Contribution
In the degradation part of Microcystin-LR, we characterized BBA_K1378001, added new data for the existing parts.
In order to obtain higher expression and improve the degradation effect, we replaced the original promoter J23110 with a much stronger constitutive promoter J23119.
Figure 1. Schematic of pKMV-mlrA-C1. We replaced the original promoter J23110 with a stronger constitutive promoter J23119, and transformed the plasmid into E. coli JM109.
Method
◇1.Plasmids including pKMV-mlrA-C1 (including BBa_K1378001) and pKMV-mlrA-THN1 (including BBa_K3699001) synthesized by BGI Tec were transformed into E. coli JM109 to construct two strains, E. coli JM109 (pKMV-mlrA-C1) and E. coli JM109 (pKMV-mlrA-THN1).
Figure 2. Plasmid Profile.pKMV-mlrA-C1 (including BBa_K1378001) and pKMV-mlrA-THN1 (including BBa_K3699001).
◇2.Culture these two strains for 16 h, and then transfer 150 mL of the bacterial culture for centrifugation, collect the cell pellets, and resuspended the cells by adding 20 mL of PB buffer.
◇3.Take 10 mL for ultrasonic fragmentation, take the supernatant as the crude extract of cells, and use it for toxin degradation experiment; take another 10 mL bacterial resuspend solution, and directly use it for toxin degradation experiment.
◇4.Take 1.5 mL centrifuge tubes, add the toxin to the final concentration of about 20 ng/mL, carry out the toxin degradation experiment at 37℃. Samples were taken every 6 h for measuring the concentration.
Figure 3. Enzyme in centrifuge tube.
Testing
We used ELISA kits to detect toxin concentrations in samples. (Kits ordered from Zhenke Biotec)
Assay procedure：
◇1.Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
◇2.Add standard: Set Standard wells, testing sample wells. Add standard 50 μL to standard well.
◇3.Add Sample: Add testing sample 10 μL then add Sample Diluent 40 μL to testing sample well; Blank well doesn’t add anything.
◇4.Add 100 μL of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37℃.
◇5.Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400 μL) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
◇6.Add chromogen solution A 50 μL and chromogen solution B 50 μL to each well. Gently mix and incubate for 15 minutes at 37℃。Protect from light.
◇7.Add 50 μL Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
◇8.Read the Optical Density（OD）, at 450 nm using a microtiter plate reader within 15 minutes.
Figure 3. Enzyme in centrifuge tube.
Result
We measured the concentration of toxin in the reaction system every 6 hours (Fig. 5 and Fig. 6).
Figure 5. Degradation result of pKMV-mlrA-C1 cell culture.
Figure 6. Degradation result of pKMV-mlrA-C1 cell extract.
·As can be seen from the graph, within 24 h, the toxins were degraded by 32.13% (cell culture) and 35.32% (cell extract). The latter is more efficient in degradation.
·Comparing the degradation rates of 18 h and 24 h, we can find that the degradation efficiency basically reaches the maximum value when the reaction is carried out for 18 h.
Due to the different experimental conditions, we cannot compare this result with the previous data.
However, this result successfully indicated that the MlrA enzyme we designed was able to degrade MC-LR and reduce its toxicity.
Reference
[1]Bourne D G . Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.[J]. Applied & Environmental Microbiology, 1996, 62.
[2]Ren YY. Synthesis, expression and purification of microcystin-degrading enzyme Mlr A gene optimized by Lactococcus lactis preference codon. [D]. Jilin University, 2015.