Difference between revisions of "Part:BBa K4335012"
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<h2>Usage</h2> | <h2>Usage</h2> | ||
− | pCrU6.3 was constructed into plasmid pTX2038 | + | pCrU6.3 was constructed into plasmid <a href="https://parts.igem.org/Part:BBa_K4335038">[pTX2038]</a> <a href="https://parts.igem.org/Part:BBa_K4335040">[pTX2040]</a> as the promoter of gRNA, and was successfully introduced into Chlamydomonas reinhardtii to obtain the positive transformant. |
<h2>Result</h2> | <h2>Result</h2> |
Latest revision as of 07:50, 11 October 2022
pCrU6.3
pCrU6.3 is a previously characterized RNA polymerase III promoter from the chromosome 8 of Chlamydomonas reinhardtii.
The first prerequisite for CRISPR/Cas9 experiments is sgRNA transcription under the control of the RNA polymerase III promoter (RNAPII) . In general, the RNAPII transcription factor binding site is intrinsic to the transcriptional sequence and therefore not amenable to sgRNA transcription. U6snRNA is one of the few examples of sgRNAs with proximal RNAPII promoter elements. Therefore, we used previously characterized U6snRNA sequences from chromosome 8.
Usage
pCrU6.3 was constructed into plasmid [pTX2038] [pTX2040] as the promoter of gRNA, and was successfully introduced into Chlamydomonas reinhardtii to obtain the positive transformant.Result
We selected PSY1 gene as the target gene to detect the sgRNA transcription driven by U6.3 promoter according to the method of { ref. 2} . 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. 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.In addition, we found that the promoter had never appeared in the iGEM database before, so we will use the results in the literature to append to it for future reference. (the following results are from literature [2])
Reference
[1Jakab, G., Mougin, A., Kis, M., Pollák, T., Antal, M., Branlant, C., and Solymosy, F. (1997). Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex. Biochimie 79: 387–395]
[2] 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 48
Illegal PstI site found at 121 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 48
Illegal PstI site found at 121
Illegal NotI site found at 453 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 48
Illegal PstI site found at 121 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 48
Illegal PstI site found at 121 - 1000COMPATIBLE WITH RFC[1000]