Difference between revisions of "Team:Virginia/Protocols"

 
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            <div>General Template Page</div>
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          <div>Lab Notebook</div>
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        <div class="sectionTitle" id="Intro">Introduction</div>
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            Below are our Benchling Protocol pages that show in detail the protocols we utilized in the construction of Manifold. For more details on how these protocols were utilized, see the <a href="https://2020.igem.org/Team:Virginia/Notebook">Notebook</a> page. For information on the planned use of these protocols, see the <a href="https://2020.igem.org/Team:Virginia/Experiments">Experiments</a> page.
 
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          <div class="sectionTitle" id="Section 1">Section 1</div>
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        <div class="sectionTitle" id="September">Protocols</div>
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                The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) with encapsulated dsDNA scaffolds <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div> that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/d/d6/T--Virginia--Protocols--0.8_Percent_Agarose_Gel_Creation_and_Pour.pdf" target="_blank">Agarose Gel Creation and Pour</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/8/80/T--Virginia--Protocols--Agarose_Gel_Electrophoresis.pdf" target="_blank">Agarose Gel Electrophoresis</a><br/>
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          <div class="sectionTitle" id="Section 2">Section 2</div>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/6/6b/T--Virginia--Protocols--Culture_from_Agar_Stab_%28Inoculation%29.pdf" target="_blank">Culture from Agar Stab (Inoculation)</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/c/c6/T--Virginia--Protocols--DpnI_Incubation.pdf" target="_blank">DpnI Incubation</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/5/5c/T--Virginia--Protocols--Glycerol_Stock.pdf" target="_blank">Glycerol Stock</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/0/02/T--Virginia--Protocols--Golden_Gate_Assembly_with_BsaI.pdf" target="_blank">Golden Gate Assembly with BsaI</a><br/>
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              The invention consists of a protein shell comprising one or more proteins, one or more nucleic acid scaffolds of which there can be multiple copies, anabolic and/or catabolic enzymes specific to the desired biosynthesis pathway each containing a nucleic acid binding domain, recognition sequences for the utilized nucleic acid binding domains, nucleic acid spacers, and a linkage between the nucleic acid scaffolds and the protein shell. The protein shell (10) can take the form of any closed or open surface that comprises one or more repeating protein units (12). Examples of valid shells include bacterial microcompartments such as the Pdu, Eut, and carboxysome microcompartments, as well as modified,  but not necessarily closed, surfaces composed of mutated versions of these microcompartment shell proteins. The nucleic acid scaffolds (18) comprise multiple recognition sequences (22) and spacers (32) and can be made from any form of nucleic acid, including: deoxyribonucleic acid, ribonucleic acid, and synthetic nucleic acids such as xeno nucleic acids and peptide nucleic acids among others. These scaffolds are attached to the protein shell. The pathway enzymes are biological proteins whose exact sequences are dependent on the given use case of the invention, but which all contain a nucleic acid binding domain either internal to their structure, or at their N or C terminus.  
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/9/94/T--Virginia--Protocols--Golden_Gate_Assembly_with_BsmBI.pdf" target="_blank">Golden Gate Assembly with BsmBI</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/1/19/T--Virginia--Protocols--NEB_Time-Saver_qualified_Restriction_Enzyme_Digestion.pdf" target="_blank">NEB Time-Saver qualified Restriction Enzyme Digestion</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/3/3a/T--Virginia--Protocols--NEBuilder_Hifi_Mutagenesis.pdf" target="_blank">NEBuilder Hifi Mutagenesis</a><br/>
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             <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/9/9d/T--Virginia--Protocols--PCR_Purification_Kit_%28GenScript%29.pdf" target="_blank">PCR Purification Kit (GenScript)</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/4/45/T--Virginia--Protocols--Phusion_PCR_Protocol.pdf" target="_blank">Phusion PCR Protocol</a><br/>
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              <b>Fig 1.</b> Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.
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             <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/6/61/T--Virginia--Protocols--QIAGEN_HiSpeed_Plasmid_Purification.pdf" target="_blank">QIAGEN HiSpeed Plasmid Purification</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/3/31/T--Virginia--Protocols--QIAprep_Spin_Miniprep_Kit.pdf" target="_blank">QIAprep Spin Miniprep Kit</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/5/5a/T--Virginia--Protocols--Restriction_Enzyme%28s%29_Digest.pdf" target="_blank">Restriction Enzyme(s) Digest</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/3/3b/T--Virginia--Protocols--Site_Directed_Mutagenesis.pdf" target="_blank">Site Directed Mutagenesis</a><br/>
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              Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used,  if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).<br/><br/><br/>
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             <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/7/7f/T--Virginia--Protocols--Standard_Assembly_%28Column_3_-_pduD_%2B_GFP%29.pdf" target="_blank">Standard Assembly (Column 3 - pduD + GFP)</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/2/25/T--Virginia--Protocols--Standard_Assembly_%28Combining_Column_1_and_3%29.pdf" target="_blank">Standard Assembly (Combining Column 1 and 3)</a><br/>
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            <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/0/04/T--Virginia--Protocols--Transforming_Competent_Cells.pdf" target="_blank">Transforming Competent Cells</a><br/>
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             <img class="icon" src="https://static.igem.org/mediawiki/2020/d/dd/T--Virginia--images--PageIcon.svg"/><a href="https://static.igem.org/mediawiki/2020/f/ff/T--Virginia--Protocols--TRIzol_DNA_Extraction.pdf" target="_blank">TRIzol DNA Extraction</a><br/>
 
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Latest revision as of 03:32, 28 October 2020

Manifold

HOME
Main Abstract
PROJECT
Description Design Experiments Results Modeling Engineering Implementation Notebook Safety
PARTS
Parts
OUTREACH
Human Practices Public Engagement Collaborations Contributions Entrepreneurship
TEAM
Members Attributions
AWARDS
Awards Medals
RESOURCES
Sources Nucleic Acids Protocols
Index:
Lab Notebook
Introduction
Below are our Benchling Protocol pages that show in detail the protocols we utilized in the construction of Manifold. For more details on how these protocols were utilized, see the Notebook page. For information on the planned use of these protocols, see the Experiments page.
Protocols
Agarose Gel Creation and Pour
Agarose Gel Electrophoresis
Culture from Agar Stab (Inoculation)
DpnI Incubation
Glycerol Stock
Golden Gate Assembly with BsaI
Golden Gate Assembly with BsmBI
NEB Time-Saver qualified Restriction Enzyme Digestion
NEBuilder Hifi Mutagenesis
PCR Purification Kit (GenScript)
Phusion PCR Protocol
QIAGEN HiSpeed Plasmid Purification
QIAprep Spin Miniprep Kit
Restriction Enzyme(s) Digest
Site Directed Mutagenesis
Standard Assembly (Column 3 - pduD + GFP)
Standard Assembly (Combining Column 1 and 3)
Transforming Competent Cells
TRIzol DNA Extraction
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