In our experiment, we extracted Cas12b and LysPBC5. The following are the general steps of our protein expression, extraction, and purification process. Single colonies of the BL21 strain on plates were inoculated into an LB medium containing kanamycin and cultured. To induce protein expression, IPTG was added when the OD600 value of the culture reached approximately 0.6. Bacterial precipitates were collected by centrifugation and the bacteria were lysed with lysate, lysozyme and ultrasonic cell lyser, respectively. The lysed mixture was separated by centrifugation and the supernatant was isolated. The supernatant of bacterial lysis treated with His-tag purification resin and the non-denaturing lysate was loaded into the empty column tube of the affinity chromatography column, and the flow-through solution was collected and saved for subsequent analysis. Collect the liquid after washing with the non-denaturing washing buffer for subsequent analytical assays. Elute the target protein with a non-denaturing elution buffer and collect the purified protein sample labeled by His tag. To test whether the target proteins (Cas12b and LysPBC5) were successfully expressed and the amount of expression, these proteins purified by the above operation were analyzed by SDS-PAGE and western blotting.
Western blotting has taken up a major part of all experiments included in the project. Through countless times of attempting and revising, we gradually accumulate the knowledge of a flawless WB result. To begin with, the preparation of protein gel sets the first barrier for us. Unsolidified gel, unproportionate stacking-to-resolving ratio, titled lane after pulling out the comb, and impurity force us to start over again dozens of times. Next, during sample loading, due to dramatic differences between different protein samples, overexposure and diffusion are obtained for dense samples while only lightly darkened areas are observed for samples with lower concentrations.
Figure 1.SDS-PAGE result of LysPBC5 and Cas12b without ProQC system.
We then dilute and determine the optimal final concentrations for later use. After electrophoresis, Coomassie Brilliant Blue (CBB) was added to dye the gel. During early trying, the gel is stained over time that following decolorization couldn’t wash off the blue stain left on the gel. Then, when it comes to membrane transfer, the major difficulty encountered is the heat emitted during the transfer. Although cooling packs are placed both in and out of the transfer box, the temperature of the liquid ended warm after transferring, and thus slight residue of the marker is observed on the gel. Moreover, the Ponceau S dye is also applied to determine the protein residue on the gel, though it seems like it lacks the sensitivity to tell the potential minimal residue after decolorization. After, the membrane is blocked and incubated with primary and secondary antibodies.
Figure 2. SDS-PAGE, transmembrane, and the Western blotting result of LysPBC5 and Cas12b without ProQC system.
Lastly, electrochemiluminescence imaging is conducted to produce final Western Blotting images at different exposure times. Despite several setbacks in WB, its fundamental mechanism, that the imaging marks only refer to the His-tag protein, has already proven that we obtained the correct protein, Lys and Cas, instead of other proteins of the same size.
During imaging, a few issues lie in the way. The most notable issue shown in the figure is the hollow strains in the middle of the lane of protein LysPBC5. This is a consequence of the high protein concentration of purified LysPBC5. High protein concentration leads to high primary antibody levels, and thus causes a high concentration of secondary antibodies with Horseradish Peroxidase to stack in the middle of the band, which leads to fast depletion of fluorescence substrates in the center.
Another issue is that several shorter bands are present in each lane of protein. Considering that only proteins with His-tag are able to bind with the primary antibody and both protein has His-tag on their N-terminal, these shorter bands are probably formed by incomplete polypeptides of LysPBC5 or Cas12B. The presence of incomplete, non-functional protein would significantly decrease the purity of our product and the efficiency of the expression system. Considering that price is a major consideration of potential users of our product, we applied the protein quality control system (ProQC system) to solve this problem.
First, 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 3. 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
We hypothesized that HBLA cannot be amplificated from B.subtilis because B.subtilis didn’t have the HBLA gene. The result from PCR are shown below (Fig. 4). Bands from left to right are B.cereus-16s, B.thuringiensis -16s, B.subtilis-16s, DNA marker, B. cereus-HBLA, B.thuringiensis -HBLA, and B.subtilis-HBLA. The three bands on the left of the marker showed that 16s can be amplificated in B. cereus, B.thuringiensis, and B.subtilis. However, the three bands on the right side demonstrated that HBLA can only be amplificated in B. cereus and B.thuringiensis, no B.subtilis, which proved the original hypothesis. In this case, we can exclude the chances to detect B.subtilis by using PCR for the HBLA gene.
Figure 4.The result of Regular PCR for 16S rDNA and HBLA gene.（Bands from left to right are 1:B. cereus-16s, 2:B.thuringiensis-16s, 3:B.subtilis-16s, DNA marker, 4:B. cereus-HBLA, 5:B.thuringiensis-HBLA, 6:B.subtilis-HBLA.）
We do colony PCR in order to find whether PCR can be used directly to detect the 16s gene without extraction of DNA. The result of colony PCR is shown below (Fig. 5). Except for the markers at both ends, the bands from left to right are B. cereus-PCR, B. cereus-Colony PCR, B.subtilis-PCR, B.subtilis-Colony PCR, B.thuringiensis-PCR, B.thuringiensis-Colony PCR, and positive control group(AMPR). It verifies that colony PCR can amplify the 16s gene from bacteria but cannot distinguish the three types of bacteria.
Figure 5.The result of colony PCR for 16S rDNA gene(The bands from left to right are 1:B. cereus-PCR, 2:B. cereus-Colony PCR, 3:B.subtilis-PCR, 4:B.subtilis-Colony PCR, 5:B.thuringiensis-PCR, 6:B.thuringiensis-Colony PCR, 7:positive control group AMPR)
Except for the marker on the right side, the bands from left to right are water, B.thuringiensis, B.subtilis, and B.cereus, and water individually (Fig. 6). The results of LAMP demonstrate that LAMP can amplify 16s genes from B.cereus and B.thuringiensis, but not B.subtilis. The band of B.subtilis only shows primer dimer which is the same as the bands of water with primers. In this case, the chances of detecting B.subtilis are excluded from detecting B. cereus by using LAMP. Hence, using LAMP which needs more restrictive conditions than PCR can effectively detect the target gene.
Figure 6. The result of LAMP for 16S rDNA gene(The bands from left to right are 1: water, 2:B.thuringiensis, 3:B.subtilis, 4:B.cereus)
We used the purified Cas12b protein, sgRNA transcribed in vitro, and amplified 16S rDNA gene to perform the HOLMES assay. The relative fluorescent intensity at 556nm with an excitation wavelength of 535nm of each template was measured. However, the difference in relative fluorescent intensity is very small, and the group without a template becomes the group with the highest fluorescent intensity.
Graph 2.relative fluorescent intensity of HOLMES reaction system with different templates.
The failure in HOLMES might have several causes. Due to the limitation of apparatus and reagants, we can’t do an RNA electrophoresis to verify the length and purity of our sgRNA. Also, the Cas12b protein’s concentration and purity might not be high enough. (the Cas12b protein used in this experiment is without the ProQC system)
In order to repeat and obtain the standard curve, several attempts are made. Although we successfully determine its luminescence peak at the wavelength of 562nm, precision wasn’t able to achieve at the same time. we created 9 levels of ATP concentration ranging from 10-4 to 10-12M. Yet multiple testing has yielded the same unsuccessful result that later was proven when higher levels of concentrations were used to examine the light intensity that the equipment essentially wasn’t capable of reading luminescence under 10-6.
Figure 7. relative fluorescent level of different ATP concentrations under various sensitivities.
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. 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 2).
Table 1. plate layout of kinetics assay
The result (Fig.8) 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 8. crystal violet titration curve.
We used protein expressed by BBa_K4204021 to characterize the effectiveness of the ProQC system. The western blotting result of LysPBC5 with the ProQC system shows significant improvement in the integrity of the protein. LysPBC5 is diluted four 4 times before being loaded. Rows 7 to 10, which correspond to eluted LysPBC5 with the ProQC system, are clearly visible in the membrane picture obtained using ECL imaging (Fig. 9), demonstrating that the LysPBC5 protein with the ProQC system is being produced appropriately and that few proteins are lost during the washing process (lanes 1-6 that contains wash through are empty or nearly empty). Western blot analysis reveals that only the correct and precise target bands are being expressed, with the mass of protein being around 40kDa (the calculated mass of LysPBC5 is 38 kDa) and no aberrant or interfering stripes in the result. Overall, the WB result with the ProQC system shows that it is useful in improving the full-length expression rate of LysPBC5.
Figure 9.The WB result of LysPBC5 protein with protein quality control system
(Lanes 1-6, 7-10, and 11-13 contain wash-through, eluted protein, and cell lysate flow-through, respectively)
