Difference between revisions of "Part:BBa K4800055"

 
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<partinfo>BBa_K4800055 short</partinfo>
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<partinfo>BBa_K4800013 short</partinfo>
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We used pACYCDuet-sfp-MmCAR-YahK as a template and amplified the PT7-YciA-Ptrc-sfp-MmCAR-Yahk fragment by PCR, and homologous recombination of PT7-YciA-Ptrc-sfp-MmCAR-Yahk with the linearized pRSFDuet vector was carried out by the In-fusion cloning method to yield plasmid of pRSFDuet-sfp-MmCAR-YahK.
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The plasmid of pRSFDuet-sfp-MmCAR-YahK were constructed to obtain the functional modules for 1,5-pentanediol.We co-transformed the plasmid of pTrc99a-davB-davA-GabT and pRSFDuet-sfp-MmCAR-YahK into a lysine producing strain <i>E. coli</i> NT1003 to  produce 1,5-PDO from glucose.
 
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===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4800055 SequenceAndFeatures</partinfo>
 
  
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===Functional Parameters===
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<partinfo>BBa_K4800055 parameters</partinfo>
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>Document</title>
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    <p style="font-size: 150%; font-weight: bold;">Build:
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    <div align="center">
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        <p><centre><img src="https://static.igem.wiki/teams/4800/wiki/parts/bba-k4800013-1.png" width="50%" height="30%"></centre></p>
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        <strong>Figure 1. The schematic of plasmid pRSFDuet-sfp-MmCAR-YahK.</strong>
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    <p>The genes of 1,5-PDO synthesis module including MmCAR and YahK were subcloned into the plasmid pACYCDuet together under the control of trc  promoter to yield plasmid pACYCDuet-sfp-MmCAR-YahK. In detail, based on the sequence of an existing part of <partinfo>BBa_K1655000</partinfo> , it was codon-optimized and synthesized to obtain the fragment of sfp-MmCAR, which was then ligated into the pACYCDuet plasmid under the control of trc  promoter. The Ptrc-YahK fragment was amplified with plasmid pTrc99a-YahK as the template, and In-Fusion cloning was then used to ligate into the above vectors to yield plasmid pACYCDuet-sfp-MmCAR-YahK. The plasmid were transformed into <i>E. coli</i> Trans-T1, and corrected colonies were verified by PCR, and sequencing.
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    </p >
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  <p style="font-size: 150%; font-weight: bold;">Test:
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    <div align="center">
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        <p><centre><img src="https://static.igem.wiki/teams/4800/wiki/parts/bba-k4800013-1.png" width="50%" height="30%"></centre></p>
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    </div>
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        <strong>Figure 2.

Revision as of 10:07, 12 October 2023

_NOTOC__ YcjQ sgRNA

The plasmid of pRSFDuet-sfp-MmCAR-YahK were constructed to obtain the functional modules for 1,5-pentanediol.We co-transformed the plasmid of pTrc99a-davB-davA-GabT and pRSFDuet-sfp-MmCAR-YahK into a lysine producing strain E. coli NT1003 to produce 1,5-PDO from glucose.

Document

Build:

Figure 1. The schematic of plasmid pRSFDuet-sfp-MmCAR-YahK.

The genes of 1,5-PDO synthesis module including MmCAR and YahK were subcloned into the plasmid pACYCDuet together under the control of trc promoter to yield plasmid pACYCDuet-sfp-MmCAR-YahK. In detail, based on the sequence of an existing part of BBa_K1655000 , it was codon-optimized and synthesized to obtain the fragment of sfp-MmCAR, which was then ligated into the pACYCDuet plasmid under the control of trc promoter. The Ptrc-YahK fragment was amplified with plasmid pTrc99a-YahK as the template, and In-Fusion cloning was then used to ligate into the above vectors to yield plasmid pACYCDuet-sfp-MmCAR-YahK. The plasmid were transformed into E. coli Trans-T1, and corrected colonies were verified by PCR, and sequencing.

Test:

Figure 2.