Difference between revisions of "Part:BBa K5311006"

Revision as of 20:34, 29 September 2024

CAP 10-3 Endolysin

We hypothesized that the incorporation of an endolysin into our Cutibacterium acnes design would help to address both safety concerns and improve the release of the Cry proteins, maximizing therapeutic delivery. Since the bacteria will be in direct contact with the skin, using an endolysin enables controlled cell lysis, reducing the risks of having a GMO in prolonged contact with the skin.

We will combine its implementation with a regulation mechanism by using an RNA thermometer that controls the endolysin expression, activating it at the regular skin temperature (around 37°C) to ensure precise timing, and facilitating the distribution of our product, as it wouldn’t start producing the endolysin until it reached the skin. Additionally, the endolysin helps facilitate the release of therapeutic Cry proteins which is another of the key components of our project, breaking down the bacterial cell walls and enhancing the effectiveness of the treatment against skin infestations. This dual function ensures safety while improving protein release.

Sequencing

Sequence and Features

Biology

In the case of the endolysin derived from the CAP 10-3 bacteriophage, its action specifically targets the degradation of the cell wall of Cutibacterium acnes, a Gram-positive bacterium with a cell wall rich in peptidoglycan, which is a crucial structural component. This endolysin is classified as an N-acetylmuramoyl-L-alanine amidase, which cleaves the bond between the N-acetylmuramic acid and the L-alanine residue in the peptidoglycan backbone.

By breaking down the peptidoglycan, the endolysin disrupts the rigid structure of the cell wall, leading to a loss of osmotic integrity. As a result, the bacterial cell can no longer withstand the internal pressure, causing rapid cell lysis. This mechanism is particularly effective against Gram-positive bacteria like C. acnes because they have a thick peptidoglycan layer that is more accessible to endolysins, unlike Gram-negative bacteria, which possess an additional outer membrane that restricts endolysin access.[1]

Advantages of Endolysins Against C. acnes:

• High Specificity: The CAP 10-3 Endolysin is known for its high specificity towards C. acnes, often recognizing unique peptidoglycan motifs present in the bacterial cell wall. This ensures that they selectively lyse the pathogenic bacteria without affecting other beneficial members of the skin microbiota [2].
• Low Risk of Resistance: Unlike traditional antibiotics, which typically target essential processes such as protein synthesis or DNA replication, endolysins act on the structural components of the bacterial cell wall, such as the peptidoglycan layer. The peptidoglycan is a highly conserved and essential component that is less prone to mutation, reducing the likelihood of bacteria developing resistance [3].
• Safety and reduced side effects: Endolysins have a favorable safety profile compared to traditional antibiotics, as they are proteins that are rapidly degraded in the human body without inducing toxic side effects. Furthermore, since they specifically target bacterial cell wall components, they do not interact with human cells, making them safe for topical or systemic applications [4].

Usage

Pending

Characterization

As we have described previously one of the main objectives on using endolysin was to implement a regulation mechanism with an RNA Thermometer that allows transcription once the ideal temperature is reached (37°C). Previous to this transformation we succesfully characterized the behavior of this part on C. acnes strain KPA171202. In order to do that designed our construct with a His-tag thanks to this we were able to perform a Western Blot that allowed us to quantify the expression in both of our chassis of interest C. acnes and E. coli and we also conducted an experiment to quantify the effect of endolysin on the growth of C. acnes.

Western Blot :

The Western Blot performed by our team was conducted to characterize the endolysin expression with the combination of different chassis and different promoters. Since we also wanted to evaluate how was the protein being expressed we conducted the western blot with both pellet and supernatant. Finally on the Western Blot the wells were organized with the following order:

• Sample 1: Not rellevant for this part documentation
• Sample 2: E. coli NZY 5α with Endolysin+MG10 (pellet)
• Sample 3: C. acnes with Endolysin+MG10 (pellet)
• Sample 4: C. acnes with Endolysin+MG26 (pellet)
• Sample 5: Not relevant for this part documentation
• Sample 6: C. acnes with Endolysin+MG10 (supernatant)
• Sample 7: C. acnes with Endolysin+MG26 (supernatant)
• Sample 8: Not relevant for this part documentation

Where MG10 and MG26 are constitutive promoters that allow expression on C acnes (while MG10 also allows it in E. coli) For E. coli, proteins are primarily intracellular, requiring cell lysis to isolate the proteins from the pellet. For C. acnes, we anticipated protein expression both within the cell and in the supernatant. Thus, we evaluated both pellet and supernatant samples to capture the full protein profile.

For the loading of the SDS-PAGE we used the SeeBlue Pre-Stained Standard Western blot ladder which has its reference atta