Difference between revisions of "Team:Bielefeld-CeBiTec/Results/unnatural base pair/uptake and biosynthesis"

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Uptake and Biosynthesis

Computational Analysis of PtNTT2

Zhang et al. used an N-terminal truncated version of PtNTT2, lacking the first 65 amino acids, since they observed some kind of toxicity resulting from the native N-terminal sequence (Zhang et al., 2017). For our project, we analyzed the amino acid sequence of PtNTT2 using the prediction software Phobius (Käll et al., 2007). Using Phobius, we analyzed the signal peptide and the transmembrane topology of PtNTT2. The analysis revealed that the native signal peptide is formed by amino acids 1 30, which means that Zhang et al. removed more than the native signal peptide for their experiment. The results of the prediction are shown in figure (1). Analysis of the transmembrane topology of the transporter, which is integrated into the plastid membrane in its native algal cell, shows iterative non-cytoplasmatic, transmembrane and cytoplasmic regions. The topology might indicate, that the transporter will be integrated into the inner membrane when expressed in E. coli.

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.

Plasmid Design

For the analysis and characterization of PtNTT2, a total of eleven plasmids were designed and cloned based on initial research and the computational analysis. The coding sequence of PtNTT2 was codon optimized using the IDT Codon Optimization Tool [REF] and ordered as two gBlocks. Using overlap extension PCR, the two gBlocks were put together and inserted into pSB1C3 using Gibson Assembly. The truncated versions of the transporter as well as the versions with new signal peptides were constructed using primers and Gibson assembly. Based on BBa_B0034 and BBa_K2082221 the fusion proteins were designed and cloned. A schematic overview of the design of the different plasmids is shown in figure (2). All plasmids except pSB1C3-PtNTT2 were tagged with the same cMyc-GFP construct for imaging and western blots.

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

Given that Zhang et al., 2017 reported some kind of toxicity resulting from the N-terminal sequence of PtNTT2, we first investigated if we could also observe the same toxicity associated with the N-terminal sequence. We also tested whether our own versions of the transporter result in better growth and reduced toxicity compared to the native transporter. Therefore, after cloning the plasmids in E. coli DH5α and verifying the correct assembly via sequencing, all plasmids were transformed into E. coli BL21(DE3). The presence of the correct plasmids was again verified by colony PCR. The first cultivations were carried out in shake flasks in LB media. A total cultivation volume of 50 mL was used. The cultures were incubated at 37 °C and 180 rpm. All cultures were inoculated with an OD₆₀₀ of 0.01. OD₆₀₀ was measured every hour during lag and stationary phase and every 30 minutes during the exponential phase. The optical density was measured using an Eppendorf Photometer and standard cuvettes. E. coli BL21(DE3) without a plasmid and E. coli BL21(DE3) harboring pSB1C3-PtNTT2 were used as negative controls. Two biological replicates of each strain were tested and three technical replicates were measured for each timepoint.

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.

E. coli BL21(DE3) without any plasmid shows the best growth, with the highest specific growth rate and the highest final OD₆₀₀ of 5.178 ± 0.046. The second negative control, E. coli BL21(DE3) harboring pSB1C3-PtNTT2 (BBa_K2201004) reached the second highest OD₆₀₀ with 4.638 ± 0.029. Of the functional PtNTT2 variants, strains harboring pSB1C3-PlacUV5-PtNTT2(66-575) (BBa_K2201001) and pSB1C3-PlacUV5-pelB-SP-PtNTT2 (BBa_K2201006) reached the highest ODs with 4.397 ± 0.062 and 4.171 ± 0.051, respectively. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) (BBa_K2201005) showed similar growth to the two previous strains during the lag phase and early exponential phase, but reaching a lower OD₆₀₀ of 3.802 ± 0.135. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 (BBa_K2201000) and E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 (BBa_K2201007) grew significantly weaker compared to all previous strains, with a lag phase nearly four hours long and final ODs of 2.499 ± 0.134 and 2.735 ± 0.150, respectively. The difference in growth between the two negative controls can be explained by the metabolic burden caused by plasmid replication and expression of the chloramphenicol resistance. Therefore, E. coli BL21(DE3) pSB1C3-PtNTT2 is the more accurate control, since all samples contain the same plasmid backbone and were also grown in LB media supplemented with chloramphenicol. The difference in growth between strains expressing the native, full length transporter and the truncated version PtNTT2(66-575), observed by Zhang et al. 2017, could also be shown. This negative effect might be associated with the native signal peptide of PtNTT2, which E. coli might not be able to process correctly. Another explanation for the weak growth could be that the native transporter variant has a higher activity compared to the other variants. If the activity is too high, this might lead to a toxic effect and to the observed weak growth.

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 µ_max. The graphical determination of µmax for the shake flask cultivation is shown in figure (4).

Figure (4): Graphical determination of µ_max.
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 µ_max.

Unsurprisingly, the highest specific growth rate can be observed for the negative controls E. coli BL21(DE3) and E. coli BL21(DE3) pSB1C3-PtNTT2 with values of 1.201 ± 0.070 h-1 and 1.212 ± 0.029 h-1, respectively. The lowest specific growth rate could be observed for E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 with a value of 0.946 ± 0.030 h-1. Based on the specific growth rate, the minimal doubling time was calculated using the following equation:

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	µ_max [h^-1]	t_d [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

Due to the high price of unnatural nucleoside triphosphates, performing experiments such as cultivations in a small volume is desirable. Therefore, we validated whether cultivations in micro well plates can achieved the same results as cultivations performed in shake flasks and optimized the method [LINK]. The optimal conditions for cultivations in a small volume were determined as follows: 12 well plates with a cultivation volume of 1 mL, 37 °C and 600 rpm. To analyze the growth of the differentPtNTT2 variants in a micro cultivation, the shake flask cultivation was repeated in a 12 well plate. All cultures were inoculated with an OD₆₀₀ of 0.1. Three biological replicates of each strain were tested and three technical replicates were measured at each time point. The results are shown in figure (5).

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.

The results of the micro cultivations show the same growth pattern observed in the shake flask cultivations. E. coli BL21(DE3) and E. coli BL21(DE3) pSB1C3-PtNTT2 reached the highest ODs with 5.487 ± 0.038 and 4.337 ± 0.01, respectively. E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 reached a final OD₆₀₀ of 4.110 ± 0.005, followed by E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) with a final OD₆₀₀ of 4.035 ± 0.051. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) reached the third highest OD₆₀₀ of allPtNTT2 variants with a final OD₆₀₀ of 3.865 ± 0.008. The lowest final ODs were again reached by E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2, reaching a final OD₆₀₀ of 2.28 ± 0.337 and 1.623 ± 0.481. Therefore, we were able to demonstrate that the results of the shake flask cultivations can be transferred to a smaller format, such as a micro cultivation in 1 mL. The reproduction of the results in a 50 times smaller volume is important for further experiments.

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, µ_max 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.

All cultures show extended doubling times when compared to the shake flask cultivations. This is not surprising, since the oxygen transfer is much better in shake flasks compared to well plates. Nonetheless, similar final OD₆₀₀ values were reached in the micro cultivations. Furthermore, the same differences in growth between the different strains can be observed, with E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 and E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 growing the weakest.

Table (4): Maximum specific growth rate and minimum doubling time for all cultures cultivated in 12 well plates.

Strain	µ_max [h^-1]	t_d [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.

The results obtained by iGEM team UNIFI validate our results, since the same difference in growth can be observed between the truncated version PtNTT2(66-575) and PtNTT2. Also in a 96 well plate, E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 reached only 49.5 % of the final cell density of E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) (40.2 % for the cultivation carried out in 12 well plates). After 27 hours, the final OD₆₀₀ of the strain carrying the full-length version of PtNTT2 was 0.329 ± 0.037, while the final OD₆₀₀ of the strain expressing the truncated version PtNTT2(66-575) was 0.664 ± 0.033. These results clearly indicate that it is highly beneficial for higher cell densities and better growth to cultivate in larger well plates. Furthermore, shaking at higher frequencies can improve the growth, as show in the validation of micro cultivations [LINK]. The graphical determination of the maximum specific growth rates is shown in figure (7).

Figure (7): Graphical determination of the maximum specific growth rates for the cultivations carried out in 96 well plates by the iGEM team UNIFI.

Graphical determination of the specific growth rates and the minimum doubling times revealed significantly weaker growth of the cultures in 96 well plates compared to 12 well plates or shake flasks. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) reached the highest specific growth rate with 0,11 ± 0,002 h-1. E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 showed much weaker growth with a maximum specific growth rate of 0,041 ± 0,004 h-1. The maximum specific growth rates and minimum doubling times are summarized in table (5).

Table (4): Maximum specific growth rate and minimum doubling time for all cultures cultivated in 12 well plates.

Strain	µ_max [h^-1]	t_d [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

Compared to the shake flask cultivations and the micro cultivations in 12 well plates, far lower maximum specific growth rates and much higher minimum doubling times were reached.

Verification of the Function of PtNTT2

To verify that PtNTT2 fulfills its desired function, a simple experiment was designed. Usually, the function of nucleotide transporters is determined using radioactively labeled nucleotides. Given that we did not want to work with radioactively labeled nucleotides and that not every lab has access to suitable equipment, we focused on verifying the function without using radioactively labeled nucleotides. Under phosphate starvation, E. coli secretes phosphatases to utilize the phosphates from nucleotides as phosphate source. Subsequently, the nucleosides can also be taken up by E. coli. On the other hand, cells expressing PtNTT2 and integrating it into the inner membrane should be able to take up nucleoside triphosphates directly. By taking up nucleoside triphosphates directly from the media, the cells can directly take up three phosphates and the nucleobase. Given that the uptake of NTPs by PtNTT2 is facilitated by counter exchange of NDPs, NDPs are exported and consequently converted to NMPs by extracellular phosphatases. The experiment we designed consists of two parts and is based on the disability of E. coli to take up nucleoside triphosphates from the surrounding media. The first part consists of cultivations performed in MOPS minimal media which is supplemented with either 1,32 mM K₂HPO₄ or 1 mM ATP. Calculation of the Odds-ratio as shown in equation (A) allows to evaluate how beneficial expression of PtNTT2 is for the cell if extracellular ATP represents the sole phosphate source. The second part of the experiment consists of liquid chromatography – mass spectrometry (LC-MS) measurements for the quantification of AMP in the supernatant. Combined, these methods provide a way to investigate the function of PtNTT2 without the application of radioactively labeled nucleotides. Furthermore, these experiments might also serve as a way for future iGEM teams to easily characterize the function of membrane proteins.
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 final OD₆₀₀ values varied widely, with E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 reaching the highest OD₆₀₀ of 3.907 ± 0.018. The lowest OD₆₀₀ was reached by E. coli BL21(DE3) pSB1C3-PlacUV5-PtNTT2 with a value of 1.537 ± 0.045. All final optical densities at 600 nm are shown in table (5).
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.

In ATP supplemented media, the wildtype strain reached the highest OD₆₀₀ with 4.967 ± 0.143. Of the transporter variants, E. coli BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 again reached the highest OD₆₀₀ and E. coli BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 the lowest. The results for both cultivations are summarized in table (5).

Table (5): Final OD₆₀₀ values for all cultivations carried out in MOPS media with 1,32 mM K₂HPO₄.

Strain	Final OD_{600, K₂HPO₄} [-]	Final OD_{600, 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

The maximum specific growth rate was graphically determined for all cultures as shown in figure (10).

Figure (10): Graphical determination of the maximum specific growth rates for all cultures cultivated in MOPS media with 1.32 mM K₂HPO₄.

Based on the maximum specific growth rates, the minimum doubling time was calculated. The results are shown in table (6). Once again, the strains expressing the native transporter and the transporter with a TAT signal peptide showed the weakest growth. The best growth characteristics were achieved by the strains expressing the truncated versions PtNTT2(66-575) and PtNTT2(31-575), and PtNTT2 with a pelB signal peptide.

Table (6): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1.32 mM K₂HPO₄.

Strain	µ_max [h^-1]	t_t [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.

The determined values for µmax and the minimal doubling times are shown in table (7).

Table (7): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1 mM ATP.

Strain	µ_max [h^-1]	t_t [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

Based on the results of the two cultivations, the relative beneficial effect (RBE) of PtNTT2 was calculated using equation (2). For each measurement point n, the optical density of the cultivation of a transporter variant in ATP supplemented media was divided by the optical density of the reference E. coli BL21(DE3) pSB1C3-PtNTT2 in ATP supplemented media. The same was done for the cultures in K₂HPO₄ supplemented media. The value for ATP was then divided by the value for K₂HPO₄. The sum of the quotient for all measurement points n was then divided by n to obtain the final value for the relative beneficial effect of PtNTT2 shown in figure (12). The final error was calculated through error propagation of the standard error of each measured optical density.

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.

Of all transporter variants, the native PtNTT2 showed the highest beneficial effect with a value of 1.360 ± 0.161. The second highest value was reached by PtNTT2(31-575) with 1.294 ± 0.107. PtNTT2(66-575) reached a value of 1.166 ± 0.105, pelB-SP-PtNTT2 one of 1.073 ± 0.114 and TAT-SP-PtNTT2 one of 0.732 ± 0.116. This data suggests that the expression of different PtNTT2 variants, especially of the native PtNTT2, is beneficial for the cell when cultivated in MOPS minimal media supplemented with ATP as the sole phosphate source. Given that the reference strain does not express PtNTT2, the expression of PtNTT2 must have a beneficial effect for the cells since they grow better compared to the reference in ATP when compared to the reference in K₂HPO₄. Therefore, the beneficial effect is larger than the metabolic burden associated with recombinant protein expression. Consequently, the transporter exhibits a function beneficial to the cell in ATP supplemented media, meaning it can facilitate the direct uptake of ATP from the media.

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-								<th style="width: 100%;" class="header">Strain</th>
+								<th style="width: 60%;" class="header">Strain</th>
-								<th style="width: 100%;" class="header">µ<sub>max</sub> [h<sup>-1</sup>]</th>
+								<th style="width: 20%;" class="header">µ<sub>max</sub> [h<sup>-1</sup>]</th>
-								<th style="width: 100%;" class="header">t<sub>d</sub> [h]</th>
+								<th style="width: 20%;" class="header">t<sub>d</sub> [h]</th>
 							</tr>
 						</thead>
@@ Line 420: / Line 420: @@
 </div>
+<div class="contentbox">
+	<div class="content">
+	<h3> Verification of the Function of <i>Pt</i>NTT2 </h3>
+	<article>
+	To verify that <i>Pt</i>NTT2 fulfills its desired function, a simple experiment was designed. Usually, the function of nucleotide transporters is determined using radioactively labeled nucleotides. Given that we did not want to work with radioactively labeled nucleotides and that not every lab has access to suitable equipment, we focused on verifying the function without using radioactively labeled nucleotides.
+Under phosphate starvation, <i>E.&nbsp;coli</i> secretes phosphatases to utilize the phosphates from nucleotides as phosphate source. Subsequently, the nucleosides can also be taken up by <i>E.&nbsp;coli</i>. On the other hand, cells expressing <i>Pt</i>NTT2 and integrating it into the inner membrane should be able to take up nucleoside triphosphates directly. By taking up nucleoside triphosphates directly from the media, the cells can directly take up three phosphates and the nucleobase. Given that the uptake of NTPs by <i>Pt</i>NTT2 is facilitated by counter exchange of NDPs, NDPs are exported and consequently converted to NMPs by extracellular phosphatases.
+The experiment we designed consists of two parts and is based on the disability of <i>E.&nbsp;coli</i> to take up nucleoside triphosphates from the surrounding media. The first part consists of cultivations performed in MOPS minimal media which is supplemented with either 1,32 mM K<sub>2</sub>HPO<sub>4</sub> or 1 mM ATP. Calculation of the Odds-ratio as shown in equation (A) allows to evaluate how beneficial expression of <i>Pt</i>NTT2 is for the cell if extracellular ATP represents the sole phosphate source. The second part of the experiment consists of liquid chromatography – mass spectrometry (LC-MS) measurements for the quantification of AMP in the supernatant. Combined, these methods provide a way to investigate the function of <i>Pt</i>NTT2 without the application of radioactively labeled nucleotides. Furthermore, these experiments might also serve as a way for future iGEM teams to easily characterize the function of membrane proteins.
+<br>
+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, <i>E.&nbsp;coli</i> BL21(DE3) and <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PtNTT2, were cultivated in MOPS minimal media containing either 1,32 mM K<sub>2</sub>HPO<sub>4</sub> 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 K<sub>2</sub>HPO<sub>4</sub> as the sole phosphate source.
+<div class="figure large">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/64/T--Bielefeld-CeBiTec--microcultivation_K2HPO4.jpeg">
+			<p class="figure subtitle"><b>Figure (8): Cultivation of all transporter variants in MOPS media with K<sub>2</sub>HPO<sub>4</sub> acting as the sole phosphate source.</b><br> 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. </p>
+		</div>
+The final OD<sub>600</sub> values varied widely, with <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 reaching the highest OD<sub>600</sub> of 3.907 ± 0.018. The lowest OD<sub>600</sub> was reached by <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2 with a value of 1.537 ± 0.045. All final optical densities at 600 nm are shown in table (5).
+<br>
+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).
+	<div class="figure large">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/1/14/T--Bielefeld-CeBiTec--microcultivation_ATP.jpeg">
+			<p class="figure subtitle"><b>Figure (9): Cultivation of all strains in MOPS media with 1 mM ATP acting as the sole phosphate source.</b><br> Three biological replicates were cultivated and three technical replicates measured for each time point.</p>
+		</div>
+		In ATP supplemented media, the wildtype strain reached the highest OD<sub>600</sub> with 4.967 ± 0.143. Of the transporter variants, <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 again reached the highest OD<sub>600</sub> and <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 the lowest. The results for both cultivations are summarized in table (5).
+	<p class="figure subtitle"><b>Table (5): Final OD<sub>600</sub> values for all cultivations carried out in MOPS media with 1,32 mM K<sub>2</sub>HPO<sub>4</sub>. </b><br> </p>
+<table style="margin:auto">
+						<thead>
+							<tr>
+								<th style="width: 60%;" class="header">Strain</th>
+								<th style="width: 20%;" class="header">Final OD<sub>600, K<sub>2</sub>HPO<sub>4</sub></sub> [-]</th>
+								<th style="width: 20%;" class="header">Final OD<sub>600, ATP</sub> [-]</th>
+							</tr>
+						</thead>
+						<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) </td>
+								<td>2.923 ± 0.028</td>
+								<td>4.967 ± 0.143</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PtNTT2 </td>
+								<td>3.507 ± 0.048</td>
+								<td>3.673 ± 0.091</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2 </td>
+								<td>1.537 ± 0.045</td>
+								<td>3.033 ± 0.028</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) </td>
+								<td>3.560 ± 0.011</td>
+								<td>3.347 ± 0.032</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) </td>
+								<td>3.797 ± 0.065</td>
+								<td>3.580 ± 0.006</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 </td>
+								<td>3.907 ± 0.018</td>
+								<td>3.710 ± 0.177</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 </td>
+								<td>3.307 ± 0.029</td>
+								<td>2.177 ± 0.007</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+					</table>
+The maximum specific growth rate was graphically determined for all cultures as shown in figure (10).
+<div class="figure large">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/3/3c/T--Bielefeld-CeBiTec--%C2%B5maxK2HPO4.jpeg">
+			<p class="figure subtitle"><b>Figure (10): Graphical determination of the maximum specific growth rates for all cultures cultivated in MOPS media with 1.32 mM K<sub>2</sub>HPO<sub>4</sub>.</b><br></p>
+		</div>
+			Based on the maximum specific growth rates, the minimum doubling time was calculated. The results are shown in table (6). Once again, the strains expressing the native transporter and the transporter with a TAT signal peptide showed the weakest growth. The best growth characteristics were achieved by the strains expressing the truncated versions <i>Pt</i>NTT2(66-575) and <i>Pt</i>NTT2(31-575), and <i>Pt</i>NTT2 with a pelB  signal peptide.
+	<p class="figure subtitle"><b>Table (6): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1.32 mM K<sub>2</sub>HPO<sub>4</sub>.</b><br> </p>
+<table style="margin:auto">
+						<thead>
+							<tr>
+								<th style="width: 60%;" class="header">Strain</th>
+								<th style="width: 20%;" class="header">µ<sub>max</sub> [h<sup>-1</sup>]</th>
+								<th style="width: 20%;" class="header">t<sub>t</sub> [h]</th>
+							</tr>
+						</thead>
+						<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) </td>
+								<td>0.444 ± 0.053</td>
+								<td>1.561 ± 0.199</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PtNTT2 </td>
+								<td>0.499 ± 0.050</td>
+								<td>1.389 ± 0.100</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2 </td>
+								<td>0.385 ± 0.044</td>
+								<td>1.800 ± 0.114</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) </td>
+								<td>0,568 ± 0.057</td>
+								<td>1.220 ± 0.100</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) </td>
+								<td>0.532 ± 0.022</td>
+								<td>1.303 ± 0.041</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 </td>
+								<td>0.549 ± 0.017</td>
+								<td>1.263 ± 0.031</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 </td>
+								<td>0.463 ± 0.028</td>
+								<td>1.497 ± 0.060</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+					</table><br>
+The graphical determination of the maximum specific growth rates of the cultures cultivated in ATP supplemented media is shown in figure (11).
+	<div class="figure large">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/b/bf/T--Bielefeld-CeBiTec--%C2%B5maxATP.jpeg">
+			<p class="figure subtitle"><b>Figure (11): Graphical determination of the maximum specific growth rates of all cultivations performed in MOPS media and 1 mM ATP. </b><br> </p>
+		</div>
+The determined values for µmax and the minimal doubling times are shown in table (7).
+	<p class="figure subtitle"><b>Table (7): Maximum specific growth rates and minimal doubling times of the cultivations in MOPS media with 1 mM ATP.</b><br> </p>
+<table style="margin:auto">
+						<thead>
+							<tr>
+								<th style="width: 60%;" class="header">Strain</th>
+								<th style="width: 20%;" class="header">µ<sub>max</sub> [h<sup>-1</sup>]</th>
+								<th style="width: 20%;" class="header">t<sub>t</sub> [h]</th>
+							</tr>
+						</thead>
+						<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) </td>
+								<td>0.673 ± 0.012</td>
+								<td>1.030 ± 0.018</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PtNTT2 </td>
+								<td>0.600 ± 0.021</td>
+								<td>1.155 ± 0.035</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2 </td>
+								<td>0.463 ± 0.035</td>
+								<td>1.497 ± 0.076</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(66-575) </td>
+								<td>0.644 ± 0.069</td>
+								<td>1.076 ± 0.107</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-PtNTT2(31-575) </td>
+								<td>0.428 ± 0.091</td>
+								<td>1.620 ± 0.213</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-pelB-SP-PtNTT2 </td>
+								<td>0.518 ± 0.043</td>
+								<td>1.338 ± 0.083</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+<tbody>
+							<tr>
+								<td><i><i>E.&nbsp;coli</i></i> BL21(DE3) pSB1C3-PlacUV5-TAT-SP-PtNTT2 </td>
+								<td>0.334 ± 0.047</td>
+								<td>2.075 ± 0.141</td>
+								<td align="right"></td>
+							</tr>
+						</tbody>
+					</table>
+Based on the results of the two cultivations, the relative beneficial effect (RBE) of <i>Pt</i>NTT2 was calculated using equation (2). For each measurement point n, the optical density of the cultivation of a transporter variant in ATP supplemented media was divided by the optical density of the reference <i>E.&nbsp;coli</i> BL21(DE3) pSB1C3-PtNTT2 in ATP supplemented media. The same was done for the cultures in K<sub>2</sub>HPO<sub>4</sub> supplemented media. The value for ATP was then divided by the value for K<sub>2</sub>HPO<sub>4</sub>. The sum of the quotient for all measurement points n was then divided by n to obtain the final value for the relative beneficial effect of <i>Pt</i>NTT2 shown in figure (12). The final error was calculated through error propagation of the standard error of each measured optical density.
+<br>
+<br>
+<br>
+<div class="figure large">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/4/40/T--Bielefeld-CeBiTec--RBE-1mM.jpeg">
+			<p class="figure subtitle"><b>Figure (12): Relative beneficial effect of the different <i>Pt</i>NTT2 variants.</b><br> 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.</p>
+		</div>
+Of all transporter variants, the native <i>Pt</i>NTT2 showed the highest beneficial effect with a value of 1.360 ± 0.161. The second highest value was reached by <i>Pt</i>NTT2(31-575) with 1.294 ± 0.107. <i>Pt</i>NTT2(66-575) reached a value of 1.166 ± 0.105, pelB-SP-PtNTT2 one of 1.073 ± 0.114 and TAT-SP-PtNTT2 one of 0.732 ± 0.116.
+This data suggests that the expression of different <i>Pt</i>NTT2 variants, especially of the native <i>Pt</i>NTT2, is beneficial for the cell when cultivated in MOPS minimal media supplemented with ATP as the sole phosphate source. Given that the reference strain does not express <i>Pt</i>NTT2, the expression of <i>Pt</i>NTT2 must have a beneficial effect for the cells since they grow better compared to the reference in ATP when compared to the reference in K<sub>2</sub>HPO<sub>4</sub>. Therefore, the beneficial effect is larger than the metabolic burden associated with recombinant protein expression. Consequently, the transporter exhibits a function beneficial to the cell in ATP supplemented media, meaning it can facilitate the direct uptake of ATP from the media.
+	</article>
+	</div>
+</div>
 </div>