Difference between revisions of "Part:BBa K4335020"
 
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BBa_ K4335020 is composed of a promoter, 3 × Flag is composed of NLS, SpCas9 protein and a terminator
 
BBa_ K4335020 is composed of a promoter, 3 × Flag is composed of NLS, SpCas9 protein and a terminator

Latest revision as of 08:23, 11 October 2022


HSP70A Promoter+Flag+NLS+SpCas9+NLS+Rbcs2 Term

BBa_ K4335020 is composed of a promoter, 3 × Flag is composed of NLS, SpCas9 protein and a terminator

HSP70A is a heat and light inducible promoter for use in Chlamydomonas reinhardtii × Flag serves as the label of our Cas9 protein and connects NLS to guide the protein to locate in the nucleus

Usage

HSP70A Promoter+3 × Flag+NLS+SpCas9+NLS+Rbcs2 Term is one of the core functional elements of plasmid pTX2038 and plasmid pTX2040. Its main function is to express Cas9 protein with special functions, and to act with pCrU6.3+insert site+SpScaffold+polyT Term [BBa_K4335021] to cut specific genes of the nuclear gene group of Chlamydomonas reinhardtii, so as to knock out lipid related negative regulatory genes

Result

We selected PSY1 gene as the target gene to detect the sgRNA transcription driven by U6.3 promoter according to the method of [1] . The plant endo synthase gene (PSY1) is involved in chlorophyll synthesis, and disruption of PSY1 produces white colonies that are easy to detect and count.

PSY1 was selected as the target gene.Detection of chlamydia U6 promoter (CrU6.3) drive sgRNA transcription.Rhine chlamydomonas wild type and guide

The color of the algae transferred into PSY1 was significantly lighter than that of the wild type, and the individuals of white algae also appeared. We can confirm that Cas9 plays a role in Chlamydomonas Rhein and that the Cru6.3 promoter plays a normal role.

Due to the time factor, we can not further analyze the function of Cas9 system. We will prove that Cas9 protein has successfully synthesized and cleaved the specific gene sequence by protein purification, DNA extraction and sequencing in Chlamydomonas reinhardtii.

Reference

[1] Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. Plant Cell 29, 2498–2518 (2017). Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 466
    Illegal PstI site found at 1012
    Illegal PstI site found at 2434
    Illegal PstI site found at 2638
    Illegal PstI site found at 2668
    Illegal PstI site found at 3880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 70
    Illegal PstI site found at 466
    Illegal PstI site found at 1012
    Illegal PstI site found at 2434
    Illegal PstI site found at 2638
    Illegal PstI site found at 2668
    Illegal PstI site found at 3880
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 473
    Illegal BglII site found at 1268
    Illegal BglII site found at 1958
    Illegal BglII site found at 4037
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 466
    Illegal PstI site found at 1012
    Illegal PstI site found at 2434
    Illegal PstI site found at 2638
    Illegal PstI site found at 2668
    Illegal PstI site found at 3880
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 466
    Illegal PstI site found at 1012
    Illegal PstI site found at 2434
    Illegal PstI site found at 2638
    Illegal PstI site found at 2668
    Illegal PstI site found at 3880
    Illegal NgoMIV site found at 1300
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 3693