Difference between revisions of "Part:BBa K4002004"
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Name: pHCas9-Nours | Name: pHCas9-Nours | ||
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pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. This part is inserted into plasmid to get pHCas9-Nours plasmid (Figure 1). | pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. This part is inserted into plasmid to get pHCas9-Nours plasmid (Figure 1). | ||
[[File:T--Xiamen City--BBa K4002004-Figure1.png|500px|thumb|center|Figure 1. Schematic map of pHCas9-Nours plasmid.]] | [[File:T--Xiamen City--BBa K4002004-Figure1.png|500px|thumb|center|Figure 1. Schematic map of pHCas9-Nours plasmid.]] | ||
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1. Construction of CRISPR expression plasmids | 1. Construction of CRISPR expression plasmids | ||
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[[File:T--Xiamen City--BBa K4002004-Figure5.png|500px|thumb|center|Table 2. Measurement of PgaA concentration and unit of activity in various samples.]] | [[File:T--Xiamen City--BBa K4002004-Figure5.png|500px|thumb|center|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. | 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. | ||
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Latest revision as of 08:21, 21 October 2021
pHCas9-Nours
pHCas9-Nours
Profile
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).
Experimental approach
1. Construction of CRISPR expression plasmids
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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3177
- 1000COMPATIBLE WITH RFC[1000]