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The survivability of P. putida is better than E. coli in a high concentration of Cd2+ proved by a cell viability assay

Aim

The aim of this experiment is to assess and compare the survivability of E. coli and P. putida with or without expressing metallothionein (MT) under different concentrations of cadmium. The strains we used include wild-type E. coli BL21(EC), E. coli BL21 expressing metallothionein (ECMT), wild-type P. putida KT2440 (PP), and P. putida KT2440 expressing MT (PPMT). This experiment proved that P. putida KT2440 survived better than E. coli BL21 in a high concentration of cadmium (> 30 µM), which is chosen as our chassis. Meanwhile, the expression of MT increases the survivability of both bacteria.

Experimental design

Bacteria preparation:

  • Three colonies of each sample were picked and inoculated in 5 mL of LB without antibiotics for 16 hours at 37°C.
  • The overnight culture is subcultured at a 1:50 ratio into 50 mL LB at 37°C until OD600 reaches ~1.0.
  • The expression of MT in E. coli was induced by IPTG during subculturing for 4 hours.
  • The subcultures are diluted 100 times before cadmium is added.
  • OD600 was measured after incubating for 9 hours.

Cadmium dilution:

  • A serial dilution was done on the cadmium stock solution using LB as the diluent.
  • 0 µM to 480 µM was used as the range of final concentration of cadmium solution, as referenced from a study (Kim et al., 2020).

Plate reading:

  • OD600 was measured using a Flexstation 3 Multi-Mode Microplate Reader.
  • Kinetic mode was used to obtain data points at a 20-minute interval for 19 hours and 20 minutes.

Results

The average OD600 was calculated from triplicates, and a heat map was plotted with the concentration of cadmium solution against the OD600 of different types of bacteria.

Figure 1.

Figure 1. Heat map of cadmium concentration against OD600 of different types of bacteria.

Figure 2.

Figure 2. Bar chart of OD600 of EC and PP at 30 µM Cd2+. Student’s t-test ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3.

Figure 3.

Figure 3. Bar chart of OD600 of different types of bacteria at 240 µM Cd2+. Student’s t-test ∗P < 0.05, n = 3.

From the gradient in colour between EC and ECMT (30 µM to 240 µM), it can be observed that ECMT has a higher OD600 than EC, which justifies that MT does increase the survival of bacteria in cadmium solutions (Figure 1, 3). What’s more, at 30 µM Cd2+, P. putida has a significantly larger OD600 than E. coli (Figure 2).

Hence, it is concluded that the metallothionein protein increases the survivability of the bacteria, and P. putida is a more advantageous candidate as a chassis to remove cadmium. However, we still need to prove the successful expression of MT in both E. coli and P. putida (see next experiment).

Future Plan

Different parameters, such as different pH levels, citrate concentrations, and IPTG induction time, can be applied in the experiment design and further characterize the survivability of each bacterial strain.

The successful expression of MT in E. coli and P. putida verified by Western Blot

Aim

The aim of this experiment is to confirm the expression of metallothionein (MT) in E. coli BL21 and P. putida KT2440.

Experimental design

MT is constructed with a His-tag, so anti-His-tag mouse monoclonal antibody (sc-8036, Santa Cruz) is used as the primary antibody, and goat anti-mouse IgG-Horseradish Peroxidase (HRP) (sc-2005, Santa Cruz) is used as the secondary antibody.

Protein Sample Preparation:

  • Both E. coli and P. putida were inoculated overnight for approximately 14-16 hours (E. coli was grown at 37°C while P. putida at 30°C).
  • The expression of MT in E. coli was induced with IPTG for 4 hours.
  • Bacteria were centrifuged, and the cell pellet was lysed with RIPA lysis buffer.
  • Supernatant was collected, and cell pellets were resuspended in the remaining supernatant in tubes.
  • 6X SDS Loading buffer was added to both supernatant and cell pellet resuspension, and the samples were boiled.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

  • A polyacrylamide gel was set up and run (4% stacking and 12% separating gels).

Western Blot:

  • Anti-His-tag mouse monoclonal primary antibody.
  • Goat anti-mouse IgG-HRP secondary antibody.

Results

Figure 4.

Figure 4. Western blot result of E. coli with metallothionein (ECMT) induced by different concentrations of IPTG. Ponceau S is used as the loading control.

Figure 5.

Figure 5. Western blot result of P. putida with metallothionein (PPMT). An expected band of 23 kDa was shown.

From Figure 4, it was observed that the production of metallothioneins increased with increasing amounts of IPTG, as indicated by the stronger detected signals. It also proved the successful expression of MT in E. coli . In addition, MT was also detected in P. putida after electroporation of the plasmid (Figure 5). We successfully expressed the fusion proteins with our circuits in both chassis – E. coli and P. putida. However, the MT was shown in the cell precipitation. Based on our design, Lpp-OmpA-MT should be expressed on the cell membrane. One explanation is that MT is attached to some membrane debris and is not soluble. Next, we need to prove the membrane localization of MT (see next experiment).

Future Plan

We can quantify the Western blot bands with standard curves to get the protein expression level.

Lpp-OmpA-MT is expressed on the bacterial cell membrane verified by TEV protease cleavage

Aim

The aim of this experiment is to verify the expression of the fusion proteins on the bacterial surface and ensure they are in the correct orientation. This experiment is done with TEV protease and performing Western Blot to detect the cleaved peptides.

Experimental design

Bacteria preparation:

  • 1 colony of E. coli BL21 with metallothionein (ECMT) was picked and inoculated in 5 mL of LB without antibiotics at 37°C overnight.
  • The next day, the OD600 absorbance of the overnight culture was checked with an absorbance reader.
  • Based on the absorbance, the overnight culture was diluted to an OD600 of ~0.5-0.6.
  • The diluted overnight culture was then subcultured with 5 mL of fresh LB with antibiotics and IPTG to induce protein expression.
  • The subculture was incubated for 1 to 2 hours at 37°C for more growth.
  • The subculture was then centrifuged to harvest the cell pellet.
  • The cell pellet was washed twice with 50 mM Tris-HCl, pH 7.5.
  • The cell pellet was then resuspended in the same buffer, and TEV protease was added at a ratio of 1 unit per OD unit.
  • The cells were incubated overnight at 4°C with slow rotation on a belly dancer.

Sample preparation:

  • The cells were centrifuged, and the supernatant and pellet were collected.
  • SDS-PAGE and Western blot were performed to see the cleaved metallothionein proteins.

Expected Results

Cleaved MT of ~7 KDa will be detected in the supernatant.

Results

Figure 6.

Figure 6. Western blot result of ECMT TEV cleavage test

Figure 7.

Figure 7. Western blot result of PPMT TEV cleavage test

After incubation of bacterial culture with TEV protease overnight at 4°C, supernatant and cell pellets were separated to detect the cleaved MT in the supernatant as expected. However, we could not detect the cleaved MT with a size of 7 kDa in the supernatant but found the whole MT of 23 kDa, one band at around 17 kDa, and one band at the bottom (Figure 6). To note, the separating gel of SDS-PAGE was 12%, where the range of target proteins’ molecular weights is from 10 to 250 kDa. This suggests that the bands at the bottom can be the cleaved MT but not separated well by SDS-PAGE. Interestingly, the results show the presence of TEV protease at 37 kDa when the literature expected its size to be 27 kDa. Considering the reproducibility of the bands and the different composition of the SDS-PAGE gel, we concluded that the 37 kDa band is TEV protease. The results show TEV protease cleavage works, but the cleaved MTs and the enzymes are found in the sample precipitation, which suggests some interactions happened to pull down TEV protease and cleaved MTs. We suspect that some lipopolysaccharide (LPS) or lipoproteins may interact with them.

Future Plan

Based on the results to date, we observed that cleaved MTs are found in the precipitation after TEV protease cleavage. We suspect that the cleaved MTs interact with some LPS or proteins on the bacteria surface, because TEV protease is also in the precipitation. More repeats need to be conducted. Furthermore, the cell membrane can be separated through the membrane fractionation to strengthen our conclusion of the localization of fusion proteins. In addition, Tricine-SDS-PAGE will be used to separate the cleaved peptides or the percentage of the crosslinker will be increased.

The functionality of MT is proved by cadmium binding assay - ICP-OES

Aim

The aim of this experiment is to confirm the function of the metallothionein expressed in E. coli BL21, to the metal, cadmium, by using Inductively-Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Experimental design

Bacteria preparation:

  • E. coli BL21 expressing metallothionein (ECMT) and wild-type E. coli BL21 (ECWT) were picked and inoculated in 5 mL of LB with the appropriate antibiotic selection for 16 hours at 37°C and 250 RPM shaking incubator.
  • Due to past empirical results showing reduced growth (overnight OD ~0.8-0.9), each inoculum consisted of 2-3 colonies to increase the growth rate.

Sample preparation:

  • OD600 of the overnight culture was measured. Readings reached ~1.0-1.5.
  • The overnight culture was subcultured into three groups: ECMT with IPTG (20mL), ECMT without IPTG (20mL), and ECWT (20mL).
    • The overnight culture was subcultured at a 1:20 ratio into 20 mL of LB at 37°C until OD600 reached ~0.5-0.7.
    • Each group had 3 subcultures for triplicate. A total of 9 cultures were prepared.
    • 2 mM of IPTG was added for induction (ECMT with IPTG) and incubated for 4-5 hours at 37°C in a 250 RPM Shaking Incubator.
  • After incubation, each subculture was separated into 4 Falcon tubes with 5 mL each. (Total of 9 tubes).
  • The subculture was washed twice with 1x PBS. Each wash was done at 3000 x g for 10 - 15 minutes.
  • The cell pellets were resuspended and stored in 1 mL 1x PBS solution overnight due to working hour restrictions.
  • The next day, cells were centrifuged at 3000 x g for 15 minutes to re-pellet. Supernatant was removed.
  • 1 mg/mL Cadmium Solution was first diluted in 2-5% Nitric Acid to produce a 240 µM Cd2+ solution. From there, dilutions of 0, 20, 40, 60, 80, 100, and 120 µM Cd2+ Solution.
  • Cell pellets were resuspended in 3-4 mL of 60 µM Cd2+ solution and incubated at 37°C in a shaking incubator at 230 RPM.
  • Half the samples (3 tubes of each group) were immediately centrifuged at 15000 x g for 15 minutes to sample for t0 readings.
  • Samples were also then filtered through a 0.22 micron PES Filter.

tO/N samples were prepared in parallel by allowing the incubation in 60 µM Cd2+ solution to occur overnight.

Expected Results

There will be a decrease in the concentration of Cd2+ solution in the samples after incubation.

Results

Figure 8.

Figure 8. ICP-OES result of E. coli with different treatments in a 60 µM Cd2+ solution. n = 2-3.

After the concentration of Cd2+ was detected by ICP-OES, we normalized the data with OD600 to see the difference in the concentrations of the three bacterial samples. E. coli with MT shows a higher decrease in cadmium concentration, which proposes its potential to bind Cd2+ and extract it from the solutions.

Future Plan

We observed a dramatic decrease of cadmium concentrations with ECMT. However, the variation among samples caused the result not to be significant (Figure 8). Therefore, we will repeat the experiment to obtain more data to make it consistent and significant. The experiment will also be conducted in P. putida with the identical sample preparation and experiment design to verify the functionality of the metallothioneins in P. putida' s main circuit. A higher decrease of cadmium concentrations is expected compared to E. coli, because P. putida has a better survivability in high Cd2+ solutions (Figure 1).

Biosensor and Kill Switch Results

Experimental data for Biosensor and Kill Switch can be found below.

References

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