Difference between revisions of "Team:Shanghai HS ID/Contribution"

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        <div class="sub-title">Contribution</div>
 +
        <div class="article-title">BBa_K4099000</div>
 +
        <div class="article-content"><b>== Profile ==</b></div>
 +
        <div class="article-title">
 +
            Name: sgRNA<br />
 +
            Base Pairs: 112bp<br />
 +
            Origin: Lactobacillus casei, genome<br />
 +
            Properties: A piece of RNA
 +
        </div>
 +
        <div class="article-content"><b>== Usage and Biology ==</b></div>
 +
        <div class="article-content">BBa_K4099000 is a piece of RNAs that function as guides for RNA- or DNA-targeting
 +
            enzymes, which they form complexes with.</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/b/b4/T--Shanghai_HS_ID--contribution01.jpg" alt="" />
 +
            <span>Figure1. Principle diagram of CRISPR-Cas9.</span>
 +
        </div>
 +
        <div class="article-content">The sgRNA and HR are inserted in the pLCNICK vector. (Figure 3).</div>
 +
        <div class="article-title">BBa_K4099001</div>
 +
        <div class="article-content"><b>== Profile ==</b></div>
 +
        <div class="article-title">
 +
            Name: pNCas9<br />
 +
            Base Pairs: 4107bp<br />
 +
            Origin: Streptococcus pyogenes, Addgene<br />
 +
            Properties: A dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system
 +
        </div>
 +
        <div class="article-content"><b>== Usage and Biology ==</b></div>
 +
        <div class="article-content">
 +
            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
 +
        </div>
 +
        <div class="article-title">BBa_K4099004</div>
 +
        <div class="article-content"><b>== Profile ==</b></div>
 +
        <div class="article-title">
 +
            Name: LSEI-2094<br />
 +
            Base Pairs: 1599bp<br />
 +
            Origin: Lactobacillus casei, genome<br />
 +
            Properties: A coding sequence of restriction endonuclease LSEI-2094
 +
        </div>
 +
        <div class="article-content"><b>== Usage and Biology ==</b></div>
 +
        <div class="article-content">
 +
            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.
 +
        </div>
 +
        <div class="article-title">BBa_K4099006</div>
 +
        <div class="article-content"><b>== Profile ==</b></div>
 +
        <div class="article-title">
 +
            Name: pNCas9-LSEI-2094<br />
 +
            Base Pairs: 14379 bp<br />
 +
            Origin: Synthetic<br />
 +
            Properties: CRISPR technology to kick out the DNA segment, LSEI-2094 gene.
 +
        </div>
 +
        <div class="article-content"><b>== Usage and Biology ==</b></div>
 +
        <div class="article-content">
 +
            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.
 +
        </div>
 +
        <div class="article-content">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.
 +
        </div>
 +
        <div class="article-content">
 +
            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.
 +
        </div>
 +
        <div class="article-content"><b>== Construct design ==</b></div>
 +
        <div class="article-content"> 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. </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/0/0d/T--Shanghai_HS_ID--contribution02.jpg" alt="" />
 +
        </div>
 +
        <div class="article-content">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.</div>
 +
        <div class="article-content">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).</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/6/6d/T--Shanghai_HS_ID--contribution03.jpg" alt="" />
 +
            <span>Figure 3. Schematic map of pNCas9-LSEI-2094 expression plasmids.</span>
 +
        </div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/2/22/T--Shanghai_HS_ID--contribution04.jpg" alt="" />
 +
            <span>Figure 4. A: DNA profile of the plasmid pLCNICK; B: DNA Profile of LSEI-2094+ upstream and downstream
 +
                homologous arms.</span>
 +
        </div>
 +
        <div class="article-title">BBa_K4099008</div>
 +
        <div class="article-content"><b>== Profile ==</b></div>
 +
        <div class="article-title">
 +
            Name: pIB165<br />
 +
            Base Pairs: 4139 bp<br />
 +
            Origin: Escherichia coli, Addgene<br />
 +
            Properties: A E. coli - Streptococci shuttle plasmid for gene expression.
 +
        </div>
 +
        <div class="article-content"><b>== Usage and Biology ==</b></div>
 +
        <div class="article-content">
 +
            The plasmid pIB165 is a E. coli-Streptococci shuttle plasmid for gene expression in streptococci with P23
 +
            promoter.
 +
        </div>
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            <p class="margin-bottom-10"><i>WeChat Official Account: 乳酸LAB</i></p>
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            <p>Contact Email：<i>jhxchen@gmail.com</i></p>
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<h1>Contribution </h1>
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<H3>
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Bronze Medal Criterion #4
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</H3>
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<p>Make a useful contribution for future iGEM teams. Use this page to document that contribution.
+
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<br><br>
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Please see the <a href="https://2021.igem.org/Judging/Medals">2021 Medals Page</a> for more information.
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<p>If you are making a contribution by adding information to an existing Part or creating a new Part, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria. You can use this page to link to that part and include additional information about your contribution.</p>
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Revision as of 17:39, 19 October 2021

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.
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