Our engineering success contains two parts, which characterize LysPBC5 and the crystal violet induction system. Both of them encountered a problem with the initial design and two rounds of the engineering cycle were done to achieve the final success.
Depended by both ways of detection, LysPBC5 is the most important protein in our project. We cloned part BBa_K4204000 to expression vector pET28a(+) to express it in E.coli. After purifying the protein, we performed two experiments complimentary to each other to examine the lysing ability of LysPBC5.
The crystal violet induction system is a cheap and sensitive expression system that could greatly lower the price of our protein product by lowering the price of the inducer, thus we designed BBa_ K4204016 to prove that it’s practically applicable and titrated its induction curve.
In the first experiment, we use OD600 to reflect the concentration of living bacteria. 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.
Graph 1. The Change in B. cereus OD600 with Respect to Endolysin Concentration
Through the first experiment, we found that it’s hard to o 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.
Figure 1. 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
To characterize the crystal violet induction system, we designed and build BBa_ K4204016, a crystal violet-induced sfGFP expression system, in which sfGFP is used as a reporter, and the relative fluorescent level per cell under a certain concentration of crystal violet could represent the expression level of the induction system under that inducer concentration.
However, after we constructed our initial design on pET28a(+), it failed to show any fluorescent even when the concentration of crystal violet reaches the maximum induction concentration recorded in the literature [1]. Thus we consulted our secondary PI Wanji Li and he finds out that the ribosome binding site is 17 bp far from the start codon, causing the site with the highest translation activity to shift to an uncommon start codon upstream and cause the open reading frame to shift. Thus the expressed protein is not functional and won’t have green fluorescent.
After redoing a golden gate assembly to fix the problem, we did a kinetics assay to characterize the induction curve of the crystal violet induction system. 8 different concentrations of crystal violet are tested with 6 repeats each. The detailed plate setup is below (Table 1).
Table 1. plate layout of kinetics assay
The result (shown in figure 2.) shows that the minimal concentration of crystal violet required to get observable protein expression is 0.02µM, and the expression level reaches a maximum when crystal violet concentration reaches 0.2µM. For higher concentrations of crystal violet, the expression level per strain decreases by about 25% while the exponential phase of strain growth delays, probably due to the burden of expression and the slight cytotoxicity of crystal violet. Overall, the optimal concentration of crystal violet for induction is 0.2µM.
Figure 2. crystal violet titration curve.
[1] 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
[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
