Part:BBa_K4002007

Cas9+gRNA+HR-L-endo-pgaA-HR-R

Cas9+gRNA+HR-L-endo-pgaA-HR-R

Profile

Name: Cas9+gRNA+HR-L-endo-pgaA-HR-R

Usage and Biology

Saccharomyces cerevisiae is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. In fruit wine production, Saccharomyces cerevisiae uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are specific regions in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes, function as an adaptive immune system in prokaryotes. While the specific ‘adaptive’ nature of this immunity is still under investigation, it is known that exogenous DNA is processed by Cas proteins into short (~30 base pair) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to form spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs or pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and act to guide them to initiate the sequence-specific cleavage of the target sequence.

Figure 1. The principle of CRISPR Cas9.

Construct design

pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. sgRNA is a sequence of RNA participant in CRISPR. HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 2).

Figure 2. Cas9+gRNA+HR-L-endo-pgaA-HR-R box.

The profiles of every basic part are as follows:

BBa_K4002000

Usage and Biology

This is a coding sequence of left homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region. system (T6SS).

BBa_K4002001

Usage and Biology

This is a coding sequence of right homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region.

BBa_K4002005

Usage and Biology

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase.

BBa_K4002004

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.

BBa_K4002003

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.

Experimental approach

1. Construction of CRISPR expression plasmids The PgaA is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid residues present in polygalacturonan in plant cell walls and therefore facilitate plant cell wall breakdown.  In the production of fruit wine, pectinase has been used to destroy the pectin in the cell wall in order to improve the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes.

Figure 3. Construction CRISPR expression plasmids. (A) Schematic representation of CRISPR expression vectors; (B) Agarose gel electrophoresis of pHCas9-Nours (lanes 1 and 2) and pYES2–gRNA-hyg-MCS (lanes 3 and 4) plasmids.

We sought to integrate PgaA gene into S. cerevisiae genome based on the CRISPR technology in order to obtain yeast strains that not only produces alcohol but can also decompose pectin. To this end, we designed two plasmids expressing Cas9 and gRNA (Fig. 3A), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmids of pHCas9-Nours and pYES2–gRNA-hyg-MCS were extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments.

2. Construction of repair template The repair template DNA containing PgaA gene (Fig. 4A) was generated by the overlap-PCR method. Firstly, the DNA fragments of upstream and downstream homologous regions were amplified with ~20 bp ends overlapping to the PgaA gene, producing ~500 bp PCR products (Fig. 4B). Secondly, the two fragments were annealed to the 5’- and 3’-ends of PgaA. Finally, the annealed products were further amplified using end primers of HR-L and HR-R, which resulted in a fragment of 1.5 kbas verified by agarose gel electrophoresis and DNA sequencing (Figs. 4C and 4D).

Figure 4. Construction of repair template. (A) Schematic representation of repair template; (B) Agarose gel electrophoresis of PCR products; (C) DNA sequencing result analysis.

3. Yeast strain transformation and positive transformants verification The constructed CRISPR plasmids and repair template DNA were chemically transformed into the S. cerevisiae 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. 5A, 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 S. cerevisiae genome (Fig. 5B).

Figure 5. Verification of PgaA containing transformants. (A) Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis.

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. 6, 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 6. 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.

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.

Improvement of an existing part

Compared to the old part BBa_K1051900, G1 phase magic with cell synchronization device and CRISPRi induced alternative splicing device, we design a new part BBa_K4002007, which contains the HCas9 protein. The HCas9 protein is a human optimized Streptococcus pyogenes Cas9. And its N and C terminal are inserted with SV40 NLS peptide.

The group iGEM13_Shenzhen_BGIC_ATCG wish to grasp the usage of cell cycle tools, and they wanted to direct refined actions in a cell by CRISPR Cas9 technology.

Our team design the new composite part BBa_K4002007 to express HCas9, with sequence different from the old one BBa_K1051900 (Figure 7). We thought of using CRISPR-Cas9 technology to achieve heterologous expression of endo-pgaA, an endo-galacturonase gene from Aspergillus niger SC323, in fruit wine yeast to obtain a strain that can both degrade pectin and ferment alcohol.

Figure 7. The blast results about the sequence of our new part BBa_K4002007 and the old one BBa_K1051900.

Our project will help explore the development of multifunctional fruit wine yeast, reduce the production cost of fruit juice and fruit wine, and contribute to the development of food industry production and modern brewing engineering.

References

1.Nishimasu, H., et al. Cell. 2014

2.James E.D., et al. Nucleic Acids Res. 2013

3.https://www.asme.org/topics-resources/content/8-ways-crisprcas9-can-change-world

4.https://www.nature.com/articles/s41599-019-0319-5

5.https://www.sciencedirect.com/topics/food-science/fruit-wine

6.崔凯宇,李迎秋.果胶酶生产和应用的研究进展[J].江苏调味副食品,2016(01):11-13.

7.Yang J , Luo H , Jiang L , et al. Cloning, expression and characterization of an acidic endo-polygalacturonase from Bispora sp. MEY-1 and its potential application in juice clarification[J]. Process Biochemistry, 2011, 46(1):272-277.

8.李烨青. 真菌来源的嗜热果胶酶基因挖掘及其催化效率的改造[D].江西农业大学,2017.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4971
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 688
  Illegal BglII site found at 1625
  Illegal BglII site found at 6156
  Illegal BglII site found at 6714
  Illegal BamHI site found at 2443
  Illegal XhoI site found at 3981
  Illegal XhoI site found at 4584
  Illegal XhoI site found at 6674
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3177
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 6413
  Illegal BsaI.rc site found at 5915
  Illegal BsaI.rc site found at 6785