To solve the problem described in our description page, we plan to make a convenient, cheap, and broadly applicable detection kit. The kit uses endolysin to lys B.cereus cells and detect the ATP or DNA it released. Based on the result of our integrated human practice which reminds us to lower the price of the protein product, we decided to apply the protein quality control (ProQC) system to lower the price of our protein in terms of increasing the amount of functional protein and apply the crystal violet induction system to lower the price of inducer.
Endolysins are hydrolytic enzymes that bacteriophages induced the bacteria to express at the end of the lytic cycle. Different from ordinary antibiotic medicines that destroy harmful bacteria indiscriminately, endolysin is more effective and precise since it is coded for the targeted bacteria cell wall. It is a two-domain protein, containing a CBD (cell-wall binding domain) that recognizes and binds to specific cell wall structures, and the EAD (enzymatically active domain) that later breaks the cell wall.
Figure 1. The structure of LysPBC5. LysPBC5 forms a homodimer structure as shown in the figure. Chain A is marked pink while chain B is marked blue.
The decision of choosing endolysins is enlightened by previous research that chemically conjuncted CBD with metal ions to label B.cereus strains and then separate them using a magnetic field. The separated strains are then lysed and the ATP they released is detected [1]. Although this method works, it is still tedious and lab-dependent. To find a highly specific way that could lyse the cell without a separation process, we identified endolysin LysPBC5. Among the endolysins we find, it is the most accurate one: CBD of LysPBC5 recognizes the peptidoglycan structure of B.cereus via conjoined tandem SH3b domains rather than interacts with the peptide crosslinks [2].
After applying lysPBC5 to the sample to detect, It could specifically recognize B.cereus PG (peptidoglycan structure) domain on the cell wall and break it. After the burst of the cell wall, ATP and the B.cereus genome are released for further detection.
Table 1. Host range of Bacteriophage PBC5, lytic range of LysPBC5, and binding range of LysPBC5-CBD [2].
For the sake of amplifying the specific DNA fragment for the HOLMES technique, Loop-mediated isothermal amplification (LAMP) is applied to achieve the high efficiency of amplification and detection. LAMP is a newly developed thermostatic nucleic acid amplification approach for gene diagnosis, which was published by Japanese scholar Tsugunori Notomi in Nucleic Acids Res in 2000. The hallmark of LAMP is high specificity and sensitivity, simultaneously simple operation, and low requirements for experimental facilities.
Whilst the LAMP reaction, gene amplification is performed by repeating two types of extension reactions that occur through the loop region (i.e., the template extends itself from a stem-loop structure formed at the 3’end, followed by new primers that bind to and extend the loop region). Notably, paired internal and external primers are used. Each internal primer has a sequence complementary to a strand of the amplified region at the 3-terminus and identical to the internal region of the same strand at the 5-terminus. The extension reaction is repeated sequentially by DNA polymerase-mediated strand-substitution synthesis, and the basic principle of this method is to generate significant quantities of DNA amplification products with complementary sequences and alternating structural repeats [3].
Figure 2. basic process of LAMP
The target gene of LAMP (which the HOLMES system will detect later) was set as the gene of 16srRNA [4] (which has known SNP between strains in the B.cereus group that is detectable for HOLMES) and HBLA [5] (a toxin-related gene in our chosen strain). Primers targeted on these genes are designed and the amplification result could be seen on our result page.
One-HOur Low-cost Multipurpose highly Efficient System (HOLMES) is a technique for detecting the presence of a specific sequence of DNA under the condition of a low target concentration. In our project, we are trying to use this technique to detect the presence of certain unique sequences in the B. cereus genome after it is subjected to cell lysis.
The fundamental principle for the HOLMES system is detecting DNA sequences and releasing detectable fluorescence signals. It is composed of the CRISPR RNA (crRNA), the Cas12b protein, and the ssDNA probe which is comprised of a fluorescence group and a quenching group each at one end. After adding the target sequence (mostly amplification product) to a tube with the HOLMES system, under the guidance of the crRNA in the system, the Cas12b protein will bind to the target sequence. After performing the cis-activity which breaks the target DNA strand, the ssDNA probe in the system will be trans-cleaved by the Cas12b enzyme and thus generate fluorescence able to be detected with apparatus. The fluorescence enables the identification of the presence of the target sequence [5].
Figure 3. Principle of HOLMES
To Lower the detection limit, we employed the Loop-Mediated Isothermal Amplification (LAMP) method to amplify the target sequence. The employment of the LAMP method will lower the lowest detection limit and enable us to detect bacterial presence at lower concentrations, as it decreases the lowest target concentration significantly from 1nM to 10-8 nM. The procedure for LAMP and the target gene is described in detail in the previous section.
Another way of detecting B.cereus is to detect the ATP lysed B.cereus released. The determination of ATP is based on the reaction of luciferase catalyzing luciferin with the presence of ATP. ATP Bioluminescence Assay Kit CLS II (Sigma Aldrich) is adopted in terms of all measurements of ATP concentration during the project. This kit could be further freeze-dried and fixed on nitrocellulose paper to get ATP test strips [6].
Theoretically, after the addition of lysis protein Lys-PBC5, our target bacteria Bacillus Cereus would break and release its content which includes ATP. The ATP released will react with luciferin (catalyzed by luciferase) and emit fluorescence with an emission peak of 562nm [7]. Through experiments, we would be able to infer the amount of ATP contained by graded concentrations of B.cereus so as to draw the standard curve of ATP content within any concentration of the bacteria.
To lower the price of the inducer, we employed a Crystal Violet induction (Jungle Express) system for protein production in our project. This system originated from Enterobacter lignolyticus, a rainforest strain, in which EilR, the repressor protein, regulates the expression of EilA, a protein responsible for pumping out multiple ionic chemicals. [8] Cationic dyes release EilR from its operator. Due to the sensitive response of EilR to uM levels of crystal violet, Crystal violet is used for its rapid induced transcription and high potency and efficacy, which indicates its positive cooperativity.
Crystal violet induction system provides high protein expression levels at very low costs due to its high sensitivity and low price of crystal violet. Thus although crystal violet has minor cytotoxicity as a nucleic acid dye, its toxicity is ignorable due to the low concentration required for induction. In the application of Jungle Express for protein production in E. coli, compared to traditional Pt7 promotors, CV has only minimal effects on the growth rate at the required concentrations for full induction. In combining this operator with E. coli phage early promotors and using cationic dye crystal violet as low-cost inducers, EilR functions as a cheap, effective, and sensitive regulatory component [8]. Thus we used the crystal violet induction system to replace the Lac operon on our expression vector pET28a(+).
Figure 4. gene circuit of Crystal Violet induction system
The longer an expressed mRNA is, the higher its risk for truncation due to the halting and dissociation of bacterial RNA polymerase. Truncated mRNA lack part of the coding frame and the terminator, thus will cause the translation of nonfunctional polypeptides. The translation of mRNA has a high energy cost; however, the energy cannot be recycled. The high truncated mRNA generation rate means a lot of energy is wasted. Given that when genes are overly expressed for engineering purposes, the ribosome-rescue systems of bacteria themselves (A system that enables bacteria to avoid errors during translation) are already insufficient, it is vital for us to prevent ribosome wastes. As a result, we want to solve the problem by imitating post-transcriptional regulation to prevent abnormal protein synthesis. (Limiting translation to fully transcribed mRNAs).
We found the ProQC system. The system contains a toehold switch at the 5' end of an mRNA and a corresponding trigger (Cis-trigger) sequence with reverse complementarity to the toehold switch at the 3' end of the mRNA. The switch binds with its complementary sequence and blocks the RBS inside a neck-ring structure; this blocks transcription initiation. When Cis-trigger appears, the switch will bind with the trigger preferentially because the Cis-trigger has a higher affinity with the switch. Thus, the switch and the trigger RNA pairs, deform the neck-ring structure to free the RBS and circularized the mRNA regardless of the gene of interest. The exposed RBS enables translation initiation. Thus only the full-length mRNAs with trigger sequence at its 3’end are selectively used as the templates for translation [9].
Thus, we apply the new Quality control system to guarantee full-length protein expression in our project (Cas12b and LysPBC5).
Figure 5. ProQC system and its functional mechanism
[1] Park, C., Kong, M., Lee, J.-H., Ryu, S., & Park, S. (2018). Detection of bacillus cereus using bioluminescence assay withcell wall-binding domain conjugated magnetic nanoparticles. BioChip Journal, 12(4), 287–293.https://doi.org/10.1007/s13206-018-2408-8
[2] Lee, K. O., Kong, M., Kim, I., Bai, J., Cha, S., Kim, B., Ryu, K.-S., Ryu, S., & Suh, J.-Y. (2019). Structural basis for cell-wall recognition by bacteriophage PBC5 endolysin. Structure, 27(9).https://doi.org/10.1016/j.str.2019.07.001
[3] Notomi, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), 63e63. https://doi.org/10.1093/nar/28.12.e63
[4] Goto, K., Omura, T., Hara, Y., & Sadaie, Y. (2000). Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. The Journal of General and Applied Microbiology, 46(1), 1–8. https://doi.org/10.2323/jgam.46.1
[5] Heinrichs, J. H., Beecher, D. J., MacMillan, J. D., & Zilinskas, B. A. (1993). Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus. Journal of Bacteriology, 175(21), 6760–6766. https://doi.org/10.1128/jb.175.21.6760-6766.1993
[6] Calabretta, M. M., Álvarez-Diduk, R., Michelini, E., Roda, A., & Merkoçi, A. (2020). Nano-lantern on paper for smartphone-based ATP detection. Biosensors and Bioelectronics, 150, 111902. https://doi.org/10.1016/j.bios.2019.111902
[7] ATP Bioluminescence Assay Kit CLS II Reagent set for the quantitative detection of ATP by luciferase driven bioluminescence 1600 MTP-assays. (n.d.). Retrieved October 9, 2022, from https://www.sigmaaldrich.cn/deepweb/assets/sigmaaldrich/product/documents/118/678/11699695001bul.pdf
[8] Ruegg, T. L., Pereira, J. H., Chen, J. C., DeGiovanni, A., Novichkov, P., Mutalik, V. K., Tomaleri, G. P., Singer, S. W., Hillson, N. J., Simmons, B. A., Adams, P. D., & Thelen, M. P. (2018). Jungle Express is a versatile repressor system for tight transcriptional control. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-05857-3
[9] Yang, J., Han, Y.H., Im, J. et al. Synthetic protein quality control to enhance full-length translation in bacteria. Nat Chem Biol 17, 421–427 (2021). https://doi.org/10.1038/s41589-021-00736-3
