Part:BBa_K4002004

pHCas9-Nours

pHCas9-Nours

BBa_K4002004

Name: pHCas9-Nours

Base Pairs: 4837bp

Origin: Streptococcus pyogenes, Addgene

Properties: An endonuclease enzyme associated with the CRISPR.

Usage and Biology

This is a sequence coding pHCas9 protein. This protein is a dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system. The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. The genes that encode the Cas9 protein and sgRNA were introduced into a cell and programmed to change its target gene. sgRNA has regions that are complementary to the target sequence. A complex consisting of sgRNA and Cas9 protein is formed inside the cell and binds to target sites.

Construct design

pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. This part is inserted into plasmid to get pHCas9-Nours plasmid (Figure 1).

Figure 1. Schematic map of pHCas9-Nours plasmid.

1. Construction of CRISPR expression plasmids

Figure 2. Agarose gel electrophoresis of pHCas9-Nours (lanes 1 and 2).

We designed a plasmid expressing Cas9 (Fig. 2). The agarose gel electrophoresis results indicated that the plasmid of pHCas9-Nours was extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments. We transformed the plasmids of pHCas9-Nours, pYES2–gRNA-hyg-MCS and repair template DNA into fruit wine yeast.

Proof of function

Pectinase activity assay The pectinase activities of PgaA were determined using the dinitrosalicylic acid (DNS) colorimetric method. Briefly, in the presence of PgaA, pectin can be degraded into galacturonic acids, which reacts with DNS to form a compound with a maximum absorption at 540 nm. Thus, the activity of PgaA can be calculated by measuring the absorbance of the reactants with a spectrophotometer. For accurate quantification, a standard curve was generated using a series of concentrations of pectinase standards. As shown in Table. 1 and Fig. 3, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.

Table 1. Measurement of standard pectinase activities at different concentrations.

Figure 3. Standard curve of pectinase.

With this standard curve, we next determined the concentration of PgaA from recombinant S. cerevisiae strains. Samples from the culture media, total cell lysates and the soluble portion of cell lysates were collected and subjected to DNS colorimetric assay. As shown in Table. 2, the concentration of PgaA in the culture media of sample -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml, respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml), suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most of the PgaA in cells are soluble.

Table 2. Measurement of PgaA concentration and unit of activity in various samples.

We successfully prepared genetically engineered wine yeast strain which contains pectinase in its genome. The pectinase produced from yeast well degrade pectin into small sugars.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 688
  Illegal BglII site found at 1625
  Illegal BamHI site found at 2443
  Illegal XhoI site found at 3981
  Illegal XhoI site found at 4584
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3177
  
• 1000
  COMPATIBLE WITH RFC[1000]