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).
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 X.
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.
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 ofPtNTT2 leads to a reduced final cell density and slower growth. Furthermore, the different variants ofPtNTT2 differ highly, indicating that some variants ofPtNTT2 negatively affect the growth rate and final cell density.