Difference between revisions of "Part:BBa K4002003"

(Proof of function)
(Proof of function)
 
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== Profile ==
 
== Profile ==
 
Name: pYES2-gRNA-hyg-MCS
 
Name: pYES2-gRNA-hyg-MCS
 +
 
Base Pairs: 388 bp
 
Base Pairs: 388 bp
 +
 
Origin: From article, Addgene
 
Origin: From article, Addgene
 +
 
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..]]
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[[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. 2 and Fig. 3, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.  
+
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 2. Measurement of standard pectinase activities at different concentrations.]]
+
[[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-Figure3.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. 3, 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.  
+
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 3. Measurement of PgaA concentration and unit of activity in various samples.]]
+
[[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.
 
 
 
 
 
 
 
 
 
 
  
  

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.

Figure 1. Schematic map of pYES2-gRNA-hyg-MCS plasmid.

Experimental approach

1. Construction of CRISPR expression plasmids

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. 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.

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. 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.

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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 134
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]