Difference between revisions of "Part:BBa K4335021"
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<h2>Result</h2>
 
<h2>Result</h2>
 
<h3>Verification of the effect of Golden Gate inserting specific sgRNA<h3>
 
<h3>Verification of the effect of Golden Gate inserting specific sgRNA<h3>
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The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.
 
The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.
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     <figure>
 
     <figure>
 
         <img src="https://static.igem.wiki/teams/4335/wiki/zhanghang-insert-site-grna-scaffold.jpg" width="100%" style="float:center">
 
         <img src="https://static.igem.wiki/teams/4335/wiki/zhanghang-insert-site-grna-scaffold.jpg" width="100%" style="float:center">

Revision as of 03:54, 11 October 2022


pCrU6.3+insert site+SpScaffold+polyT Term

BBa_ K4335021 consists of a terminator, a cloning site, SpScaffold and a terminator PCrU6.3 is a previously characterized RNA from chromosome 8 of Chlamydomonas reinhardtii [1]

Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.

sgRNA

Usage

pCrU6.3+insert site+SpScaffold+polyT Term is one of the core functional components of plasmid pTX2038 and pTX2040 Its main function is to express specific sgRNA, and combine with HSP70A Promoter+3×Flag+NLS+SpCas9+NLS+Rbcs2 Term [BBa_K4335020]works together to cut specific genes in the nuclear genome of Chlamydomonas reinhardtii, so as to knock out negative lipid related regulatory genes.

Result

Verification of the effect of Golden Gate inserting specific sgRNA

The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.

Gel run of samples from colony PCR.Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs is designed.

Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 48
    Illegal PstI site found at 121
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 48
    Illegal PstI site found at 121
    Illegal NotI site found at 453
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 48
    Illegal PstI site found at 121
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 48
    Illegal PstI site found at 121
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 551
    Illegal BsaI.rc site found at 533