Difference between revisions of "Team:Stanford-Brown-RISD/Parts"

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<h1>Parts</h1>
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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<h3>Note</h3>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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<h3>Adding parts to the registry</h3>
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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ADD PARTS
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<h3>Inspiration</h3>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h3>What information do I need to start putting my parts on the Registry?</h3>
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<img src="https://static.igem.org/mediawiki/2018/thumb/d/d4/T--Stanford-Brown-RISD--Title_PartsOverview.png/1600px-T--Stanford-Brown-RISD--Title_PartsOverview.png">
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<p>The information needed to initially create a part on the Registry is:</p>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</li>
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<li>Sequence</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Long Description (Longer description of what the DNA does)</li>
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<li>Design considerations</li>
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<h1> Stanford-Brown-RISD Key Parts (32 submitted in total) </h1>  
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<table style="background-color: #081328;">
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868000"> BBa_K2868000 </a>  </td>
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<td style="width:80%"> Composite Part T7 CsgA-6xhistag expression </td>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. This composite part consists of a T7 promoter with RBS (BBa_K525998), followed by a coding region for csgA (BBa_K1583000) with a His-Tag expressed at the end, an ochre stop codon, and then a BglII cut site followed by a T7 terminator (BBa_K731721). The construct constitutively produces the amyloid-forming E.coli curlin-precursor protein csgA when restriction ligated into a backbone plasmid of choice. The his-tag allows for the protein product to be purified with standard his-tag protein purification methods. Because of the T7 promoter and RBS, for protein production this construct must be used in T7 cell lines like BL21 that produce T7 RNA polymerase. The SalI and BglII cut sites also allow for the replacement with a desired coding region after the construct is successfully in a plasmid backbone. </td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868011"> BBa_K2868011 </a>  </td>
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<td style="width:80%"> Composite Part T7 CBD-6xhistag expression </td>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. This part contains the genetic machinery to produce a chitin binding protein that is linked with a his-tag, with a T7 promoter and TE1 terminator. </td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868014"> BBa_K2868014 </a>  </td>
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<td style="width:80%"> HHTC-Re </td>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Advait Patil. This is a version of the peptide computationally designed by Kozisek et al. (Chemistry, 2008) to bind to copper (HHTC). This metal binding domain has had amino acids replaced for specificity, and has a flexible GSGGSG linker attached to allow for spacing for proper folding of the constituent domains in any fusion proteins being created. </td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868015"> BBa_K2868015 </a>  </td>
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<td style="width:80%"> Composite Part HHTC_RE-CBD x2 Copper Binding Expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are two metal binding domains, allowing for two copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility.</td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868018"> BBa_K2868018</a>  </td>
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<td style="width:80%"> Composite Part  HHTC_RE-CBD x3 Copper Binding Expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are three metal binding domains, allowing for three copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility. </td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868019"> BBa_K2868019 </a>  </td>
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<td style="width:80%"> Composite Part HHTC_RE-CBD x6 Copper Binding Expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are six metal binding domains, allowing for six copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility. </td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868021"> BBa_K2868021 </a>  </td>
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<td style="width:80%"> Composite Part FP151 6xhistag T7 expression </td>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. An IPTG inducible T7 promoter, then medium strength RBS, then a SalI cut-site, for the mfp151 DNA sequence which has an added 6x polyhistidine tag at the end, followed by a taa stop codon, then a bglII cut-site, and finally a and a T7 terminator.</td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868024"> BBa_K2868024 </a>  </td>
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<td style="width:80%"> Composite Part Csga-CBD-6xhistag Fusion T7 expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. T7 promoter with RBS, followed by a SalI cut-site, then coding region for csgA, followed by a flexible linker, followed by the NEB chitin binding domain, which is followed by the multi-tag of Flag, lumio, and 6x polyhistidine, and a stop codon, then a BglII cut-site, and finally a T7 terminator.</td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868028"> BBa_K2868028 </a>  </td>
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<td style="width:80%"> Composite Part CBD4x T7 expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. T7 promoter, a salI cut-site, then a start codon then a Flag, Lumio, 6x polyhisitine tag, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus chitin binding domain, then another linker, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus chitin binding domain, stop codon, a bglII cut-site, and finally t7 terminator.</td>
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<td style="width:20%"> <a href="http://parts.igem.org/Part:BBa_K2868032"> BBa_K2868032 </a>  </td>
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<td style="width:80%"> Composite Part Tyrosinase+Co-Factor Arabinose induced Co-expression </td>
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</tr>
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<td style="width:20%"> </td>
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<td style="width:80%; font-size: 70%;"> Designed by Cale Lester. Arabinose inducible promoter, medium RBS, his tagged tyrosinase coding, stop codon, tactag spacer, medium strength RBS, co-factor his-tagged coding region, stop codon, T7 terminator. </td>
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<h6>Validated Part – <a href="http://parts.igem.org/Part:BBa_K2868024">BBa_K2868024</a></h6>
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<p>csgA-CBD Expression system T7 promoter with RBS, followed by a SalI cut-site, then coding region for csgA, followed by a flexible linker, followed by the NEB chitin binding domain, which is followed by the multi-tag of Flag, lumio, and 6x polyhistidine, and a stop codon, then a BglII cut-site, and finally a T7 terminator.</p>
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<p>This composite part is for the expression of the fusion protein we designed, csgA-CBD. Expression requires a T7 RNA polymerase because of the T7 constitutive promoter. It is a csgA curlin precursor of E.Coli coding region linked to a Bacillus circulans chitin binding domain with a 6x polyhistidine tag.</p>
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<p>CsgA has well documented amyloid formation behavior and is a main component of E.Coli biofilms, so we theorized that if it were fused to a chitin binding domain the resulting fusion protein would exhibit exceptional adhesive properties to chitin while also remaining cohesive with other csgA-CBD proteins. These traits are extremely attractive for use as a biological adhesive on mycelium substrates, as the fungal cell wall is largely composed of chitin.</p>
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<p>We were able to successfully express our fusion csgA-CBD protein using this composite part on a pSB1C3 plasmid backbone. We then purified the resulting csgA-CBD protein using standard his-tag purification protocols on Ni-NTA resin spin columns, followed by isoelectric precipitation. The final pure protein product tied for the strongest bonding strength on a mycelium substrate out of the proteins we tested, and had the best ratio of strength on mycelium to strength on cardboard. To read more about our design process, laboratory procedures, testing data, and results surrounding this part, please read our wiki page on the subject <a href="https://2018.igem.org/Team:Stanford-Brown-RISD/Results">here</a></p>
  
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Latest revision as of 03:44, 18 October 2018

HOME
TEAM
Team Members
Collaborations
PROJECT
Description
Design
Experiments
Results
Notebook
Mission Architecture
InterLab
Model
Demonstrate
Improve
Attributions
PARTS
Parts Overview
Basic Parts
Composite Parts
Part Collection
SAFETY
HUMAN PRACTICES
Human Practices
Education & Engagement
AWARDS
Applied Design
Measurement
Model
Software
JUDGING FORM ⇗

Stanford-Brown-RISD Key Parts (32 submitted in total)


BBa_K2868000 Composite Part T7 CsgA-6xhistag expression
Designed by Cale Lester. This composite part consists of a T7 promoter with RBS (BBa_K525998), followed by a coding region for csgA (BBa_K1583000) with a His-Tag expressed at the end, an ochre stop codon, and then a BglII cut site followed by a T7 terminator (BBa_K731721). The construct constitutively produces the amyloid-forming E.coli curlin-precursor protein csgA when restriction ligated into a backbone plasmid of choice. The his-tag allows for the protein product to be purified with standard his-tag protein purification methods. Because of the T7 promoter and RBS, for protein production this construct must be used in T7 cell lines like BL21 that produce T7 RNA polymerase. The SalI and BglII cut sites also allow for the replacement with a desired coding region after the construct is successfully in a plasmid backbone.
BBa_K2868011 Composite Part T7 CBD-6xhistag expression
Designed by Cale Lester. This part contains the genetic machinery to produce a chitin binding protein that is linked with a his-tag, with a T7 promoter and TE1 terminator.
BBa_K2868014 HHTC-Re
Designed by Advait Patil. This is a version of the peptide computationally designed by Kozisek et al. (Chemistry, 2008) to bind to copper (HHTC). This metal binding domain has had amino acids replaced for specificity, and has a flexible GSGGSG linker attached to allow for spacing for proper folding of the constituent domains in any fusion proteins being created.
BBa_K2868015 Composite Part HHTC_RE-CBD x2 Copper Binding Expression
Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are two metal binding domains, allowing for two copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility.
BBa_K2868018 Composite Part HHTC_RE-CBD x3 Copper Binding Expression
Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are three metal binding domains, allowing for three copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility.
BBa_K2868019 Composite Part HHTC_RE-CBD x6 Copper Binding Expression
Designed by Advait Patil. This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are six metal binding domains, allowing for six copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility.
BBa_K2868021 Composite Part FP151 6xhistag T7 expression
Designed by Cale Lester. An IPTG inducible T7 promoter, then medium strength RBS, then a SalI cut-site, for the mfp151 DNA sequence which has an added 6x polyhistidine tag at the end, followed by a taa stop codon, then a bglII cut-site, and finally a and a T7 terminator.
BBa_K2868024 Composite Part Csga-CBD-6xhistag Fusion T7 expression
Designed by Cale Lester. T7 promoter with RBS, followed by a SalI cut-site, then coding region for csgA, followed by a flexible linker, followed by the NEB chitin binding domain, which is followed by the multi-tag of Flag, lumio, and 6x polyhistidine, and a stop codon, then a BglII cut-site, and finally a T7 terminator.
BBa_K2868028 Composite Part CBD4x T7 expression
Designed by Cale Lester. T7 promoter, a salI cut-site, then a start codon then a Flag, Lumio, 6x polyhisitine tag, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus chitin binding domain, then another linker, followed by Bacillus circulans chitin binding domain, flexible linker, S. griseus chitin binding domain, stop codon, a bglII cut-site, and finally t7 terminator.
BBa_K2868032 Composite Part Tyrosinase+Co-Factor Arabinose induced Co-expression
Designed by Cale Lester. Arabinose inducible promoter, medium RBS, his tagged tyrosinase coding, stop codon, tactag spacer, medium strength RBS, co-factor his-tagged coding region, stop codon, T7 terminator.
Validated Part – BBa_K2868024

csgA-CBD Expression system T7 promoter with RBS, followed by a SalI cut-site, then coding region for csgA, followed by a flexible linker, followed by the NEB chitin binding domain, which is followed by the multi-tag of Flag, lumio, and 6x polyhistidine, and a stop codon, then a BglII cut-site, and finally a T7 terminator.

This composite part is for the expression of the fusion protein we designed, csgA-CBD. Expression requires a T7 RNA polymerase because of the T7 constitutive promoter. It is a csgA curlin precursor of E.Coli coding region linked to a Bacillus circulans chitin binding domain with a 6x polyhistidine tag.

CsgA has well documented amyloid formation behavior and is a main component of E.Coli biofilms, so we theorized that if it were fused to a chitin binding domain the resulting fusion protein would exhibit exceptional adhesive properties to chitin while also remaining cohesive with other csgA-CBD proteins. These traits are extremely attractive for use as a biological adhesive on mycelium substrates, as the fungal cell wall is largely composed of chitin.

We were able to successfully express our fusion csgA-CBD protein using this composite part on a pSB1C3 plasmid backbone. We then purified the resulting csgA-CBD protein using standard his-tag purification protocols on Ni-NTA resin spin columns, followed by isoelectric precipitation. The final pure protein product tied for the strongest bonding strength on a mycelium substrate out of the proteins we tested, and had the best ratio of strength on mycelium to strength on cardboard. To read more about our design process, laboratory procedures, testing data, and results surrounding this part, please read our wiki page on the subject here