Part:BBa_K2144001

Coding sequence for trunctated Lysostaphin 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 540
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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 lysostaphin's 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 maturation 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]

Results:

This biobrick was characterized and by 2016 Stockholm iGEM team. Double digested lysostaphin gene was 3A assembled with digested T7 promoter and backbone pSB1C3, forming pSB1C3-T7-Lys. 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-Lys. (1) Ladder (6) Lysostaphin Colony 1 (7) Lysostaphin Colony 2

Following protein expression, SDS PAGE was run to investigate the solubility of recombinant lysostaphin by determining the presence of expressed protein, either dissolved in the supernatant or insoluble in the pellet. The expressed protein's expected size is 27kDa and the SDS PAGE shows clear bands of around 30kDa for all transformed samples, both in the supernatant and pellets. Therefore, we cannot draw a clear conclusion regarding lysostaphin's solubility but have indications that it can be found in both the soluble and insoluble cell lysate fractions.

Figure 2. SDS PAGE of Expressed Lysostaphin

To confirm the lytic activity of lysostaphin in E. coli, a modified Kirby Bauer test of the cell lysate was performed on E. coli TOB1, a biofilm producing strain. The zone of inhibition from induced samples were larger than that of uninduced samples, which demonstrates the bacteriolytic activity from the cells lysate and implies that the expressed lysostaphin is enzymatically active.

Figure 3. Kirby Bauer Test of Expressed Lysostaphin on E. coli TOB1

The bactericidal effect of expressed lysostaphin against bacterial species commonly found in wound infections, S. aureus and P. aeruginosa was quantitatively evaluated. A bactericidal test was performed by adding different amounts of cell lysates to bacterial cultures and analysing the optical density (OD) at various time points to detect changes in bacterial density. The results illustrated bacteriolytic activity of lysostaphin on S. aureus upon treatment of both soluble and insoluble fractions from cell lysates to bacterial cultures. The positive control clearly indicates the inhibition of bacterial growth and cell lysates with concentration as low as 1µg/mL showing a similar effect. Lysate containing lysostaphin on also showed bactericidal activity on P. aeruginosa with concentrations of 30µg/mL. This confirms that lysostaphin can be potent inhibitors of bacterial growth found in chronic wounds.

Figure 4. Bactericidal test results of cell lysates containing lysostaphin on S. aureus

Figure 5. Bactericidal test results of cell lysates containing lysostaphin on P. aeruginosa

Finally, to confirm the enzymatic function and characterization of the BioBrick, the inhibition and dispersal of S. aureus and P. aeruginosa biofilm formation was demonstrated using crystal violet assay. The average absorbance of each sample treatment was graphically displayed, indicating that the biofilm is reduced with increased volume of lysostaphin treatment. The t-test statistically showed P-values of <0.001 indicated significant differences in biofilm formation for both supernatant and pellet samples. These conclusive results imply the inhibition of S. aureus biofilm formation, adding to the bacteriolytic function of lysostaphin and underlining the rationale for including lysostaphin in our project focusing on dispersing biofilm in chronic wounds.

Figure 6. Biofilm inhibition test of lysostaphin on S. aureus. Lys 2 (S) refers to the soluble fraction of cell lysate, Lys 2 (P) to the insoluble fraction.

References:

1: Sabala, I., Jagielska, E., Bardelang, P. T., Czapinska, H., Dahms, S. O., Sharpe, J. A., James, R., Than, M. E., Thomas, N. R. & M. Bochtler. Crystal structure of antimicrobial peptidase lysostaphin from Staphylococcus simulans FEBS Journal. 281, 4112-4122 (2014).

2: Wu, J. A., Kusuma, C., Mond, J. J. & Kokai-Kun, J. F. Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces. Antimicrob. Agents Chemother. 47, 3407–14 (2003).

3: Thumm, G. & Gotz, F. Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus. Mol. Microbiol. 23, 1251–1255 (1997).

4: Recsei, P. A., Gruss, A. D. & Novick, R. P. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Biochemistry 84, 1127–1131 (1987).