Updated on 2021-11-18: You might found this file, which we prepared for the Judging Session, helpful to understand our goals, approaches and what have been achieved.
Updated on 2021-11-10: we were nominated for the Best Measurement award.
# Overview
We have developed a generalizable method for measuring bacterial autolysis. During recombinant protein production, overexpression of exogenously sometimes toxic proteins in E. coli imposes a metabolic burden on the host cells, which further triggers the bacterial stress response, leading to severe cellular autolysis, reduced biomass growth, and decreased recombinant protein in production. Previously, no feasible method to assess the autolysis rate of E. coli in culture. Here, we use the leaked plasmid as the indicator for lysed bacteria, and optimized protocols to the extra plasmid from bacterial culture media, comparing with those that could be extracted from bacteria pellets (intact cells). Next, we examine the autolysis during IPTG induction for BL21 (DE3) bacteria, with or without gp5.7 expression. Further, we asked people in a nearby lab to try our method and to give us their feedback for further improvement
# Optimization
Initially, we tried to make a mixture of EDTA, NaAc, KAc, and acetic acid in a certain ratio and add a certain concentration of purified plasmids to it to simulate the environment of supernatant after bacteriophage lysis during plasmid extraction. And then we used silica gel membranes to absorb plasmid. However, we could not get any plasmid because the system was too complicated. Considering that the silica gel membrane would selectively adsorb nucleic acids at high salt (e.g. 4M guanidine hydrochloride) and low pH (e.g. pH 5.0), and would release nucleic acids at low salt and high pH (e.g. pH 8.0), we added Buffer A (20 M guanidine hydrochloride, 0.3 M MES-NaOH, pH 5.0) directly to convert the supernatant after centrifugation to high salt and low pH state, so that the plasmids in the supernatant could be adsorbed by the silica gel membrane.
To further improve the adsorption efficiency, we tried to activate the silica membrane in advance using Buffer PB (5 M guanidine hydrochloride, 30% isopropanol) before using the silica membrane to adsorb the plasmid. In addition, we simulated the supernatant conditions after centrifugation of the bacterial broth with the configured 2×YT medium spiked with Amp antibiotics and compared the results with TE simulations and ddH2O simulations, and found that the supernatant purified by TE simulations and ddH2O simulations yielded approximately twice as many plasmids as 2×YT medium, and we speculate that the adsorption of proteins to plasmids contributed to this result.
Figure 1. Purified plasmids (9 μg each) were diluted in different media, then purified using silica membranes and eluted in
30 μL Tris-EDTA buffer.
We then used Phenol chloroform : isoamyl : alcohol (25:24:1) solution to remove the protein interference. In addition, to measure the efficiency of silica gel membrane and ethanol precipitation to obtain pure plasmids each, we compared the two methods using silica gel membrane and ethanol precipitation and found that the ethanol precipitation method was unstable, so we finally chose to use phenol chloroform isoamyl alcohol precipitation followed by silica gel membrane to purify the plasmids.
Figure 2. Phenol chloroform : isoamyl : alcohol (25:24:1) solution was used to remove proteins that may absorb plasmids
In order to measure the ability of silica membranes to adsorb low concentrations of plasmids, we tested the ability of silica membranes to adsorb plasmids using a certain concentration of purified plasmids.
Figure 3. The minimal amount of plasmids DNA (diluted in an equal volume of bacterial culture media) could be purified using
silica membranes
At this point, we have been able to purify the plasmids dispersed in the supernatant after centrifugation of the bacterial broth. In order to facilitate the use of our protocol by other teams, we present a well-described protocol.
# Protocol
Take 1 mL of the bacterial solution to be measured, add it to centrifuge tube ①, draw 2/3 (i.e. 666 μL) of the bacterial solution, add it to centrifuge tube ②, and centrifuge tube ① and centrifuge tube ② simultaneously at 10,000 rpm (11,500 × g) for 1 min.
- Determination of plasmid concentration in the autolyzed portion of the bacterial solution.
1-1. Carefully transfer the supernatant of centrifuge tube ① to another centrifuge tube ③ with a pipette, add an equal volume of phenol : chloroform : isoamyl alcohol = 25:24:1 mixture, mix by reversing up and down 2-3 times, centrifuge at 12,000 rpm (13,000 × g) for 2 min, carefully aspirate 500 mL of supernatant to another centrifuge tube ④ in another centrifuge tube with 125 mL of Buffer A .
1-2. Place the adsorption column in a 2 ml collection tube, add 600 μL of Buffer PB to the column, and centrifuge at 12,000 rpm (13,000 × g) for 1 min, pour off the waste solution from the collection tube and put the column back into the collection tube.
1-3. Transfer the liquid from centrifuge tube ④ to the adsorption column and centrifuge at 12,000 rpm (13,000 × g) for 1 min. pour off the waste liquid from the collection tube and put the column back into the collection tube.
1-4. Add 600 μl of Buffer PW2 (10 mM Tris-HCl, pH 7.5, 80% ethanol)(check that it has been diluted with anhydrous ethanol) to the column and centrifuge at 12,000 rpm (13,000 × g) for 30 - 60 sec. Discard the waste solution and place the column back into the collection tube.
1-5. Repeat steps 1-4.
1-6. Place the column back into the collection tube. centrifuge at 12,000 rpm (13,000 × g) for 1 min to dry the column.
1-7. Place the column in a new sterilized 1.5 ml centrifuge tube. Add 30 μL of Elution Buffer (10 mM Tris, 1 mM EDTA, pH 8.0 ) to the center of the membrane of the column and let stand at 55°C for 2 min, then centrifuge at 12,000 rpm (13,000 × g) for 1 min to elute the DNA.
1-8. Discard the adsorption column to obtain the autolyzed portion of the plasmid.
- Determination of plasmid concentration in the non-autolyzed portion of the bacterial solution.
2-1. Carefully aspirate the supernatant from centrifuge tube ①, add 250 μL Buffer P1 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 μg/ml RNase A (From TIANGEN: RT405-12 100mg/ml) )(please check if Buffer P1 has been added to RNase A first), and mix well with a pipette or vortex shaking.
2-2. Add 250μL Buffer P2 (200 mM NaOH, 1% SDS) to centrifuge tube ① and mix gently by inverting up and down 8-10 times to make the bacteria fully lysed.
2-3. Add 350 μL of Buffer P3 (4.2 M guanidine hydrochloride, 0.9 M potassium acetate pH 4.8 (adjust with acetate)) to centrifuge tube ① and immediately neutralize Buffer P2 by gently turning it up and down 8-10 times. a white flocculent precipitate should appear. centrifuge at 12,000 rpm (13,000 × g) for 10 min.
(Note: Buffer P3 should be inverted and mixed immediately after addition to prevent localized precipitation from affecting the neutralization effect. (If there is still a small white precipitate in the supernatant, take the supernatant after centrifugation again.
2-4. Place the adsorption column in a 2 ml collection tube, add 600 μL Buffer PB [2] to the column, centrifuge at 12,000 rpm (13,000 × g) for 1 min, pour off the waste solution in the collection tube, and put the column back into the collection tube.
2-5. Carefully transfer the supernatant from centrifuge tube ① to the adsorption column by pipetting, taking care not to aspirate the precipitate, and centrifuge at 12,000 rpm (13,000 × g) for 30 - 60 sec. Pour off the waste solution from the collection tube and put the column back into the collection tube.
2-6. Add 600 μl of Buffer PW2 (check that it has been diluted with anhydrous ethanol) to the adsorption column. centrifuge at 12,000 rpm (13,000 × g) for 30 - 60 sec. discard the waste solution and return the column to the collection tube.
2-7. Repeat steps 2-6.
2-8. Place the column back into the collection tube. centrifuge at 12,000 rpm (13,000 × g) for 1 min to dry the column.
2-9. Place the column in a new sterilized 1.5 ml centrifuge tube. Add 30 μL of Elution Buffer to the center of the membrane of the column and let it stand for 2 min at 55°C. Centrifuge at 12,000 rpm (13,000 × g) for 1 min to elute the DNA.
2-10. Discard the adsorption column to obtain the non-autolyzed portion of the plasmid.
- Calculation of autolysis rate
The concentration of autolyzed fraction of plasmid was measured by Nanodrop or grayscale analysis of gels as C1 (μg/μL) and the concentration of non-autolyzed fraction of plasmid as C2 (μg/μL), and the autolysis rate was calculated as
# Result
We tested our protocol in DH5α bacteria transformed with a low-copy plasmid (pET52b) and a high-copy plasmid (pmScarlet-C1), respectively (Figure 4) and we got good results for both low-copy and high-copy plasmids. What's more, we found the logarithmic phase of bacteriophage growth has a positive correlation with the autolysis rate
Figure 4. Determination of OD600 and autolysis percentage of DH5α strains transformed with pET52b and pmScarlet-C1 plasmids
under normal culture conditions
Since our experiment requires induced expression of the target protein in BL21 (DE3) bacteria by IPTG, however, the induction process is highly susceptible to cellular autolysis due to the dual stress of reduced native expression and overexpression of the recombinant protein（Figure5）. We tried to find a balance between the two to determine the most suitable time to harvest the protein.
Figure 5. IPTG induction will lead to slower growth rate, lower stationary phase and higher autolysis rate
It is clear from the results that the viability of the bacteriophage decreases after IPTG induction. We recommend harvesting the bacteria at about 12 hours of incubation, which will keep the product maximized and save resources
# User Experience
We knew it was important that our protocol could be used by other people or other teams, so we invited the team in the lab next door to use our protocol and got relatively good results the same with ours.
Figure 6. The memeber from the next lab was using our protocol and prasied it.