Team:Shanghai HS ID/Contribution

Shanghai_HS_ID

Contribution

BBa_K4099000

== Profile ==

Name: sgRNA
Base Pairs: 112bp
Origin: Lactobacillus casei, genome
Properties: A piece of RNA

== Usage and Biology ==

BBa_K4099000 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with.

Figure1. Principle diagram of CRISPR-Cas9.

The sgRNA and HR are inserted in the pLCNICK vector. (Figure 3).

BBa_K4099001

== Profile ==

Name: pNCas9
Base Pairs: 4107bp
Origin: Streptococcus pyogenes, Addgene
Properties: A dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system

== Usage and Biology ==

BBa_K4099001 is a coding sequence of Cas9, an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence

BBa_K4099004

== Profile ==

Name: LSEI-2094
Base Pairs: 1599bp
Origin: Lactobacillus casei, genome
Properties: A coding sequence of restriction endonuclease LSEI-2094

== Usage and Biology ==

This part is a coding sequence of LSEI-2094 from Lactobacillus casei. Restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

BBa_K4099006

== Profile ==

Name: pNCas9-LSEI-2094
Base Pairs: 14379 bp
Origin: Synthetic
Properties: CRISPR technology to kick out the DNA segment, LSEI-2094 gene.

== Usage and Biology ==

This part is a coding sequence of LSEI-2094 from Lactobacillus casei. Restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

Restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

The plasmid, pNCas9-LSEI-2094 equipped with a CRISPR-Cas9 complex in order to kick out the DNA segment, LSEI-2094 gene. This gene is involved in the synthesis of an enzyme that is essential in the restriction-modification system. After this modification, the restriction enzyme could be temporarily inactivated so that the transferred exogenous DNA could successfully avoid the restriction effect of the host bacteria restriction system.

== Construct design ==

Figure 2 shows the design of a CRISPR-Cas9-based gene knockout vector in L. casei ATCC 334. Single plasmid CRISPR-Cas9 system is applied, namely gRNA, Cas9 effector protein, and repair template on one plasmid.

Figure 2. CRISPR-Cas9-based gene knockout vector design in Lactobacillus casei ATCC 334. P is the promoter (promotor); T is the terminator (terminator); pNCas9 is the Cas effector protein; HA-L and HA-R are the left homology arm and the right homology arm, respectively.

sgRNA+HR+pNCas9 backbone is a key functional factor that kicks out the DNA segment, LSEI-2094 gene. The sgRNA and HR are inserted in the pLCNICK vector. (Figure 3 and 4).

Figure 3. Schematic map of pNCas9-LSEI-2094 expression plasmids.

Figure 4. A: DNA profile of the plasmid pLCNICK; B: DNA Profile of LSEI-2094+ upstream and downstream homologous arms.

BBa_K4099008

== Profile ==

Name: pIB165
Base Pairs: 4139 bp
Origin: Escherichia coli, Addgene
Properties: A E. coli - Streptococci shuttle plasmid for gene expression.

== Usage and Biology ==

The plasmid pIB165 is a E. coli-Streptococci shuttle plasmid for gene expression in streptococci with P23 promoter.