Contribution
BBa_K4002002
== Profile BBa_K4002002==
Name: HXK1
Base Pairs: 1458 bp
Origin: Saccharomyces cerevisiae, genome
Properties: An enzyme catalyze the phosphorylation of hexose
== Usage and Biology ==
This is a sequence coding enzyme HXK1. This enzyme can catalyze the phosphorylation of hexose to hexose 6-phosphate (D-glucose 6-phosphate and D-fructose 6-phosphate, respectively). Besides, it can mediate the initial step of glycolysis by catalyzing phosphorylation of D-glucose to D-glucose 6-phosphate.
BBa_K4002003
== 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.
BBa_K4002004
== Profile ==
Name: pHCas9-Nours
Base Pairs: 4837bp
Origin: Streptococcus pyogenes, Addgene
Properties: An endonuclease enzyme associated with the CRISPR.
== 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_K4002005
== Profile ==
Name: endo-pgaA
Base Pairs: 1113bp
Origin: Aspergillus niger SC323, genome
Properties: An enzyme degradation of pectin
==== 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. This part as a repair template DNA was connected with homology arm of HXK1.
BBa_K4002006
== Profile ==
Name: HR-L-endo-pgaA-HR-R
Base Pairs: 2135 bp
Origin: Ssynthetic
Properties: CRISPR technology repair template for build a type of multi-functional yeast
== 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. Pectin is a significant carbohydrate component that comprises plant cell walls. The brewer's yeast uses sugar in the fruit juice to produce alcohol, and pectinase destroys the pectin located in the cell wall to improve the juice yield and eliminate the cloudiness of the fruit wine. HR-L and HR-R are homology arm, refers to the flanking sequence on both sides of the HXK1.
== Construct design ==
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 1 and 2).
Figure 1. HR-L-endo-pgaA-HR-R box.
Figure 2. Schematic map of HR-L-endo-pgaA-HR-R.
BBa_K4002007
== Profile ==
Name: Cas9+gRNA+HR-L-endo-pgaA-HR-R
Base Pairs: 7360 bp
Origin: Synthetic
Properties: CRISPR technology build a type of multi-functional yeast
== 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.
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