Difference between revisions of "Part:BBa K4593002"

Latest revision as of 14:19, 12 October 2023

ClyC

This part is the coding sequence of endolysin ClyC

Usage and Biology

CylC is an endolysin that specifically targets S. aureus. It is composed of two domains: a Cell Binding Domain (CBD) that recognizes cell surface ligands and attracts the enzyme to its substrate; and an Enzymatically Active Domain (EAD) that breaks down cell-wall peptidoglycan[1].

Team:BNDS-China 2023

Our project intends to develop a set of efficient methods for detecting and lysing S. aureus. ClyC is one of the tested endolysins that has a potent bactericidal capability and is applied in the lysing experiment in our project.

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 expressed 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 are 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.

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

The data highlights the potent bactericidal capability of endolysin ClyC. Over time, there's a noticeable decrease in the OD600 readings, signaling the dwindling of the S. aureus population. Interestingly, the bacterial reduction was more rapid at the 0.8uM concentration than at the 2uM and 0.2uM concentrations. This intriguing outcome could be attributed to the zero-salt environment required by the endolysin [1]. Also, the OD 600 of the mixture exhibits a significant growth just after the endolysin is added even though the resuspended bacteria in each tube are almost homogeneous, which is not explainable. Nonetheless, by the 60-minute mark, all three concentrations substantially curtailed the bacterial numbers, which successfully proved in the lytic activity.

Examining lysing ability using chromogenic plates

For further confirmation, 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'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

Sequence and Features