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To characterize the lytic efficiency of ClyC,we designed plasmid pET28a(+)_ClyC (assembled by Genscript) (Fig.1)， which allows ClyC to be expressd under the presence of IPTG.
 
To characterize the lytic efficiency of ClyC,we designed plasmid pET28a(+)_ClyC (assembled by Genscript) (Fig.1)， which allows ClyC to be expressd under the presence of IPTG.
  
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After ClyC is purified using nickel bead columns, the protein length and purity is confirmed by SDS-PAGE and stained with Coomassie Brilliant Blue.
 
After ClyC is purified using nickel bead columns, the protein length and purity is confirmed by SDS-PAGE and stained with Coomassie Brilliant Blue.
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Figure 2. SDS-PAGE Result of ClyC Purification
 
Figure 2. SDS-PAGE Result of ClyC Purification
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For a broader analysis, four S. aureus groups were diluted 200-fold and plated on chromogenic agar after 120 minutes of lysing. A 0.5uM concentration was chosen for its representativeness in evaluating ClyC endolysin's efficiency. The colony density post-overnight incubation aligns with the OD600 results, reaffirming the study's successful execution in this aspect
 
For a broader analysis, four S. aureus groups were diluted 200-fold and plated on chromogenic agar after 120 minutes of lysing. A 0.5uM concentration was chosen for its representativeness in evaluating ClyC endolysin's efficiency. The colony density post-overnight incubation aligns with the OD600 results, reaffirming the study's successful execution in this aspect
  
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Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin ClyC concentrations for 120 min.  
 
Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin ClyC concentrations for 120 min.  
 
A) water      B) elution buffer  C) 2uM ClyC      D) 0.5uM ClyC
 
A) water      B) elution buffer  C) 2uM ClyC      D) 0.5uM ClyC

Revision as of 01:20, 27 September 2023


ClyC


Usage and Biology

CylC is an endolysin that specifically lyse S.aureus. It is composed of two domains: a EAD domain that facilitates the enzymatic breakdown of peptidoglycan; a CBD domain that recognizes cell surface ligands and attracts the enzyme to its substrate [1].

Team:BNDS-China 2023

Our project intend to develop a set of efficient method of lysing S.aureus. In our project, ClyC is one of the tested endolysin that has the potent bactericidal capability. To express ClyC, this part was cloned into pET-28a and transformed into E. coli BL21 (ED3).

Characterization of lytic activity when expressed in E.coli

To characterize the lytic efficiency of ClyC,we designed plasmid pET28a(+)_ClyC (assembled by Genscript) (Fig.1)， which allows ClyC to be expressd under the presence of IPTG.


Fig. 1 The plasmid map of pET28a(+)_ClyC product

Examining protein length and purity using SDS-PAGE

After ClyC is purified using nickel bead columns, the protein length and purity is confirmed by SDS-PAGE and stained with Coomassie Brilliant Blue.

Figure 2. SDS-PAGE Result of ClyC Purification

The protein eluted displays prominent bands at about 35 kDa in both lanes, showing consistency with the calculated molecular mass (34.6 kDa). Interestingly, the final wash also shows a high purity of ClyC, suggesting that the reserved sample remains in high concentration even after the washing steps.

Examining lysing ability using spectrophotometer

To examine the sterilizing effect of varying ClyC endolysin concentrations over time, S. aureus was cultured overnight, diluted in TSB medium, and centrifuged once the OD600 reached 1.0. The cells were resuspended in reaction buffer and divided into five groups in sterilized tubes. Different concentrations of ClyC solution were added to each tube, with OD600 readings taken at 0, 15, 35, and 60 minutes post-addition, each measurement repeated thrice. The results underscore the strong bactericidal action of endolysin ClyC.


Figure 3. OD 600 of S. aureus under different concentrations of ClyC with respect to time (error bars are too small to be visible)

Examining lysing ability using chromogenic plates

For a broader analysis, four S. aureus groups were diluted 200-fold and plated on chromogenic agar after 120 minutes of lysing. A 0.5uM concentration was chosen for its representativeness in evaluating ClyC endolysin's efficiency. The colony density post-overnight incubation aligns with the OD600 results, reaffirming the study's successful execution in this aspect

Figure 4. Overnight S.aureus chromogenic plate of S.aureus treated with different endolysin ClyC concentrations for 120 min. A) water B) elution buffer C) 2uM ClyC D) 0.5uM ClyC