Difference between revisions of "Part:BBa K3044027"
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<b>The Cas9 protein</b> | <b>The Cas9 protein</b> | ||
− | This Cas9 is codon optimized for use in ''E.coli'' (for the inactive endonuclease dCas9 see <partinfo>BBa_K3044027</partinfo>) with active catalytic domains RuvC and HNH [ | + | This Cas9 is codon optimized for use in ''E.coli'' (for the inactive endonuclease dCas9 see <partinfo>BBa_K3044027</partinfo>) with active catalytic domains RuvC and HNH [https://www.addgene.org/crispr/guide/]. Cas9 has endonuclease activity and induces a double stranded break in the target DNA sequence. The protein forms a complex with the single guide RNA (sgRNA) which is responsible for target DNA identification. The sgRNA is designed according to the PAM sequence, a three nucleotide sequence in the target DNA which is crucial to Cas9 activity. The Cas9/sgRNA-system can be used for knockout of specific target genes. The Cas9 protein is expressed with an IPTG inducible promoter (<partinfo>BBa_R0011</partinfo>) to enable control of Cas9 expression. |
<b>The sgRNA design</b> | <b>The sgRNA design</b> | ||
− | The sgRNA is designed to targeted at position 75-95 on the template strand of ''gfp'' (<partinfo>BBa_E0040</partinfo>) with the sequence 5’-CAAATTTTCTGTCAGTGGAG-3’ binding adjacent to PAM sequence AGG. The sgRNA is composed of the sgRNA handle [ | + | The sgRNA is designed to targeted at position 75-95 on the template strand of ''gfp'' (<partinfo>BBa_E0040</partinfo>) with the sequence 5’-CAAATTTTCTGTCAGTGGAG-3’ binding adjacent to PAM sequence AGG. The sgRNA is composed of the sgRNA handle [https://www.ncbi.nlm.nih.gov/pubmed/24136345] and the 20 nucleotide spacer sequence that targets the gene [https://www.ncbi.nlm.nih.gov/pubmed/26670017]. The sgRNA handle binds the Cas9 protein and the 20 nucleotides base pairs to the gene sequence right before the PAM sequence. The sgRNA were expressed with a 1.00 constitutive active Anderson promotor (<partinfo>BBa_J23100</partinfo>). |
The part was tested in ''E. coli'' K12, Top10 containing a low copy number plasmid (<partinfo>pSB4C5</partinfo>) expressing ''gfp'' with a 0.86 constitutive Anderson promoter (<partinfo>BBa_K3044006</partinfo>) or with a 0.33 constitutive Anderson promoter (<partinfo>BBa_K3044002</partinfo>). The part was transformed into these ''E.coli'' expressing ''gfp''. We investigated this hypothesis by Fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The bacteria containing both the ''gfp'' plasmid and the Cas9/sgRNA plasmid were grown overnight with IPTG to fully induce the expression of Cas9. | The part was tested in ''E. coli'' K12, Top10 containing a low copy number plasmid (<partinfo>pSB4C5</partinfo>) expressing ''gfp'' with a 0.86 constitutive Anderson promoter (<partinfo>BBa_K3044006</partinfo>) or with a 0.33 constitutive Anderson promoter (<partinfo>BBa_K3044002</partinfo>). The part was transformed into these ''E.coli'' expressing ''gfp''. We investigated this hypothesis by Fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The bacteria containing both the ''gfp'' plasmid and the Cas9/sgRNA plasmid were grown overnight with IPTG to fully induce the expression of Cas9. | ||
Revision as of 01:10, 22 October 2019
sgRNA/Cas9 for gfp knockout
This part is an assembly of a sgRNA-Cas9 system that can be used for knockout of the gfp (BBa_E0040) gene expression. This system has been tested in E.coli K12, Top10
The Cas9 protein This Cas9 is codon optimized for use in E.coli (for the inactive endonuclease dCas9 see BBa_K3044027) with active catalytic domains RuvC and HNH [1]. Cas9 has endonuclease activity and induces a double stranded break in the target DNA sequence. The protein forms a complex with the single guide RNA (sgRNA) which is responsible for target DNA identification. The sgRNA is designed according to the PAM sequence, a three nucleotide sequence in the target DNA which is crucial to Cas9 activity. The Cas9/sgRNA-system can be used for knockout of specific target genes. The Cas9 protein is expressed with an IPTG inducible promoter (BBa_R0011) to enable control of Cas9 expression.
The sgRNA design The sgRNA is designed to targeted at position 75-95 on the template strand of gfp (BBa_E0040) with the sequence 5’-CAAATTTTCTGTCAGTGGAG-3’ binding adjacent to PAM sequence AGG. The sgRNA is composed of the sgRNA handle [2] and the 20 nucleotide spacer sequence that targets the gene [3]. The sgRNA handle binds the Cas9 protein and the 20 nucleotides base pairs to the gene sequence right before the PAM sequence. The sgRNA were expressed with a 1.00 constitutive active Anderson promotor (BBa_J23100). The part was tested in E. coli K12, Top10 containing a low copy number plasmid (pSB4C5) expressing gfp with a 0.86 constitutive Anderson promoter (BBa_K3044006) or with a 0.33 constitutive Anderson promoter (BBa_K3044002). The part was transformed into these E.coli expressing gfp. We investigated this hypothesis by Fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The bacteria containing both the gfp plasmid and the Cas9/sgRNA plasmid were grown overnight with IPTG to fully induce the expression of Cas9.
The fluorescence pictures of the E.coli WT, the target bacterium expressing gfp and the target bacteria that have received either the sgRNA or the Cas9/sgRNA system (figure 1A and figur 2A). The wildtype (WT) E.coli K12 Top10 is not fluorescent and the E.coli harboring the gfp gene, for both expression (gfp' 0.33 and gfp 0.86). The sgRNA is included as a control ensuring that sgRNA itself is not downregulates the fluorescent signal. In contrast, the fluorescence in the bacteria with our Cas9/sgRNA system is knocked out. The microscopy image of Cas9/sgRNA system only one bacteria is fluorescent for gfp 0.86, and no green colonies with gfp 033, illustrating a high efficiency of our system. Furthermore, the FACS histograms support the microscopy data. Thereby showing that our system can be used to knockout gfp. This biobrick can potentially be modified to target any other gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 478
Illegal BglII site found at 1552 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]