New Improved Part: BBa_K4876011 (p15A-Cas9-λ-Red)
Old Existing Part by Team iGEM17_Shenzhen_SFLS: BBa_K2200005 (Cas9)
Based on BBa_K2200005(Cas9), we constructed a new plasmid BBa_ K4876011(p15A-Cas9-λ-Red ) with replicon p15A and self-cleavage gRNA. The main purpose of p15A replicon is improved editing efficiency. Self-cutting gRNA mainly avoid the impact on the environment and protect the privacy of our products. Data supplement is carried out in the following two aspects:
1. Cas9 Protein expression in the E.coil BL21
2. Verification of editing efficiency at gntT and lacZ in Escherichia coil Nissle 1917
Cas9, originally named Csn1, is the large, multifunctional signature protein of type II CRISPR/Cas systems. It is well known even to general audiences because its RNA-guided endonuclease activity has made it a popular tool for custom editing of eukaryotic genomes.
Using CRISPR-Cas9, scientists can study the function of specific genes by selectively editing them. By observing the phenotypic changes in edited cells or organisms, it is possible to uncover the roles of genes in biological processes, enhancing our understanding of fundamental biological mechanisms1.
Many cancers are caused by genetic mutations, and CRISPR-Cas9 technology can be used to study the mechanisms of cancer development and develop new therapeutic methods. It can be used to knock out, add, or correct mutated genes to simulate and study the roles of cancer-related genes2-4.CRISPR-Cas9 technology can be applied in the field of agriculture to help improve crops. Scientists can use this technology to enhance traits such as disease resistance, salt tolerance, yield, quality, and nutritional value in crops, thereby increasing food production efficiency and quality5-8.CRISPR-Cas9 has wide applications in biotechnology and synthetic biology. It can be used to create model organisms with specific gene modifications, enabling the study of the functionality of complex biological systems. Additionally, it can be used for strain improvement in industrial production of microbes, such as the production of specific compounds or bioenergy9-11..
BBa_K4876011(p15A-Cas9-λ-Red) is composed of BBa_K2200005(Cas9), BBa _K4876003 ( λ-Red ), and BBa _K4876015 ( p15A). . The main purpose of p15A replicon is improved editing efficiency. Self-cutting gRNA mainly avoid the impact on the environment and protect the privacy of our products. Data supplement is carried out in the following two aspects:
1. A new plasmid BBa _ K4876011 ( p15A-Cas9-λ-Red ) was constructed.
2. Cas9 was expressed in E.coli (DE3).
The original Cas9 encoding plasmid has a lower copy of pSC101 origin. We selected the higher copy p15A origin to increase Cas9 expression. This allows the retention of functional Cas9 during host editing to ensure normal CRISPR-Cas system operation, reducing escape rates and improving editing efficiency. In addition, the incorporation of the λ-Red recombination system can enhance editing efficiency, so we constructed the p15A-Cas9-λ-Red plasmid (Figure 1).
Figure 1 Engineering frame of the p15A-Cas9-λ-Red plasmid.
To construct p15A-Cas9-λ-Red, we amplified p15A and Cas9-λ-Red using primers with homology arms and recombined them using a cloning kit to generate p15A-Cas9-λ-Red. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the p15A-Cas9-λ-Red plasmid (Figure 2).
Figure 2 The construction results of p15A-Cas9-λ-Red.
(A) Colony PCR results. (B) Sequencing results.
To verify Cas9 expression in E. coli Nissle 1917, we transformed the constructed p15A-Cas9-λ-Red plasmid. After inducing expression, we lysed the cells by sonication and purified Cas9. The results showed that we successfully induced Cas9 protein expression (Figure 3).
Figure 3 The SDS-PAGE result of p15A-Cas9-λ-Red protein expression.
Subsequently, we tested the induction of Cas9 expression by different concentrations of L-arabinose. The results showed that with higher concentrations of L-arabinose, the expression of Cas9 was higher, suggesting that we could control the level of Cas9 by regulating the addition of L-arabinose (Figure 4).
Figure 4 Results of Cas9 protein induction by L-arabinose.
(A) Standard curve of BSA concentration. (B) The curve of Cas9 concentration.
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