Difference between revisions of "Part:BBa K5300042"

 
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<partinfo>BBa_K5300042 parameters</partinfo>
 
<partinfo>BBa_K5300042 parameters</partinfo>
 
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        <p>We first constructed '<i>PglnK</i>-sgRNA-GFP-tracRNA-<i>PnifH</i>-<i>gfp</i>' (Regulatory sequence) through multiple overlap-PCR. Then through Gibson assembly, we connected the regulation module with PUFA1, PUFA2 with PUFA3, and PUFA4 with PUFA5 and inserted them into pUC19 respectively.We inserted all fragments of <i>pfa</i> biosynthetic gene cluster that cloned from recombinant plasmid by PCR into linearized pBBR1MCS-2 by Gibson assembly and examined by electrophoresis (Figure 1). Then we sequenced the above products and unfortunately found that there is a point mutation, which led to in a termination codon that appeared 113 amino acids early. There also existed a single frameshift mutation in <i>pfa2</i> gene. Due to the presence of mutations in the ultimate plasmid, we decided to insert the regulatory sequence and PUFA sequence into separate plasmids to verify their functions.</p>
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        <figcaption><b>Figure 1. Agarose gel electrophoresis of synthetic circuit.</b> Sample order: Marker (D5000), ultimate plasmid(Mutation present), Marker(1kb)</figcaption>
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Revision as of 20:28, 1 October 2024


PglnK-sgRNA-GFP-tracrRNA-PnifH-gfp-pfa biosynthetic gene cluster

The CRISPRi system is also available to the synthetic circuit, we used the Cas12k protein expressed in suicide circuit, which binds reversibly to target sequences under the guidance of sgRNA. When external signals reach a certain threshold, Cas12k inhibits transcription of corresponding sequence. Cas12k lacks cleavage activity, so it physically blocks transcription without cutting the target DNA sequence. As the intracellular concentration of sgRNA decreases, the bound Cas12k protein detaches from the target sequence, allowing normal transcription to resume when continuous repression is no longer needed. In the synthesis circuit, the expression of pfa biosynthetic gene cluster is also regulated by promoters nifH and glnK. Under high oxygen conditions (i.e., before the Sinorhizobium form a symbiotic relationship with the plant), the expression of pfa biosynthetic gene cluster is inhibited; when the nodule structure is formed, the low oxygen environment activates the promoter nifH. However, in the early low nitrogen environment, the CRISPRi system is activated to suppress the expression of pfa biosynthetic gene cluster. After nitrogen fertilizer is applied later, the promoter glnK is deactivated, and the pfa biosynthetic gene cluster will be expressed normally. This design ensures that PUFA is synthesized under specific conditions, preventing product loss and environmental pollution.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 13428
    Illegal NheI site found at 17592
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2785
    Illegal BglII site found at 3698
    Illegal BglII site found at 7521
    Illegal BglII site found at 8671
    Illegal BglII site found at 9543
    Illegal BglII site found at 12800
    Illegal BglII site found at 15332
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2218
    Illegal NgoMIV site found at 2347
    Illegal NgoMIV site found at 2845
    Illegal NgoMIV site found at 4710
    Illegal NgoMIV site found at 5768
    Illegal NgoMIV site found at 6325
    Illegal NgoMIV site found at 6401
    Illegal AgeI site found at 3046
    Illegal AgeI site found at 4873
    Illegal AgeI site found at 7372
    Illegal AgeI site found at 8072
    Illegal AgeI site found at 10268
    Illegal AgeI site found at 10595
    Illegal AgeI site found at 13700
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1623
    Illegal SapI site found at 13529
    Illegal SapI site found at 15500
    Illegal SapI site found at 16700
    Illegal SapI site found at 18255
    Illegal SapI.rc site found at 10824
    Illegal SapI.rc site found at 11729
    Illegal SapI.rc site found at 19033


We first constructed 'PglnK-sgRNA-GFP-tracRNA-PnifH-gfp' (Regulatory sequence) through multiple overlap-PCR. Then through Gibson assembly, we connected the regulation module with PUFA1, PUFA2 with PUFA3, and PUFA4 with PUFA5 and inserted them into pUC19 respectively.We inserted all fragments of pfa biosynthetic gene cluster that cloned from recombinant plasmid by PCR into linearized pBBR1MCS-2 by Gibson assembly and examined by electrophoresis (Figure 1). Then we sequenced the above products and unfortunately found that there is a point mutation, which led to in a termination codon that appeared 113 amino acids early. There also existed a single frameshift mutation in pfa2 gene. Due to the presence of mutations in the ultimate plasmid, we decided to insert the regulatory sequence and PUFA sequence into separate plasmids to verify their functions.

Figure 1. Agarose gel electrophoresis of synthetic circuit. Sample order: Marker (D5000), ultimate plasmid(Mutation present), Marker(1kb)