Difference between revisions of "Part:BBa K4002003"
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== Profile == | == Profile == | ||
Name: pYES2-gRNA-hyg-MCS | Name: pYES2-gRNA-hyg-MCS | ||
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Base Pairs: 388 bp | Base Pairs: 388 bp | ||
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Origin: From article, Addgene | Origin: From article, Addgene | ||
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Properties: A piece of RNA complementary to the target gene. | Properties: A piece of RNA complementary to the target gene. | ||
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== Construct design == | == Construct design == | ||
sgRNA is inserted into plasmid vector. Plasmid sequence map is shown in Figure 1. | sgRNA is inserted into plasmid vector. Plasmid sequence map is shown in Figure 1. | ||
− | [[File:T--Xiamen City--BBa K4002003-Figure1.png|500px|thumb|center|Figure 1. Schematic map of pYES2-gRNA-hyg-MCS plasmid | + | [[File:T--Xiamen City--BBa K4002003-Figure1.png|500px|thumb|center|Figure 1. Schematic map of pYES2-gRNA-hyg-MCS plasmid.]] |
== Experimental approach == | == Experimental approach == | ||
1. Construction of CRISPR expression plasmids | 1. Construction of CRISPR expression plasmids | ||
− | [[File:T--Xiamen City--BBa K4002003-Figure2.png|500px|thumb|center|Figure 2. Agarose gel electrophoresis of pYES2–gRNA-hyg-MCS (lanes 1 and 2) plasmids | + | [[File:T--Xiamen City--BBa K4002003-Figure2.png|500px|thumb|center|Figure 2. Agarose gel electrophoresis of pYES2–gRNA-hyg-MCS (lanes 1 and 2) plasmids.]] |
We designed a plasmid expressing gRNA (Fig. 2), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmid of pYES2–gRNA-hyg-MCS were extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments. | We designed a plasmid expressing gRNA (Fig. 2), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmid of pYES2–gRNA-hyg-MCS were extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments. | ||
Then we transformed the plasmids of pHCas9-Nours, pYES2–gRNA-hyg-MCS and the repair template DNA into fruit wine yeast. | Then we transformed the plasmids of pHCas9-Nours, pYES2–gRNA-hyg-MCS and the repair template DNA into fruit wine yeast. | ||
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== Proof of function == | == Proof of function == | ||
Pectinase activity assay | 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. | + | 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. |
− | [[File:T--Xiamen City--BBa K4002003-Figure4.png|500px|thumb|center|Table | + | [[File:T--Xiamen City--BBa K4002003-Figure4.png|500px|thumb|center|Table 1. Measurement of standard pectinase activities at different concentrations.]] |
− | [[File:T--Xiamen City--BBa K4002003-Figure6.png|500px|thumb|center|Figure 3. Standard curve of pectinase | + | [[File:T--Xiamen City--BBa K4002003-Figure6.png|500px|thumb|center|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. | + | 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. Unexpectedly, the concentration of PgaA in the soluble supernatants of sample 2 was higher than that of total cell lysates, this could be due to experimental mistakes. |
− | [[File:T--Xiamen City--BBa K4002003-Figure5.png|500px|thumb|center|Table | + | [[File:T--Xiamen City--BBa K4002003-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 10:15, 20 October 2021
pYES2-gRNA-hyg-MCS
pYES2-gRNA-hyg-MCS
Profile
Name: pYES2-gRNA-hyg-MCS
Base Pairs: 388 bp
Origin: From article, Addgene
Properties: A piece of RNA complementary to the target gene.
Usage and Biology
BBa_K4002003 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with. And this sequence is inserted into plasmid vector.
Construct design
sgRNA is inserted into plasmid vector. Plasmid sequence map is shown in Figure 1.
Experimental approach
1. Construction of CRISPR expression plasmids
We designed a plasmid expressing gRNA (Fig. 2), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmid of pYES2–gRNA-hyg-MCS were extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments. Then we transformed the plasmids of pHCas9-Nours, pYES2–gRNA-hyg-MCS and the 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. Unexpectedly, the concentration of PgaA in the soluble supernatants of sample 2 was higher than that of total cell lysates, this could be due to experimental mistakes.
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 134
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]