Part:BBa_K4876011

p15A-cas9

p15A-cas9

Composite Part: BBa_K4876011 (p15A-Cas9-λ-Red)

Summary

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

Construction Design/Engineering Principle

BBa_K4876011 (p15A-Cas9-λ-Red) is composed of BBa_K2200005 (Cas9), BBa_K4876005 (λ-Red), and BBa_K4876015 (p15A). The main purpose of p15A replicon is improved editing efficiency. Self-cutting gRNA mainly avoids the impact on the environment and protects 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.

Experimental Approach

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.

Characterization/Measurement

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 157
  Illegal NheI site found at 2638
  Illegal NheI site found at 10793
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4914
  Illegal BamHI site found at 5774
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5800
  Illegal NgoMIV site found at 9890
  Illegal NgoMIV site found at 10799
  Illegal AgeI site found at 243
  Illegal AgeI site found at 567
  Illegal AgeI site found at 6123
  Illegal AgeI site found at 12651
  Illegal AgeI site found at 13456
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 10918
  Illegal SapI.rc site found at 8309
  Illegal SapI.rc site found at 8551