Difference between revisions of "Part:BBa K4886007"

 
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===Usage and Biology===
 
===Usage and Biology===
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Experiments and results
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1.Ancestral sequence reconstruction (ASR) method
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FireProt-ASR (https://loschmidt.chemi.muni.cz/fireprotasr/) was used by Ms. Yang to carry out ASR. The phosphoketolase (F/Xpk) sequence (BBa_K4119076) derived from Clostridium acetobutylicum was used as the input sequence. No essential residues were selected. Percent sequence identity was set to 30%-70%. Clustering identity was set to 0.9. Evolutionary model was set to WAG. RAxML (Randomized Axelerated Maximum Likelihood) was chosen as the phylogenetic tree inference tool. Bootstraps were set to 500. The ancestral sequence of phosphoketolase (F/Xpk) predicted by ASR was named F/Xpk(ASR).
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2.Plasmid construction
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On the basis of plasmid pMTL-Pthl-F/Xpk(BD), F/Xpk(BD) fragment was replaced with F/Xpk(ASR). Using pMTL-Pthl-F/Xpk(BD) as the template and X-pMTL-F and X-pMTL-R as the primers, X-pMTL-Pthl linearized vector (5461 bp) was amplified. Using PUC57 vector plasmid as the template and P-F/Xpk(ASR)-F and P-F/Xpk(ASR)-R as the primers, F/Xpk(ASR) fragment (2436 bp) was amplified. The F/Xpk (ASR) gene fragment and the X-pMTL-Pthl linearized vector were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies using CX-FXpk-F-1 and CX-FXpk(BD)-R JP750 as the primers (957 bp). The positive colonies were transferred and the plasmid was extracted. After gene sequencing verification, the recombinant plasmid was obtained: pMTL-Pthl-F/Xpk(ASR).
  
 
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Revision as of 13:20, 10 October 2023


F/Xpk(ASR)

This part is an ancestral F/Xpk gene predicted by ancestral sequence reconstruction (ASR), based on FXpk sequence (BBa_K4119076) derived from Clostridium acetobutylicum ATCC824. F/Xpk encodes phosphoketolase which is an enzyme with both the Fpk and Xpk activity. The enzyme catalyzes the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), and the conversion of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 779
    Illegal PstI site found at 1334
    Illegal PstI site found at 1928
    Illegal PstI site found at 1964
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 779
    Illegal PstI site found at 1334
    Illegal PstI site found at 1928
    Illegal PstI site found at 1964
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 779
    Illegal PstI site found at 1334
    Illegal PstI site found at 1928
    Illegal PstI site found at 1964
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 779
    Illegal PstI site found at 1334
    Illegal PstI site found at 1928
    Illegal PstI site found at 1964
  • 1000
    COMPATIBLE WITH RFC[1000]