Difference between revisions of "Part:BBa K2201004"

 
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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 [&#945; 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.
 
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===Usage and Biology===
 
===Usage and Biology===
  
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Phaeodactylum tricornutum, a diatom of the genus Phaedactylum, 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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2201004 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K863005 parameters</partinfo>
 
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==Computational Analysis of <i>Pt</i>NTT2==
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==Plasmid Design==
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==Cultivations==
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===Shake Flask Cultivation===
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===Microcultivations===
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==Verification of the Fuction==
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===Relative Beneficial Effect===
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===HPLC===
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==Confocal Laser Scanning Microscopy==
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==Isolation of <i>Pt</i>NTT2 from the Inner Membrane==

Revision as of 13:16, 30 October 2017


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

Plasmid Design

Cultivations

Shake Flask Cultivation

Microcultivations

Verification of the Fuction

Relative Beneficial Effect

HPLC

Confocal Laser Scanning Microscopy

Isolation of PtNTT2 from the Inner Membrane