Team:Bielefeld-CeBiTec/Results/unnatural base pair/uptake and biosynthesis

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 X. 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 functionalPtNTT2 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 versionPtNTT2(66-575), observed by Zhang et al. 2017, could also be shown. This negative effect might be associated with the native signal peptide ofPtNTT2, 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.