Team:Melbourne/Project

From 2014.igem.org

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<h1 >Project</h1>
<h1 >Project</h1>
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<p><h2>PROTOCOLS
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<h2>Project description and results</strong></h2>
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</h2>
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<p><h3>PREPARING CELLS FOR TRANSFORMATION (A)</h3></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/6/6c/MELBOURNE_A1V1_Preparing_Competent_Cells_Protocol.pdf">A1V1 Preparing Competent Cells</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/6/6c/MELBOURNE_A1V1_Preparing_Competent_Cells_Protocol.pdfA2V1_Preparation_of_Liquid_Broth_Media_Protocol.pdf">A2V1 Preparation of Liquid Broth Media</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/b/b4/MELBOURNE_A3V2_Making_Agar_Plates_Protocol.pdf">A3V2 Making Agar Plates</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/f/f2/MELBOURNE_A4V1_Measuring_OD_Protocol.pdf">A4V1 Measuring OD</a></p>
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<p>&nbsp;</p>
<p>&nbsp;</p>
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<p><h3>PREPARING  PLASMIDS FOR TRANSFORMATION (B)</h3></p>
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<h3><strong>Introduction and theory</strong></h3>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/6/6f/MELBOURNE_B1V2_PCR_Protocol.pdf">B1V2 PCR Protocol</a></p>
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<p>Synthetic peptide chemists have long produced  peptide-based materials in vitro. Star-shaped peptides are a promising type of  biomaterial being explored in the field of nanomedicine (Sulistio et al.,  2012). Star peptides can have several biomedical uses such as acting as drug  delivery vehicles (Sulistio et al., 2011) or linkers for other  biomacromolecules. Star peptides generally take the form of several linear  peptide arms linked together in a central core. One way of linking these linear  peptide arms together is to used covalent bonds such as disulfides. Typically,  disulfide bonds are formed synthetically by taking several linear arms and  treating them with an oxidant in vitro. Here, we introduce a new approach to  forming star peptides by using <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and  disulfide bond forming machinery of <em>E. coli</em> can be used to form disulfide linked star peptide and key star peptide  precursors.</p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/f/f5/MELBOURNE_B2V4_DNA_Gel_Electrophoresis_Protocol.pdf">B2V4 DNA Gel Electrophoresis</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/a/a8/MELBOURNE_B3V3_DNA_Gel_Purification_Protocol.pdf">B3V3 DNA Gel Purification</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/9/9e/MELBOURNE_B4V3_Restriction_Digestion_Protocol.pdf">B4V3 Restriction Digestion</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/2/24/MELBOURNE_B5V2_Ligation.pdf">B5V2 Ligation</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/3/34/MELBOURNE_B6V1_RE-Based_Colony_Screening_Protocol.pdf">B6V1 RE-Based Colony Screening</a></p>
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<p>&nbsp;</p>
<p>&nbsp;</p>
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<p><h3>TRANSFORMATION  AND PROTEIN EXPRESSION (C)</h3></p>
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<h3>Synthesis approach</h3>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/a/a7/MELBOURNE_C1V2_Transformation_Protocol.pdf">C1V2 Transformation</a></p>
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<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains,  disulfide bonds are naturally formed by an array of enzymes which are part of  the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm  of the cell. Recently, however, several new strains of <em>E. coli</em> have been engineered which contain an oxidizing cytoplasm  conducive to disulfide bond formation. One example of this is the SHuffle cell  line (Lobstein et al., 2012). The cell line contained mutations to key enzymes  responsible for the reducing nature of the cytoplasm, namely thioredoxin  reductase (<em>trxB</em>) and glutathione  reductase (<em>gor</em>). Further, the Shuffle  cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to more successfully fold disulfide-bonded proteins in the cytoplasm as compared to non-mutants. We aimed to take advantage of disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in  cells. As shown in <strong>the figure below</strong>, the synthesis steps may proceed as follows:</p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/9/98/MELBOURNE_C2V2_Protein_Expression_in_E._Coli.pdf">C2V2 Protein Expression</a></p>
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<p>&nbsp;</p>
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<p><h3>PROTEIN PURIFICATION (D)</h3></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/e/e1/MELBOURNE_D1V1_Column_Purification_of_His-Tagged_Proteins.pdf">D1V1 Column Purification of His-Tagged  Proteins</a></p>
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<p>&nbsp;</p>
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<p><h3>PROTEIN  ANALYSIS (E)</h3></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/1/1b/MELBOURNE_E1V2_SDS-PAGE_Protocol.pdf">E1V2 SDS-PAGE</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/7/79/MELBOURNE_E2V2_Coomassie_Staining_Protocol.pdf">E2V2 Coomassie Staining</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/c/cb/MELBOURNE_E3V6_Western_Blotting_Protocol.pdf">E3V6 Western Blotting</a></p>
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<p>&nbsp;</p>
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<p><h3>OTHER (F)</h3></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/a/a5/MELBOURNE_F1V1_pH_Meter_Protocol.pdf">F1V1 pH Meter</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/0/0a/MELBOURNE_F2V2_Cleanup_Protocol.pdf">F2V2 Cleaning Up</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/6/65/MELBOURNE_F3V1_Autoclave_Protocol.pdf">F3V1 Large Autoclave</a></p>
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<p><a target="_blank" href="http://2014.igem.org/wiki/images/1/19/MELBOURNE_F4V1_Autoclaving_Materials_-_small_autoclave.pdf">F4V1 Small Autoclave</a></p>
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<p>&nbsp;</p>
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<h3> Project Description </h3>
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<h3> Content</h3>
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<p>Tell us more about your project. Give us backgroundUse this as the abstract of your project.  Be descriptive but concise (1-2 paragraphs) </p>
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<br>
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<h3>References </h3>
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<p>
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iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>  
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<p> You can use these subtopics to further explain your project</p>
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<ol>
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<li>Overall project summary</li>
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<li>Project Details</li>
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<li>Materials and Methods</li>
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<li>The Experiments</li>
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<li>Results</li>
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<li>Data analysis</li>
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<li>Conclusions</li>
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</ol>
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<p>
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It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.
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</p>
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<p>
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It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.
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</p>
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Revision as of 13:00, 17 October 2014

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Project

Project description and results

 

Introduction and theory

Synthetic peptide chemists have long produced peptide-based materials in vitro. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant in vitro. Here, we introduce a new approach to forming star peptides by using E. coli and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of E. coli can be used to form disulfide linked star peptide and key star peptide precursors.

 

Synthesis approach

E. coli naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. Recently, however, several new strains of E. coli have been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contained mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (trxB) and glutathione reductase (gor). Further, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to more successfully fold disulfide-bonded proteins in the cytoplasm as compared to non-mutants. We aimed to take advantage of disulfide bond forming capabilities of this strain of E. coli to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows: