In order to address the cosmetics bacterial contamination issue, we aim to develop a handy detection method for consumers to examine if there is any Staphylococcus aureus and Pseudomonas aeruginosa contamination in their products.
So our focus is on utilizing Cas12a, a multifunctional protein sourced from the microbial CRISPR-cas immune system. We verified whether Cas12a could cleaved the target sequences. We developed a DETECTR system to detect Staphylococcus aureus and pseudomonas aeruginosa. So we chose femA and gbcA, which are important genes for Staphylococcus aureus and pseudomonas aeruginosa, to constructed plasmid. Then we used Cas12a to cleave these two plasmids. Finally we did gel electrophoresis and fluorescence experiment to examine whether it worked well.
During the project, we created several new parts and new experimental data for the Part Register.
Add new experimental data to an existing Part BBa_K4304007
1) Construction of the gene femA and gbcA containing plasmids
We had the company synthesize the DNA fragments femA and gbcA and inserted them into plasmid pUC57. We transformed these two plasmids into E.coli DH5a and cultured them overnight on the solid LB culture medium. Then we confirmed that these two plasmids contained femA and gbcA by bacteria PCR.
Figure 1. pUC57-femA/pUC57-GbcA in DH5α
Figure 2. Bacteria PCR of femA and GbcA.
2) Cleavage experiment
We transformed pET28a-FnCas12a into E.coli BL21 and induced protein Cas12a to express. Then we extracted and purified the protein. Besides, we designed sgRNA of femA and GbcA. We got the sgRNA through in vitro transcription method. In order to verify whether Cas12a could recognize and cleave the DNA fragments, we mixed Cas12a, sgRNA and the plasmids containing the target DNA fragments. The reaction was incubated at 37℃ for 2hours and then we verified the results by electrophoresis.
Figure3 Gel electrophoresis comparing before and after cleavage of the target DNA sequences
We can see target DNA sequence was cleaved by Cas12a and sgRNA after cleavage. In contrast, NC femA and NC gbcA contained the target DNA sequence. It shows that the in vitro cleavage experiment was successful.
Then we tested the reaction of the different sgRNA in bacteria liquid, we draw graph to show the efficiency of the different sgRNAs. In the graph, faster decreasing slope means higher efficiency in uncoiling the plasmid. Since all the sgRNA shows decreasing slope, all of them successfully react with the plasmids, which means our experiment succeed.
Figure4. Graph of efficiency comparison of sgRNAs
Add new information to the part BBa_K4926002 and 003
1) BBa_K4926002, pUC57-femA
FemA is a gene that encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance.[1] FemA expresses the important characteristics of staphylococcus aureus. We designed the plasmid by inserting the femA into the plasmid pUC57. This plasmid could be used to imitate staphylococcus aureus and then used for testing our DETECTR system.
2) BBa_K4926003, pUC57-GbcA
GbcA is a highly conserved response regulator in Gram-negative bacteria. It is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp.[2] We designed the plasmid by inserting the femA into the plasmid pUC57. This plasmid could bind with sgRNA and recruit Cas12a to cleave the GbcA sequence. So this plasmid could be used to imitate pseudomonas aeruginosa and used for testing our DETECTR system.
[1] Vannuffel, P., Heusterspreute, M., Bouyer, M., Vandercam, B., Philippe, M., & Gala, J. L. (1999). Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species. Research in microbiology, 150(2), 129–141. https://doi.org/10.1016/s0923-2508(99)80030-8
[2] Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski, A., & Haas, D. (1997). The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Molecular microbiology, 24(2), 309–319. https://doi.org/10.1046/j.1365-2958.1997.3291701.x