Difference between revisions of "Part:BBa K2201002"
 
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<i>Phaeodactylum tricornutum</i>, a diatom of the genus <i>Phaedactylum</i>, features six putative nucleotide transporters (NTTs). Two isoforms of these NTTs have been characterized by Ast <i>et al.</i> 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 <i>et al.</i>, 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 <i>E. coli</i>. Zhang <i>et al.</i> 2017 investigated the use of <i>Pt</i>NTT2 for the uptake of the unnatural bases dNaM and dTPT3. Therefore, the expression of <i>Pt</i>NTT2 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 <i>Pt</i>NTT2 chromosomally in <i>E. coli</i> BL21(DE3) under control of the lacUV5 promoter. To demonstrate its feasibility for the uptake of nucleotides in <i>E. coli</i> from the media, uptake of [&#945; 32P]-dATP was measured. The native sequence of <i>Pt</i>NTT2 features an N-terminal signal sequence directing the subcellular localization to the plastid membrane. In <i>E. coli</i>, this signal sequence is likely to be retained, leading to a growth defect in cells expressing the native <i>Pt</i>NTT2 transporter.  
 
<i>Phaeodactylum tricornutum</i>, a diatom of the genus <i>Phaedactylum</i>, features six putative nucleotide transporters (NTTs). Two isoforms of these NTTs have been characterized by Ast <i>et al.</i> 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 <i>et al.</i>, 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 <i>E. coli</i>. Zhang <i>et al.</i> 2017 investigated the use of <i>Pt</i>NTT2 for the uptake of the unnatural bases dNaM and dTPT3. Therefore, the expression of <i>Pt</i>NTT2 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 <i>Pt</i>NTT2 chromosomally in <i>E. coli</i> BL21(DE3) under control of the lacUV5 promoter. To demonstrate its feasibility for the uptake of nucleotides in <i>E. coli</i> from the media, uptake of [&#945; 32P]-dATP was measured. The native sequence of <i>Pt</i>NTT2 features an N-terminal signal sequence directing the subcellular localization to the plastid membrane. In <i>E. coli</i>, this signal sequence is likely to be retained, leading to a growth defect in cells expressing the native <i>Pt</i>NTT2 transporter.  
 
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<b>This part is the <a href="https://parts.igem.org/Part:BBa_E0040">GFP</a> tagged version of <a href="https://parts.igem.org/Part:BBa_K2201000">BBa_K2201000</a>.As a linker<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2201181">cMyc</a> was used.</b>
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<b>This part is the <a href="https://parts.igem.org/Part:BBa_E0040">GFP</a> tagged version of <a href="https://parts.igem.org/Part:BBa_K2201000">BBa_K2201000</a>.As a linker <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2201181">cMyc</a> was used. For characterization of this part please view <a href="https://parts.igem.org/Part:BBa_K2201004">BBa_K2201004</a></b>.
 
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Latest revision as of 00:11, 1 November 2017

PtNTT2 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_K2201000.As a linker cMyc was used. For characterization of this part please view BBa_K2201004.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2445


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

Ast, M., Gruber, A., Schmitz-Esser, S., Neuhaus, H.E., Kroth, P.G., Horn, M., and Haferkamp, I. (2009). Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl. Acad. Sci. U. S. A. 106: 3621–3626.
Zhang, Y., Lamb, B.M., Feldman, A.W., Zhou, A.X., Lavergne, T., Li, L., and Romesberg, F.E. (2017). A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. 114: 1317–1322.