Difference between revisions of "Part:BBa K4204000"
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Latest revision as of 02:10, 14 October 2022
Coding sequence of endolysin LysPBC5
This part only contains the ORF of LysPBC5.
Usage and Biology
LysPBC5 is an endolysin that specifically lyse Bacillus Cereus but also has some activity against other strains in B.cereus group. It is composed of two domains: a CBD (cell-wall binding domain) that recognizes and binds to specific cell wall peptidoglycan (PG) structures, and the EAD (enzymatically active domain) that later cleaves the glycosidic bonds in PG layers to break the cell wall [1].
Team:BNDS_China 2022
Our project intend to develop a set of efficient method of detecting B.cereus. In our project, LysPBC5 is the most important protein since it's depended by both types of detection method designed.
To express LysPBC5,this part was cloned to expression vector pET28a(+) with a round of PCR and restriction enzymes XhoI and NheI. After purifying the protein, we performed two experiments complimentary to each other to examine the lysing ability of LysPBC5.
Examining lysing ability using spectrophotometer
In the first experiment, we use OD600 to reflect the concentration of living B.cereus. An ultraviolet spectrophotometer (blanked with pH8.0 tris-HCL) is used to test the OD600 of B. cereus in three different concentrations of endolysin presented below. The endolysin protein concentration is separated into non-protein, half-diluted protein, and none diluted protein. We used Tris buffer for the dilution and the blank, and the lysing time and other variables were kept the same during the experiment. The data is shown in the graph below. The OD600 tested for none-endolysin, half-diluted endolysin, and none-diluted endolysin are 0.484A, 0.320A, and 0.301A, respectively. The experiment shows that lysPBC5 is able to break down the cell wall, and the OD600 decrease as the concentration of protein increases.
Due to the limitation of experimental materials, we are not able to determine the exact concentration of LysPBC5. Thus further experiment is required to determine the optimal concentration of lysing B.cereus.
Examining lysing ability using chromogenic plates
Through the first experiment, we found that it’s hard to wipe out the possibility that the elution buffer (the solution used to elude the LysPBC5 off the column during protein purification) could lyse B.cereus. Thus an additional experiment using the chromogenic plate culturing method is performed. B.cereus culture that was mixed with LysPBC5 protein solution, elution buffer, and LB medium was diluted 100 times before being inoculated to the top 2, the bottom left, and the bottom right chromogenic plate, respectively.
The result shows that only a few colonies formed on the plate with LysPBC5 treated culture, while the colony number on the plate with elution buffer treated culture doesn’t have a significant difference from that of the control group. This proved that it is LysPBC5 that successfully lyses the B.cereus cell.
Fig. 2: chromogenic plate inoculated with B.cereus culture treated with different chemicals.
①: B.cereus culture treated with diluted LysPBC5
②: B.cereus culture treated with concentrated LysPBC5
③: B.cereus culture treated with elution buffer
④: B.cereus culture treated with LB medium
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 690
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 742
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 988