Figure (1): Results of the analysis of PtNTT2 using Phobius.
The 30 first amino acids are clearly recognized as a signal peptide. Ten transmembrane domains are predicted.
Computational Analysis of PtNTT2
Plasmid Design
Figure (2): Schematic overview of the design of the different transporter variants.
The lacUV5 promotor was used together with a strong RBS (BBa_B0034) for all parts. All variants except for pSB1C3-PtNTT2 were also tagged with GFP (BBa_E0040). cMyc was used as a linker (based on BBa_K2082221).
Table (1): Designed and cloned plasmids for the analysis and characterization of PtNTT2.
Plasmid Name | BioBrick Number | Characteristics | |
---|---|---|---|
pSB1C3-PtNTT2 | BBa_K2201004 | Only the cds | |
pSB1C3-PlacUV5-PtNTT2(66-575) | BBa_ K2201001 | cds with lacUV5 promotor and a strong RBS (BBa_B0034) | |
pSB1C3-PlacUV5-PtNTT2(31-575) | BBa_K2201005 | cds with lacUV5 promotor and a strong RBS (BBa_B0034), truncated version lacking the first 30 amino acids | |
pSB1C3-PlacUV5-pelB-SP-PtNTT2 | BBa_K2201006 | cds with lacUV5 promotor and a strong RBS (BBa_B0034), native signal peptide replaced with the pelB signal peptide | |
pSB1C3-PlacUV5-TAT-SP-PtNTT2 | BBa_K2201007 | cds with lacUV5 promotor and a strong RBS (BBa_B0034), native signal peptide replaced with a TAT signal peptide | |
pSB1C3-PlacUV5-PtNTT2-GFP | BBa_K2201002 | Fusion protein of BBa_ K2201000 with GFP (BBa_E0040), Myc epitope tag as linker (BBa_K2201181) | |
pSB1C3-PlacUV5-PtNTT2(66-575)-GFP | BBa_K2201003 | Fusion protein of BBa_ K2201001 with GFP (BBa_E0040), Myc epitope tag as linker (BBa_K2201181) | |
pSB1C3-PlacUV5-PtNTT2(31-575)-GFP | BBa_K2201011 | Fusion protein of BBa_K2201005 with GFP (BBa_E0040), Myc epitope tag as linker (BBa_K2201181) | |
pSB1C3-PlacUV5-pelB-SP-PtNTT2-GFP | BBa_K2201012 | Fusion protein of BBa_K2201006 with GFP (BBa_E0040), Myc epitope tag as linker (BBa_K2201181) | |
pSB1C3-PlacUV5-TAT-SP-PtNTT2-GFP | BBa_K2201013 | Fusion protein of BBa_K2201007 with GFP (BBa_E0040), Myc epitope tag as linker (BBa_K2201181) |
Cultivations of the Different PtNTT2 Variants
Figure (3): Shake flask cultivation of all PtNTT2 variants.
E. coli BL21(DE3) and E. coli BL21(DE3) pSB1C3-PtNTT2, not expressing PtNTT2, were used as negative controls. Two biological replicates of each strain were cultivated and three technical replicates taken for each measurement. A clear difference in the growth rates can be observed, with E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 showing the weakest growth. Both strains also show the longest lag phase, which is nearly twice as long as the lag phase of E. coli BL21(DE3). E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) and E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 show the best growth of all PtNTT2 variants, reaching the highest OD600.
Table (1): Final OD600 of all cultures.
The highest OD600 was reached by the wildtype E. coli BL21(DE3), the lowest by E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2.
Strain | Final OD600 [-] | |
---|---|---|
E. coli BL21(DE3) | 5.178 ± 0.046 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 4.638 ± 0.029 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 2.499 ± 0.134 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 4.397 ± 0.062 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 3.802 ± 0.135 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 4.171 ± 0.051 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 2.735 ± 0.150 |
To determine the maximum specific growth rate (µmax), the natural logarithm of the OD600 values was plotted against the cultivation time. The slope of the linear regression through the exponential phase gives µmax. The graphical determination of µmax for the shake flask cultivation is shown in figure (4).
Figure (4): Graphical determination of µmax.
The highest specific growth rate was determined for each culture by plotting the natural logarithm of OD600 against the cultivation time. The slope of the linear regression through the exponential phase gives µmax.
The maximum specific growth rates and minimal doubling times are show in table (2) for all cultures.
Table (2): Maximum specific growth rates and minimum doubling times for all cultures.
Strain | µmax [h-1] | td [h] | |
---|---|---|---|
E. coli BL21(DE3) | 1.201 ± 0.070 | 0.577 ± 0.058 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 1.212 ± 0.029 | 0.572 ± 0.024 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 0.978 ± 0.033 | 0.709 ± 0.034 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 1.194 ± 0.026 | 0.581 ± 0.022 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 1.143 ± 0.045 | 0.606 ± 0.039 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 1.189 ± 0.028 | 0.583 ± 0.024 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 0.946 ± 0.030 | 0.733 ± 0.032 |
These results clearly show that expression of PtNTT2 leads to a reduced final cell density and slower growth. Furthermore, the different variants of PtNTT2 differ highly, indicating that some variants of PtNTT2 negatively affect the growth rate and final cell density.
Microcultivations of the Different PtNTT2 Variants
Figure (5): Microcultivation of allPtNTT2 variants
E. coli BL21(DE3) and E. coli BL21(DE3) pSB1C3-PtNTT2 (BBa_K2201004) were again used as negative controls. The same growth pattern as in the shake flask cultivation can be observed, with E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2, E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) reaching the highest ODs, followed by E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575), E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2.
Table (3): Final OD600 of all cultures.
The highest OD600 was reached by the wildtype E. coli BL21(DE3) with 5,487 ± 0.038, the lowest by E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 with 1.623 ± 0.481.
Strain | Final OD600 [-] | |
---|---|---|
E. coli BL21(DE3) | 5.487 ± 0.038 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 4.337 ± 0.010 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 1.623 ± 0.481 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 4.035 ± 0.051 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 3.865 ± 0.008 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 4.110 ± 0.005 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 2.280 ± 0.337 |
Like for the shake flask cultivation, µmax was determined graphically (figure 5). Bases on the obtained values, the minimum doubling time was calculated. The results are summarized in table (4).
Figure (5): Graphical determination of the maximum specific growth rate µmax for the microcultivations.
Table (4): Maximum specific growth rate and minimum doubling time for all cultures cultivated in 12 well plates.
Strain | µmax [h-1] | td [h] | |
---|---|---|---|
E. coli BL21(DE3) | 1.059 ± 0.143 | 0.655 ± 0.135 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 1.016 ± 0.133 | 0.682 ± 0.131 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 0.829 ± 0.071 | 0.836 ± 0.086 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 1.023 ± 0.105 | 0.678 ± 0.103 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 1.021 ± 0.096 | 0.679 ± 0.094 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 1.047 ± 0.097 | 0.662 ± 0.093 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 0.924 ± 0.113 | 0.750 ± 0.122 |
To investigate the effect of smaller well plates, a cultivation of two of our strains was performed by the iGEM team UNIFI from Florence, Italy. The team cultivated E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) in a 96 well plate. The cultivation was performed at 37 °C and 130 rpm in 3 mL of LB media. Three biological replicates were cultivated and measured at each time point. The results are shown in figure (6).
Figure (6): Microcultivation in a 96 well plate performed by iGEM team UNIFI from Florence, Italy.
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) were cultivated in a total volume of 3 mL at 37 °C and 130 rpm. The growth difference between the two strains observed in previous cultivations could also be observed in this experiment carried out by the team from Florence. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 reached a final OD600 of 0.329 ± 0.037 while E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) reached a final OD600 of 0.664 ± 0.033.
Figure (7): Graphical determination of the maximum specific growth rates for the cultivations carried out in 96 well plates by the iGEM team UNIFI.
Table (4): Maximum specific growth rate and minimum doubling time for all cultures cultivated in 12 well plates.
Strain | µmax [h-1] | td [h] | |
---|---|---|---|
E. coli BL21(DE3) | 0.042 ± 0.004 | 16.504 ± 0.095 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 0.110 ± 0.002 | 6.301 ± 0.018 |
Verification of the Function of PtNTT2
For the first part of the experiment, two sets of cultivations were carried out in parallel. All transporter variants as well as two negative controls, E. coli BL21(DE3) and E. coli BL21(DE3) pSB1C3-PtNTT2, were cultivated in MOPS minimal media containing either 1,32 mM K2HPO4 or 1 mM ATP as sole phosphate source. Three biological replicates of each strain were cultivated in 1 mL of media in a 12 well plate at 37 °C and 600 rpm. For each measurement point, three technical replicates were measured. Figure (8) shows the growth curves of the cultivations carried out with 1,32 mM of K2HPO4 as the sole phosphate source.
Figure (8): Cultivation of all transporter variants in MOPS media with K2HPO4 acting as the sole phosphate source.
The cultivation was carried out in 12 well plates and three biological replicates were cultivated of each strain. For measurement of the optical density at 600 nm, three technical replicates were taken.
The cultivations were performed in parallel in MOPS media supplemented with 1 mM ATP as sole phosphate source. Again, three biological replicates of each strain were cultivated and three technical replicates measured for each time point. The growth curves are shown in figure (9).
Figure (9): Cultivation of all strains in MOPS media with 1 mM ATP acting as the sole phosphate source.
Three biological replicates were cultivated and three technical replicates measured for each time point.
Table (5): Final OD600 values for all cultivations carried out in MOPS media with 1,32 mM K2HPO4.
Strain | Final OD600, K2HPO4 [-] | Final OD600, ATP [-] | |
---|---|---|---|
E. coli BL21(DE3) | 2.923 ± 0.028 | 4.967 ± 0.143 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 3.507 ± 0.048 | 3.673 ± 0.091 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 1.537 ± 0.045 | 3.033 ± 0.028 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 3.560 ± 0.011 | 3.347 ± 0.032 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 3.797 ± 0.065 | 3.580 ± 0.006 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 3.907 ± 0.018 | 3.710 ± 0.177 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 3.307 ± 0.029 | 2.177 ± 0.007 |
Figure (10): Graphical determination of the maximum specific growth rates for all cultures cultivated in MOPS media with 1.32 mM K2HPO4.
Table (6): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1.32 mM K2HPO4.
Strain | µmax [h-1] | tt [h] | |
---|---|---|---|
E. coli BL21(DE3) | 0.444 ± 0.053 | 1.561 ± 0.199 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 0.499 ± 0.050 | 1.389 ± 0.100 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 0.385 ± 0.044 | 1.800 ± 0.114 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 0,568 ± 0.057 | 1.220 ± 0.100 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 0.532 ± 0.022 | 1.303 ± 0.041 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 0.549 ± 0.017 | 1.263 ± 0.031 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 0.463 ± 0.028 | 1.497 ± 0.060 |
The graphical determination of the maximum specific growth rates of the cultures cultivated in ATP supplemented media is shown in figure (11).
Figure (11): Graphical determination of the maximum specific growth rates of all cultivations performed in MOPS media and 1 mM ATP.
Table (7): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1 mM ATP.
Strain | µmax [h-1] | tt [h] | |
---|---|---|---|
E. coli BL21(DE3) | 0.673 ± 0.012 | 1.030 ± 0.018 | |
E. coli BL21(DE3) pSB1C3-PtNTT2 | 0.600 ± 0.021 | 1.155 ± 0.035 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 | 0.463 ± 0.035 | 1.497 ± 0.076 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) | 0.644 ± 0.069 | 1.076 ± 0.107 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) | 0.428 ± 0.091 | 1.620 ± 0.213 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 | 0.518 ± 0.043 | 1.338 ± 0.083 | |
E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 | 0.334 ± 0.047 | 2.075 ± 0.141 |
Figure (12): Relative beneficial effect of the different PtNTT2 variants.
As expected, the native transporter variant shows the highest positive effect since it most likely also exhibits the highest activity. Surprisingly, the two truncated versions show a higher effect than the versions with a pelB and TAT signal peptide.
Subcellular Localization of PtNTT2
Figure (13): Confocal laser scanning microscopy of the different PtNTT2 variants fused to GFP (BBa_E0040).
The pictures were taken with 100x magnification and show from A to E: E. coli BL21(DE3), E. coli BL21(DE3) pSB1C3-PtNTT2, E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2, E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575), E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575), E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2.