Part:BBa_K2144010
Coding sequence for Esp regulated by T7-promoter
Coding sequence for Esp regulated by T7-promoter. Extracellular serine protease (Esp) is secreted by Staphylococcus epidermidis. It is capable of dispersing the biofilm of S. aureus.
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:
Extracellular serine protease (Esp); secreted by S. epidermidis, this protease degrades the proteins that maintain structural and chemical integrity of the biofilm. Purified Esp inhibits biofilm formation at the preliminary stage and is able to detach a pre-existing one. Additionally, Esp reduces the virulence of S. aureus through degrading other important proteins, e.g. quorum sensing molecule receptors, enzymatic proteins for biofilm dispersal.
The encoding part of the BioBrick is derived from BBaK531005 named Esp Basic (EB) , 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:
The function of Esp degrades the proteins that maintain structural and chemical integrity of the biofilm. It also reduces the virulence of S. aureus through degrading other important proteins. Esp could be applicable for the prevention or treatment of infectious diseases caused by S. aureus.
Results:
This biobrick was characterized and validated by 2016 Stockholm iGEM team. Double digested Esp Basic (EB) gene was 3A assembled with digested T7 promoter and backbone pSB1C3, forming pSB1C3-T7-EB. The ligated product was successfully transformed into both strains of E.coli: Top10 and BL21(DE3). The colonies observed were picked and confirmed with colony PCR using primers VF2 and VR.
Figure 1. Colony PCR of E.coli TOP 10 containing pSB1C3-T7-EB. (1) Ladder (2) T7-EB-pSB1K3 Colony 1 (3) T7-EB-pSB1K3 Colony 2 (4) T7-EC-pSB1K3.
Our synthesized EB displayed potent biofilm dispersal capacity in the Biofilm assay for both S. aureus and P. aeruginosa. For P. aeruginosa the treatment with Esp was as efficient as the positive control.
Figure 5. Biofilm dispersion test of Esp on S. aureus.
| None |