Difference between revisions of "Part:BBa K3044016"

Revision as of 10:14, 17 October 2019

Conjugative sgRNA-dCas9 system

Validation of sgRNA-dCas9 system incl. an oriT-R sequence

(Characterized by SDU-Denmark 2019)

SDU 2019 has assembled an sgRNA-dCas9 system designed to repress the expression of the gfp gene (BBa_E0040). This system is made transferable by conjugation between bacteria by the implementation of the oriT-R part(BBa_J01003). However, the presence of a compatible conjugative plasmid is required for the efficient delivery.

The dCas9 is a codon optimized version of BBa_K1150000 optimized for E. coli. It forms a complex with the single guide RNA (sgRNA), and together this system can be used for silencing of specific target genes. dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [1]

This sgRNA sequence targets residue 75-95 on the template strand in the GFP sequence and have an efficiency of 59.09. When forming a complex with the dCas9, the sgRNA guides the dCas9 to the appropriate target gene. The sgRNA is designed according to the location of the PAM sequence in the target gene, because the PAM sequence is recognized by and activates the dCas9. The sgRNA consists of 100 nucleotides of which 20 nucleotides base pair with the target DNA sequence and the rest is called a dCas9 handle and interacts with the dCas9 protein.

The plasmid is made conjugative by the insertion of OriT (BBa_J01003). This makes the sgRNA-dCas9 transferable between bacteria.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 478
Illegal BglII site found at 1552
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 4547
Illegal NgoMIV site found at 4557
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 4449

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3044016 short</partinfo>
-The assembled sgRNA-dCas9 system can be used for silencing of GFP (<partinfo>BBa_E0040</partinfo>) expression. This system is made transferable between bacteria by the implementation of OriT(<partinfo>BBa_J01003</partinfo>) in the plasmid.
+===Validation of sgRNA-dCas9 system incl. an oriT-R sequence===
+(Characterized by SDU-Denmark 2019)<br>
-This dCas9 is a codon optimized version of <partinfo>BBa_K1150000</partinfo> for expression in E. coli. It forms a complex with the single guide RNA (sgRNA), and together this system can be used for silencing of specific target genes.
+<b>SDU</b> 2019 has assembled an sgRNA-dCas9 system designed to repress the expression of the <I>gfp</i> gene (<partinfo>BBa_E0040</partinfo>). This system is made transferable by conjugation between bacteria by the implementation of the oriT-R part(<partinfo>BBa_J01003</partinfo>). However, the presence of a compatible conjugative plasmid is required for the efficient delivery.<br>
+The dCas9 is a codon optimized version of <partinfo>BBa_K1150000</partinfo> optimized for <i>E. coli</I>. It forms a complex with the single guide RNA (sgRNA), and together this system can be used for silencing of specific target genes.
 dCas9 is the inactive form of the endonuclease Cas9. In contrast to Cas9, dCas9 does not cleave the DNA. Instead, it sterically blocks the elongation of transcription as a result of the sgRNA-DNA interaction. This will only repress the expression of the target gene thus avoiding destruction of the gene. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/]