Difference between revisions of "Part:BBa K3875010"
 
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<strong>Design：</strong>
 
<strong>Design：</strong>
 
Contained gene:  
 
Contained gene:  
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<i>pcd</i> (BBa_K3875005),  
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<i>pcd</i> (<a href = "https://parts.igem.org/Part:BBa_K3875005" > BBa_K3875005</a>),  
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<i>p4h</i> (BBa_K3875004),  
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<i>p4h</i> (<a href = "https://parts.igem.org/Part:BBa_K3875004" > BBa_K3875004</a>),  
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<i>trpB</i> <strong>(BBa_K187029)</strong><br>
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<i>trpB</i> <strong>(<a href = "https://parts.igem.org/Part:BBa_K187029" > BBa_K187029</a>)</strong><br>
  
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This composite part contains three genes, of which <i>p4h</i> and <i>trpB</i> are the genes involved in the last two steps of 5-HTP synthesis. <i>trpB</i> encodes the beta subunit of tryptophan synthase (react as <strong>Figure 1.</strong>).  
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This composite part contains three genes, of which <i>p4h</i> and <i>trpB</i> are the genes involved in the last two steps of 5-HTP synthesis. <i>trpB</i> encodes the beta subunit of tryptophan synthase (react as <strong>Figure 1.</strong>). <br>
  
 
<img src="https://static.igem.org/mediawiki/parts/3/3e/T--BUCT--The_reaction_trpB_gene_action.png"width="640px";height="30px"/><br>
 
<img src="https://static.igem.org/mediawiki/parts/3/3e/T--BUCT--The_reaction_trpB_gene_action.png"width="640px";height="30px"/><br>
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<img src="https://static.igem.org/mediawiki/parts/5/5b/T--BUCT--MH4_Recycle._PCD%2C_pterin-4%CE%B1-carbinolamine_dehydratase%3B_DHMR%2C_dihydromonapterin_reductase%3B_P4H%2C_phenylalanine_4-hydroxylase.png"width="640px";height="30px"/><br>
 
<img src="https://static.igem.org/mediawiki/parts/5/5b/T--BUCT--MH4_Recycle._PCD%2C_pterin-4%CE%B1-carbinolamine_dehydratase%3B_DHMR%2C_dihydromonapterin_reductase%3B_P4H%2C_phenylalanine_4-hydroxylase.png"width="640px";height="30px"/><br>
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These three genes are almost the end steps of the product 5-HTP, so we put these three genes on a single plasmid. The plasmid map is shown in <strong>Figure 3</strong>. <br>
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<img src="https://static.igem.org/mediawiki/parts/7/7f/T--BUCT--The_plasmid_contains_pcd%2C_trpB_and_p4h.png"width="640px";height="30px"/><br>
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<strong>Method:</strong>
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Three genes (<i>pcd</i>,<i> p4h</i>, <i>trpB</i>) were obtained by PCR amplification, and the recombinant plasmids were obtained after 1h reaction with pCS27 plasmids at 50 ℃ with homologous recombinase. Finally, plasmid transfer into <i>E.coli</i> DH5a.
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After bacterial culture, we extracted the plasmid from <i>E.coli</i> DH5a. Restriction enzyme HindIII and BamHI were used for enzyme digestion verification of plasmids, and the results were shown in <strong>Figure 4</strong> (succeeded). <br>
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<strong>Result:</strong>
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The result of agarose gel electrophoresis proves that our plasmid has been successfully constructed (Figure 4).<br>
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<img src="https://static.igem.org/mediawiki/parts/a/ad/T--BUCT--Enzyme_validation.png"width="640px";height="30px"/><br>
  
 
</html>
 
</html>

Latest revision as of 16:28, 19 October 2021


Usage and Biology

Design： Contained gene: pcd ( BBa_K3875005), p4h ( BBa_K3875004), trpB ( BBa_K187029)
This composite part contains three genes, of which p4h and trpB are the genes involved in the last two steps of 5-HTP synthesis. trpB encodes the beta subunit of tryptophan synthase (react as Figure 1.).

The purpose of inserting genes of PCD and P4H into the plasmid is to construct an artificial MH4 cycle, for which the main form of pterin in E.coli is MH4 [1]. And dihydromonapterin reductase (DHMR) that is from E. coli itself is together with PCD and P4H to form the MH4 cycle (as Figure. 2).

These three genes are almost the end steps of the product 5-HTP, so we put these three genes on a single plasmid. The plasmid map is shown in Figure 3.

Method: Three genes (pcd, p4h, trpB) were obtained by PCR amplification, and the recombinant plasmids were obtained after 1h reaction with pCS27 plasmids at 50 ℃ with homologous recombinase. Finally, plasmid transfer into E.coli DH5a. After bacterial culture, we extracted the plasmid from E.coli DH5a. Restriction enzyme HindIII and BamHI were used for enzyme digestion verification of plasmids, and the results were shown in Figure 4 (succeeded).
Result: The result of agarose gel electrophoresis proves that our plasmid has been successfully constructed (Figure 4).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 409
  • 1000
    COMPATIBLE WITH RFC[1000]