Difference between revisions of "Part:BBa K4586006"

Latest revision as of 13:39, 12 October 2023

Guide RNA

Part Description

Our guide RNA part is a specific RNA fragment that navigates the Cas system to the RNA or DNA target of interest according to its family and serves to make some changes, such as deletion, insertion, or alteration, to the target RNA or DNA by building complexes with them.

Usage

Our guide RNA is designed to navigate the Cas nuclease toward its specific target gene of interest, to knock-out the BAFF-R gene of auto-reactive B-Cell which is the gene responsible for the survival and maintenance of B-Cell that leads to apoptosis, and limit its pathogenic effect in RA as shown in figure 1.

Figure 1: This figure illustrates the activity of the Cas12k protein into the target autoreactive B-cells through knocking down the B-cell activating factor receptor (BAFF-R) gene that is responsible for B-cells survival and proliferation leading to their apoptosis.

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P7) including Guide RNA and Human U6 promoter, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.

We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P7).

After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.

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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+After the ligation step, we cultured the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the pCDNA3(-) plasmid vector containing insert parts.
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