Difference between revisions of "Part:BBa K3171181"

Revision as of 11:23, 21 October 2019

HisD gRNA 2

The variable components needed for a CRISPR mediated knockout are gRNA and a sequence for homologous recombination. gRNA and the homologous part have to be engineered strictly depending on the target. In this case, all parts were designed with the aim to knockout HisD in V. natriegens.

We designed 3 homologous parts Part:BBa_K3171177, Part:BBa_K3171178, Part:BBa_K3171179 that serve as a template for homologous recombination once the Cas9 enzyme has cut the target site. Consequently, it is designed according to the up- and downstream regions of the targeted sequence. 3 kbp, 1.5 kbp and 600 bp lengths for each up- and downstream of HisD were achieved using PCR and respective primers.

The gRNA enables the Cas9 enzyme to recognize and cut at the correct site. The Cas9 enzyme forms a complex with the gRNA and can then find the target sequence based on complementary base pairing. Parts Part:BBa_K3171180, Part:BBa_K3171181, Part:BBa_K3171182 have been designed to select suitable target site, considering e.g. possible off-targets or location within coding region. These parts have been ordered as single stranded oligonucleotides, annealed and ligated into a vector encoding the necessary compounds of CRISPR machinery.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K3171181 short</partinfo>
-We experimented with CRISPR with the aim to make a histidine auxotroph by knocking out HisD in both E. coli and V. natriegens. To make a histidine auxotroph by knocking out HisD in E. coli specific gRNA targets were selected for knockout of the HisD gene. We designed 3 homologous parts [[Part:BBa_K3171177]], [[Part:BBa_K3171178]], [[Part:BBa_K3171179]] in order to decide on the correct homologous part required for knockout. 3 gRNA target sites [[Part:BBa_K3171180]], [[Part:BBa_K3171181]], [[Part:BBa_K3171182]] were selected for knock out to determine the exact region for knockout.
+The variable components needed for a CRISPR mediated knockout are gRNA and a sequence for homologous recombination. gRNA and the homologous part have to be engineered strictly depending on the target. In this case, all parts were designed with the aim to knockout HisD in V. natriegens.
+We designed 3 homologous parts [[Part:BBa_K3171177]], [[Part:BBa_K3171178]], [[Part:BBa_K3171179]] that serve as a template for homologous recombination once the Cas9 enzyme has cut the target site. Consequently, it is designed according to the up- and downstream regions of the targeted sequence. 3 kbp, 1.5 kbp  and 600 bp lengths for each up- and downstream of HisD were achieved using PCR and respective primers.
+The gRNA enables the Cas9 enzyme to recognize and cut at the correct site. The Cas9 enzyme forms a complex with the gRNA and can then find the target sequence based on complementary base pairing. Parts [[Part:BBa_K3171180]], [[Part:BBa_K3171181]], [[Part:BBa_K3171182]] have been designed to select suitable target site, considering e.g. possible off-targets or location within coding region. These parts have been ordered as single stranded oligonucleotides, annealed and ligated into a vector encoding the necessary compounds of CRISPR machinery.