From 2011.igem.org

Esp

Esp is a type of serine protease, secreted by Staphylococcus epidermidis, a non-pathogenic bacterium that is usually found in human nasal and oral cavities. S. epidermidis has a close relative called Staphylococcus aureus, which is a bacterium found on the skin and in the nasal passages of up to 25% of healthy people and animals. Some strains of S. aureus cause illness by producing a heat stable toxin and it is difficult to get rid of because it can form a biofilm.

Moon et al. first isolated and biochemically characterized the Esp in 2001, discovering this “novel extracellular protease from S. epidermidis” can degrade fibrinogen, complement protein C5, and several other proteins with a strong preference for cleavage after glutamic acid residues. This mechanism was claimed, therefore, to enable S. epidermidis to evade the complement defense system. As a matter of fact, this claim was supported by another group of scientists’ discovery. Dubin et al. noticed that the gene structure of Esp and the amino acid sequence of its mature form showed close similarity with one of the S. aureus serine proteases, which have been long regarded as important virulence factors destructing tissue and blood proteins and thus contributing to enhanced invasiveness (Dubin et al. 2001).

However, Iwase et al found out Esp, in reality, hindered S. aureus colonization and infection by inhibiting biofilm formation. Although the mechanism of how Esp breaks the S. aureus biofilm is still unknown and does not resemble any bacterial interference mechanisms such as growth inhibition and bactericidal activity, Esp was observed to degrade S. aureus biofilm by changing S. aureus from the sessile to the planktonic form. Sixteen hours incubation with supernatant containing Esp resulted in a astonishing four-fold decrease of S. aureus biofilm. One thing that distinguishes Esp from the other biofilm breakers is the fact that Esp does not break biofilm by killing the bacteria that are in the biofilm, but rather releasing the bacteria from the biofilm. Therefore, it will be hard for S. aureus to build resistance to Esp.

We tested two versions of the esp gene, the wildtype gene and the optimized version for expressing in Caulobacter crecentus. The wildtype gene was acquired from colony PCRing S. epidermidis with the corresponding primers designed for esp gene. Because the esp gene is overall G/C poor with a G/C content of around 30% while our model organism Caulobacter crecentus has a G/C rich genome (60%) and presumably expresses G/C rich DNA more accurately and efficiently, we decided to optimized the gene codon without any change on amino acids sequence. This gene was synthesized by IDT. BioBrick part BBa_K531003 and Bba_K531006.