Part:BBa_K4002003

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. 2 and Fig. 3, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.

Table 2. 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. 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.

Table 3. 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]