Team:Stuttgart/Results
Results of the Escherichia Coli experiments
In the experimets, we performend, we used different variants of the Dsup protein: the wildtype protein (WT),N-term ( a His-Tag on the N-terminus),C-Term (a His-Tag on the C-terminus).
The vector and the fragments of the different Dsup variants are shown in Fig. 1. All Variants were successfully cloned into the pBAD18 vector.
Transformation
The plasmids were transformed into cells of the effective transformation strain DH5α. Afterwards, they were isolated and transformed into the expression BW25113 strain.
Expression
The transformed BW25113 cells were used to express Dsup at different temperatures. In the first run, the WT and N-term were cultivated at 37°c for 340 minutes (Fig. 2). The OD600 values were measured in regular intervals to determine the growth curve and optimal point of induction with arabinose. After 100 minutes the expression of both variants was induced with 5 mL arabinose. The process was determined at the stationary phase.
In the second run (Fig. 3), the C-term variant was cultivated over the span of 505 minutes. One culture was kept at room temperature, while the other was cultivated at 37°C like in the first run. The expression of Dsup was induced by the addition of 5 ml arabinose solution after 140 minutes. The second culture at 37 °C shows a stronger growth. The culture, incubated at room temperature, grows slower, but the protein folding might be less affected by the temperature.
The second expression of N-term was performed at 30 °C and the growth is shown in Fig. 4. The expression of the Dsup was induced after 180 min.
Purification and desalination
The purification of the C-term cell lysate was performed on a 10 mL HisTrap FF column according to the chromatogram in Fig. 5. The column was loaded with 10 mL as shown by the conductivity plateau. Then the His-Tag was eluted with imidazole in a one-step elution, resulting in three peaks. The first one at 15,5 mL wasn’t recognized by the software but was collected separately from the second peak at 16.22 mL. The collection of the second peak was stopped as soon as the conductivity started to rise at around 16 mL. The last peak at 18.34 mL wasn’t collected.
The desalination of the protein was performed by using affinity chromatography, using a HisTrap FF column (Fig. 6). The solution of the second peak was collected until the conductivity started to rise at approximately 11 mL. The same procedure was used to desalt the solution of the first peak (purified C-term sample).
For the purification of the N-term cell lysate 30 mL were applied to the column. After loading the solution on the column, the samples were eluted again, but with an imidazole gradient elution. Fig. 7 shows, that small concentrations of imidazole already elute the His-Tag of the N-term Dsup. The first peak at 104.34 mL and the second peak at 116.17 mL were collected separately.
In Fig. 8 the desalination shows several plateaus during the desalination of the second peak of the N-term purification. The desalted solution from peak 2 was collected in one Falcon tube until the conductivity started to rise at approximately 11 mL. For the sample of the first peak, the same procedure was performed and the solution was collected in a separate falcon tube.
Bradford protein assay
To evaluate the samples from the different peaks of the purification step, the protein concentration was determined. The raw data and dilution factors of the samples measured with the Bradford protein assay are shown in Tab. 1. The coefficient of determination of the BSA calibration line in Fig. 9 is 0,9849.
By converting the given equation 1 in Fig. 1 into equation 2 the protein concentration of the samples can be determined, whereas y stands for the absorbance and x defines the protein mass.
y=0,0281∙x+0,0341 (equation 1)
x=(y - 0,0341)/0,0281 (equation 2)
The mean absorption of the C-terminus was 0,37 and 20 µL of undiluted sample was used. The protein mass is determined in equation 3 and subsequently the concentration is determined in equation 4.
x=(0,37-0,0341)/0,0281=11,95 µg (equation 3)
concentration_(C-terminus raw extract)=(11,95 µg)/(20 µL)=0,60 (µg)/(µL) (equation 4)
The highest protein concentration was found in the N-terminal His-Tagged cell lysate. The C-term peak 2 solution has the highest protein concentration of the purified and desalted samples with 0.54 mg/mL before and 0.45 mg/mL after sterile filtration.
Tab. 1: Bradford protein assay of the cell lysates and purified samples
SDS-Page
The SDS-PAGE Gels in Fig. 10 show all the samples from the expression of the Dsup protein of the different genotypes. The "pellets" are the cell pellets, which were generated at the end of the expression after the last centrifugation step. After four hours a slight band at approximately 45 kDa is visible.
In Fig. 11 and Fig. 12 the cell lysates and the desalted samples are shown. The additional samples of the expression in Fig. 11 can be compared to the lysed and purified samples. After 5 h of C-term expression, multiple bands can be seen across the size standard, whereas only two bands at approximately 38 kDa are seen in the pellet. Only very weak bands are visible in the sample of the cell lysate. The protein concentration of the C-term lysate was first determined for the sample preparation for the SDS-PAGE in Fig. 12. A lesser volume of the C-term lysate as needed was applied. But the bands for the maximum volume of undiluted 7.5 µL C-term cell lysate in Fig. 12 are even weaker than in Fig. 11. There are no visible bands for the C-term peak 2 samples. The same result is shown in Fig. 12 for the other C-term peak and sterile filtered sample. At the beginning of the N-term expression in RT, only one weak band at approximately 40 kDa is visible, whereas the N-term cell lysate sample shows several bands with one outstanding band at 43 kDa. The purified N-term samples of all peaks show a clear band at approximately 43 kDa and a second weaker band at around 38 kDa. Except for peak 1 of expression 1, where the bands are less present. In Fig. 8 also the N-term peak 2 of expression 2 shows a clear band at 43 kDa as the other desalted N-term samples.
The C-term sample from the beginning of the expression in Fig. 12 shows weak bands at approximately 35 and 37 kDa. As mentioned earlier the bands of the C-term cell lysate in Fig. 8 are nearly not visible. The C-term peak 1 sample in Fig. 12 leaked between the gel and the glass plates of the gel.
Experiment
In the first irradiation experiment the Dsup expression was induced in all genotypes except in the WT n (non-induced) as a negative control. The different E. coli genotypes were irradiated with different energy levels as seen in Fig. 13 and Tab. 2. There were less than 100 colonies on each plate, even in the uniradiated controls. A clear effect of the radiation is seen at radiation levels of 2500 µl/cm2. None of the genotypes survived a irradiation of 10000 µJ/cm2. The number of colonies varies between the plates, to make predictions of a trend, more replicates should have been performed.
Tab. 2: Raw data of the first irradiation experiment with different energy levels of all genotypes. As negative control cells without the induction of the WT Dsup expression were used: WT n: WT non-induced, WT: induced WT.
Therefore the experiment was performed with one genotype using a specific condition, with replicates (Fig. 14). The number of colonies varies between the plates. The radiation seems to have no effects on the induced C-term cells, whereas the number of colonies in the non-induced C-term genotype seems to decrease. As seen in the raw data of the second irradiation experiment, the total number of colonies is too low and the variation is high, so further experiments would be necessary to prove the protective effect of the Dsup.
Tab. 3: Raw data of the second irradiation experiment of the C-term genotype. As negative control cells without the induction of the C-terminal His-tagged Dsup expression were used: C-term n: non-induced C-term, C-term i: induced C-term.
Troubleshooting
E. coli irradiation with intracellular expressed Dsup:
• Dilution: many dilution steps are susceptible for mistakes, which leads to amounts of colonies, which are not countable.
• Contamination: We wanted to eliminate the possible effects of antibiotics on cell growth, therefore we didn’t use any antibiotics. But we couldn’t place the UV radiator under the clean bench so we had several contaminations at the first trial (data not shown). The set-up should be performed under clean and sterile conditions to avoid contaminations.
Discussion
The cell growth of the expressions worked for all genotypes. The band, which can be found at approximately 43 kDa, matches with the given size of the native Dsup protein of 42.8 kDa [1]. Unfortunately, there could not be detected proteins by the SDS-PAGEs of the purified and desalted C-term Dsup, even though the highest protein concentration was measured in the C-term peak 2 samples. One reason for the missing Dsup bands could be the high protein concentration, which was detected in an elution step. When the samples are boiled before loading on the SDS-PAGE the imidazole remnants could lead to a hydrolyzation of the proteins [2]. The protein content in the C-term cell lysate seemed to decrease. The protein concentration of the C-term lysate was determined to right before the preparation of the second SDS-Page. For the preparation of the first SDS-PAGE with desalted samples, it was assumed that the C-term cell lysate has a similar protein concentration as the N-term lysate, a smaller volume was used. In Fig. 10 the maximum volume of 7.5 µL of the C-term cell lysate was used but there are even fewer bands visible than in Fig. 9. It can be assumed that the C-term samples were exposed to room temperature for too long and proteases in the cell lysate degraded some of the proteins in this sample [3]. The total loss of protein bands in the C-term cell lysate rather indicates, that the sample preparation was incorrect, than no Dsup was expressed. The high protein concentration and the high specificity of the HisTrap column contribute to the successful expression and purification. However, it cannot be ensured assuredly that the C-term Dsup protein was expressed.
The extracellular irradiation experiment showed that a high variation of the numbers of colonies occur. Most likely because of the possible mistakes, that can happen during the dilution steps. Also, it seems critical to avoid leftover suspension in the pipet tip, because there also was a high variety of colonies from the same dilution. The experiment needs to be repeated for all the genotypes with more replicates to achieve a representative result. Nevertheless, the critical irradiation energy at 2500 µJ/cm2 and a lethal energy level to all genotypes at 10000 µJ/cm2 could be observed.
[1] Chavez, C., Cruz-Becerra, G., Fei, J., Kassavetis, G. A., & Kadonaga, J. T. (2019). The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals. ELife, 8, e47682. (https://doi.org/10.7554/eLife.47682)
[2] Rapley, R. (2000). The Nucleic Acid Protocols Handbook. Springer Science & Business Media.
[3] Ali M, Suzuki H, Fukuba T, Jiang X, Nakano H, Yamane T. Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant. J Biosci Bioeng. 2005 Feb;99(2):181-6. doi: 10.1263/jbb.99.181. PMID: 16233776.
Results HeLa live cell imaging
In the pre-experiment, three tests were performed to find the best adjustments to observe the test. In these videos two blocks were performed: the first block takes place without any radiation. In the second block, the cells were stressed with rays every 15 minutes, to find out their tolerance level for phototoxicity.
In the first test (test 1: 380 nm, 7s every 15 min), all HeLa cells died after a few hours. In the following test (test 2: 380 nm, 7s every 15 min), a filter was used that allowed only 10% of the rays to pass through. Even after 72 hours, no change in proliferation was visible. The results of test 3 (380 nm, 7s every 15 min) are shown in the following two video clips.
The first video shows the first block. The cells are not stressed and proliferate constantly. The second video shows the second block of the third test. After approximate 11 hours, blebbing occurs. Probably the cells are dying due to the phototoxicity. <p>
Live cell imaging with extracellular Dsup protein
The actual experiment only shows block two, to find out, whether the Dsup has any impact. Due to time reasons, the experiment was only performed once. To test the extracellular Dsup protection potential, the protein with a His-tag at the C-terminus was used. Four different Dsup amounts (0 ng, 450 ng, 4500 ng, 0,1125 mg) were tested at the same time in a quartered confocal dish. Each quarter contains 18.000 HeLa cells. The videos below show the third test with the addition of Dsup. The rays lead to the death of the cells even with a low exposure time. Due to the results of the pre-experiments, the cells were observed with the addition of DSUP within the media. By taking a look at the positive control, no dying of the cells can be observed. The cells with an addition of 450 ng and 4500 ng of Dsup don’t show any difference in their behaviour. Only the highest concentration (0,1125 mg) of Dsup leads to apoptosis of the cells
Discussion
In the preliminary test with the untreated HeLa cells, we were able to show that they react sensitively to UVA radiation. Already 3s of 380 nm every 15 min led to the death of the cells after a few hours.
We could not prove a protective effect of the Dsup protein in this setup. However, this may have several reasons. Firstly, the preliminary experiment was carried out six weeks before the experiment with Dsup. The cells were therefore in different passage numbers. This could have a greater influence on the sensitivity of the cells than we thought. Secondly, the amount of protein might not have been high enough. Furthermore, only the C-term Dsup was tested. Possibly the His-tag has a negative influence on the folding of the protein, which is why it cannot show its potential. This assumption could have been refuted with the same procedure for the N-term. Unfortunately, there was not enough time for this. But we could show that the Dsup extracellularly has no negative influence on the proliferation of HeLa cells. The highest concentration of Dsup leads to the death of the cells, this is not the result, we have expected. But since the pre-experiment in this set-up hasn’t worked out as well and the cells were not in a good place already in the beginning, there can’t be made a clear prediction out of this result. The experiments have to be repeated to find out if Dsup has a positive impact.