Part:BBa_K2201004

Nucleotide Transporter PtNTT2 from Phaeodactylum tricornutum

Usage and Biology

Phaeodactylum tricornutum, a diatom of the genus Phaedactylum, features six putative nucleotide transporters (NTTs). Two isoforms of these NTTs have been characterized by Ast et al. 2009 and it was shown that both isoforms facilitate transport across the plastid membrane. While isoform 1 (NTT1) acts as a proton-dependent adenine nucleotide importer, NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates (Ast et al., 2009).The isoform 2 of the nucleotide transporter was shown to be a broad range (deoxy-)nucleoside transporter, facilitating the uptake of CTP, GTP, dCTP, ATP, UTP, dGTP, dATP and TTP when expressed in E. coli.Zhang et al. 2017 investigated the use of PtNTT2 for the uptake of the unnatural bases dNaM and dTPT3. Therefore, the expression of PtNTT2 was investigated in different strains, under control of different promotors, and plasmid-bound as well as integrated into the chromosome. In their final design, Zhang and colleagues integrated PtNTT2 chromosomally in E. coli BL21(DE3) under control of the lacUV5 promoter. To demonstrate its feasibility for the uptake of nucleotides in E. coli from the media, uptake of [α 32P]-dATP was measured. The native sequence of PtNTT2 features an N-terminal signal sequence directing the subcellular localization to the plastid membrane. In E. coli, this signal sequence is likely to be retained, leading to a growth defect in cells expressing the native PtNTT2 transporter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Computational Analysis of PtNTT2

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 were predicted.

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.

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 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.

Plasmid Name	BioBrick Number	Characteristics
pSB1C3-PtNTT2	BBa_K2201004	Only the cds
pSB1C3-PlacUV5-PtNTT2	BBa_K2201000	cds with lacUV5 promotor and a strong RBS ( BBa_B0034 )
pSB1C3-PlacUV5-PtNTT2(66-575)	BBa_K2201001	cds with lacUV5 promotor and a strong RBS ( BBa_B0034 ),truncated version lacking the first 65 amino acids
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 )