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| + | <section class="article p-t-30 p-b-54"> |
| + | <h1 class="content-header">Proof of Concept</h1> |
| + | |
| + | <section> |
| + | <h1 class="title">Overview </h1> |
| + | <p>Alcohol has been used for thousands of years. The application areas of alcohol penetrate through |
| + | people’s lives, ranging from cosmetics to medical treatment, and drinks, etc. The earliest evidence |
| + | of alcoholic beverages has been found dating from 5400-5500 BC. In current society, alcoholic drinks |
| + | also influence our daily life deeply. As one of the major alcoholic drinks, fruit wines have |
| + | gradually attracted more and more attention. According to data released by JD. Com (One of the |
| + | largest E-Commerce platforms in China), there have been over 300 fruit wine sellers on JD. Com in |
| + | 2019, whose sales have endured over 200% compound increase rate from 2014 to 2019. Therefore, our |
| + | project focuses on the fruit wine industry, hoping to contribute to optimizing the production |
| + | process, lower production costs, and enhance fruit wine quality. </p> |
| + | <p>The preparation process of traditional fruit wines is expensive and cumbersome. This is probably the |
| + | greatest efficiency issue facing fruit wine today in the production process. During our research, we |
| + | discovered that majority of the pectinases available on the market are complex enzymes obtained from |
| + | Aspergillus fermentation, and the price per 1 kg of pectinase varies from 100-250 RMB. The high cost |
| + | of pectinase is not conducive to the further development of the food manufacturing industry and |
| + | other fields.</p> |
| + | <p>We want to apply CRISPR-Cas technology to successfully heterologously express endo-pgaA, an |
| + | endogalacturonase gene from Aspergillus niger SC323, in fructooligosaccharide yeast. So, we can |
| + | obtain <b>a strain that is capable of both pectin degradation and alcohol fermentation</b>. This |
| + | will help to explore the development of multifunctional fruit wine yeast and reduce the costs in the |
| + | production of fruit juice and fruit wine. It will also contribute to the development of food |
| + | industry production and modern brewing engineering.</p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Supporting Experiment Results</h1> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Yeast strain transformation and positive transformants verification</h1> |
| + | <p>The constructed CRISPR plasmids and repair template DNA were chemically transformed into the <i>S. |
| + | cerevisiae</i> strains. The positive transformants were selected against YPD medium supplemented |
| + | with Nours and hygromycin. The resulting colonies were picked up and cultured. To investigate |
| + | whether the PgaA gene was integrated into yeast genome, we performed PCR experiments using the |
| + | upstream and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. |
| + | 3A, we obtained specific PCR products with expected size of ~1500 bp. The DNA fragments were then |
| + | extracted and purified for sequencing. The sequencing results finally confirmed that the PgaA gene |
| + | was successfully integrated into <i>S. cerevisiae</i> genome (Fig. 1). </p> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/7/72/T--Xiamen_City--img_proof_of_concept_1.jpg" alt="" style="width: 100%"> |
| + | <p style="text-align: center"><b>Fig. 1 Verification of PgaA containing transformants.</b> (A) |
| + | Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis.</p> |
| + | </div> |
| + | <br> |
| + | <p>To obtain an optimal culture time, we monitored the growth rate of recombinant S. cerevisiae cells |
| + | from 2 to 72 h. As shown in Table.1, the OD<sub>600</sub> of culture increased from 2 h and reached |
| + | a plateau at 48 h, indicating that the optimal culture time was 48 h.</p> |
| + | <div class="img-container"> |
| + | <span class="figure">Table. 1 Growth rate of PgaA expressing cells</span> |
| + | <img src="https://static.igem.org/mediawiki/2021/d/d0/T--Xiamen_City--img_proof_of_concept_2.jpg" alt="" style="width: 100%"> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">2. Pectinase activity assay</h1> |
| + | <p>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. 4, the concentration of enzyme correlates well |
| + | with the absorbance detected at 540 nm, applying to the Lambert-Beer law. </p> |
| + | <div class="img-container"> |
| + | <span class="figure">Table. 2 Measurement of standard pectinase activities at different concentrations</span> |
| + | <img src="https://static.igem.org/mediawiki/2021/9/99/T--Xiamen_City--img_proof_of_concept_3.jpg" alt="" style="width: 100%"> |
| + | </div> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/b/b8/T--Xiamen_City--img_proof_of_concept_4.jpg" alt="" style="width: 80%"> |
| + | <span class="figure">Fig. 2 Standard curve of pectinase.</span> |
| + | </div> |
| + | <p>With this standard curve, we next determined the concentration of PgaA from recombinant <b><i>S. |
| + | cerevisiae</i> strains</b>. 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. </p> |
| + | <div class="img-container"> |
| + | <span class="figure">Table. 3 Measurement of PgaA concentration and unit of activity in various samples</span> |
| + | <img src="https://static.igem.org/mediawiki/2021/8/82/T--Xiamen_City--img_proof_of_concept_5.jpg" alt="" style="width: 100%"> |
| + | </div> |
| + | <p>The experiment results of Pectinase activity assay indicate that we have successfully obtained a |
| + | <b>gene-edited yeast that is capable of both pectin degradation and alcohol fermentation</b>. It |
| + | will serve |
| + | as a solid foundation for our future research and development of wine yeast. Surely, we know that it |
| + | is essential to make further experiments to enhance the pectin degradation capacity of our yeast, we |
| + | have already obtained a good start. </p> |
| + | </section> |
| + | </section> |
| + | </div> |
| + | </div> |
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