [9]
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].
The following BioBricks are derived from BBa_K2144000, BBa_K2144001, BBa_K2144002 and BB_K2144003. The inserted LT-sequence (55 bp) contain the gene encoding for His-tag which enables the use of IMAC to purify the combat protein. Furthermore, the LT-sequence contain the gene encoding the LPXTG-motif which makes the conjugation between the expressed protein and spider silk possible. More information about how the conjugation works can be found here [Link to Amans Sortase-section]
T7-Nuclease-LT (BB_K2144004)
(Picture)
T7-Lysostaphin-LT (BB_K2144005)
(Picture)
T7-ESP-LT (BB_K2144006)
(Picture)
T7-Defensin-LT (BB_K2144007)
(Picture)
Basic Parts
A vision for iGEM Stockholm 2016 was to create a new part for the registry encoding for the match-making protein Sortase.
Sortase (BB_K2144008)
Sortase A is a bacterial enzyme with the ability to break and form new peptide bonds. The key feature of the enzyme 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. To learn more about Sortase A [Click Here] (Link to Sortase Description)
The sequence BB_K2144008 is isolated from S.aureus by iGEM Stockholm 2016.
References
[1] Megan R. Kiedrowski, 2012, Characterization of Staphylococcus aureus extracellular nuclease activity, Iowa Research Online, 5 February
[2] Michael E. Olson et al, 2013, Staphylococcus aureus Nuclease Is an SaeRS-Dependent Virulence Factor, American Society for Microbiology, 4 February
[3] Megan R. Kiedrowski et al, 2011, Nuclease Modulates Biofilm Formation in Community-Associated Methicillin-Resistant Staphylococcus aureus, PLOS One, 11 November
[4] Kokai-Kun JF, Walsh SM, Chanturiya T, Mond JJ (May 2003). "Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model". Antimicrobial Agents and Chemotherapy.
[5] Wu JA, Kusuma C, Mond JJ, Kokai-Kun JF (Nov 2003). "Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces".Antimicrobial Agents and Chemotherapy.
[6] Paul A. Recsei et al, 1987, Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans, Biochemistry Vol.84, 17 October
[7] Gunther Tumm et al, 1997, Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus Simulans biovar staphylolyticus, Research Gate, March
[8] Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T, Mizunoe Y. 2010. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature 465:346 –349.
[9] Chen Chen, Vengadesan Krishnan, Kevin Macon, Kartik Manne. Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus. J Biol Chem. 2013 Oct 11; 288(41): 29440–29452.
[10] Sugimoto S, Iwamoto T, Takada K, Okuda K, Tajima A, Iwase T, Mizunoe Y. Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction. J Bacteriol. 2013 Apr;195(8):1645-55.
