Part:BBa_K2201012
PtNTT2 with pelB Signal Peptide Tagged with GFP and a cMyc Linker
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.
This part is the GFP tagged version of BBa_K2201006. As a linker, cMyc was used. For characterization of this part please view BBa_K2201004.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 103
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2421
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 | 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 ), cMyc epitope tag as linker ( BBa_K2201181 ) |
| pSB1C3-PlacUV5-PtNTT2(66-575)-GFP | BBa_K2201003 | Fusion protein of BBa_ K2201001 with GFP ( BBa_E0040 ), cMyc epitope tag as linker ( BBa_K2201181 ) |
| pSB1C3-PlacUV5-PtNTT2(31-575)-GFP | BBa_K2201011 | Fusion protein of BBa_K2201005 with GFP ( BBa_E0040 ), cMyc epitope tag as linker ( BBa_K2201181 ) |
| pSB1C3-PlacUV5-pelB-SP-PtNTT2-GFP | BBa_K2201012 | Fusion protein of BBa_K2201006 with GFP ( BBa_E0040 ), cMyc epitope tag as linker ( BBa_K2201181 ) |
| pSB1C3-PlacUV5-TAT-SP-PtNTT2-GFP | BBa_K2201013 | Fusion protein of BBa_K2201007 with GFP ( BBa_E0040 ), cMyc epitope tag as linker ( BBa_K2201181 ) |
References
| None |