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Team:Imperial/EColi
From 2014.igem.org
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<div class="accordion"> | <div class="accordion"> | ||
| - | <h3> | + | <h3>Cell Strains and Media Components</h3> |
<div> | <div> | ||
| - | <p> | + | <p>Escherichia coli strain DH10B were made chemically competent following a standard protocol and were used to clone the pSB-AraC-pBAD and pSB-LacI-pLAC elements. Additionally, High Efficiency chemically competent DH10B E.coli were purchased from New England Biolabs for assembly of the Acs cellulose-producing operon, which was codon-optimized for expression in E.coli and synthesized by GenArt. Both strains were grown overnight in liquid cultures (supplemented with specific antibiotics), or on semi-solid LB-Agar plates, provided with the specific antibiotic, as listed on the table below.</p> |
| + | |||
| + | <p>Luria Broth Miller (LB) and LB Agar were purchased from ????, prepared using standard protocols and used throughout the experimental procedure. During characterization, Acs-containing strains were supplemented with 1% D-glucose, 0.1% Arabinose and 0.5mM IPTG. | ||
| + | </p> | ||
</div> | </div> | ||
| - | <h3> | + | <h3>Chemicals </h3> |
<div> | <div> | ||
| - | <p> | + | <p>Congo Red was purchased from Sigma Aldrich (Steiheim, Germany) and diluted in 1x PBS to a concentration of 350uM. The final Congo Red concentration used during system characterization steps was 20uM.</p> |
</div> | </div> | ||
| - | <h3> | + | <h3>Construct Assembly</h3> |
<div> | <div> | ||
| - | <p> | + | <h4>Cloning of pSB-AraC-pBAD</h4> |
| + | <div> | ||
| + | <p>The AraC and pBAD elements were obtained by PCR, using the Biobricks BBa_K325108, BBa_K325218 and BBa_K325219 as DNA templates, all of which follow the standard structure showed on the image below (left hand side, add legend, add figure number). | ||
| + | The primers used during this procedure were as follows:</p> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Forward: TACTAGTAGCGGCCGCTGCAG</li> |
| - | <li> | + | <li>Reverse: GCTAGCCCAAAAAAACGGGTATGGAG</li> |
| - | + | ||
</ul> | </ul> | ||
</div> | </div> | ||
| + | <p>The amplified elements were DpnI-treated following a standard procedure (New England Biolabs), and were subsequently submitted to a purification step (Qiaquick PCR clean up, Qiagen) prior to self-ligation. Three different template concentrations were tested during ligation (specifically 3ng, 7.5ng and 15 ng), and were incubated at room temperature for 1h+. The ligation mix was then transformed into chemically competent DH10B E.coli using a standard chemical transformation protocol and plated out on Chloramphenicol-specific LB plates.</p> | ||
<h3>Engineering Sub-Section 4</h3> | <h3>Engineering Sub-Section 4</h3> | ||
<div> | <div> | ||
Revision as of 01:53, 16 October 2014
Team E. coli
Overview
Zombie ipsum reversus ab viral inferno, nam rick grimes malum cerebro. De carne lumbering animata corpora quaeritis. Summus brains sit, morbo vel maleficia? De apocalypsi gorger omero undead survivor dictum mauris. Hi mindless mortuis soulless creaturas, imo evil stalking monstra adventus resi dentevil vultus comedat cerebella viventium. Qui animated corpse, cricket bat max brucks terribilem incessu zomby. The voodoo sacerdos flesh eater, suscitat mortuos comedere carnem virus. Zonbi tattered for solum oculi eorum defunctis go lum cerebro. Nescio brains an Undead zombies. Sicut malus putrid voodoo horror. Nigh tofth eliv ingdead.
Key Achievements
Introduction
Zombie ipsum reversus ab viral inferno, nam rick grimes malum cerebro. De carne lumbering animata corpora quaeritis. Summus brains sit, morbo vel maleficia? De apocalypsi gorger omero undead survivor dictum mauris. Hi mindless mortuis soulless creaturas, imo evil stalking monstra adventus resi dentevil vultus comedat cerebella viventium. Qui animated corpse, cricket bat max brucks terribilem incessu zomby. The voodoo sacerdos flesh eater, suscitat mortuos comedere carnem virus. Zonbi tattered for solum oculi eorum defunctis go lum cerebro. Nescio brains an Undead zombies. Sicut malus putrid voodoo horror. Nigh tofth eliv ingdead.
Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etia
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etia
Aims
Specification
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etia.
Engineering
Lorem ipsum dolor sit amet, consectetuer adipiscing elit.
Cell Strains and Media Components
Escherichia coli strain DH10B were made chemically competent following a standard protocol and were used to clone the pSB-AraC-pBAD and pSB-LacI-pLAC elements. Additionally, High Efficiency chemically competent DH10B E.coli were purchased from New England Biolabs for assembly of the Acs cellulose-producing operon, which was codon-optimized for expression in E.coli and synthesized by GenArt. Both strains were grown overnight in liquid cultures (supplemented with specific antibiotics), or on semi-solid LB-Agar plates, provided with the specific antibiotic, as listed on the table below.
Luria Broth Miller (LB) and LB Agar were purchased from ????, prepared using standard protocols and used throughout the experimental procedure. During characterization, Acs-containing strains were supplemented with 1% D-glucose, 0.1% Arabinose and 0.5mM IPTG.
Chemicals
Congo Red was purchased from Sigma Aldrich (Steiheim, Germany) and diluted in 1x PBS to a concentration of 350uM. The final Congo Red concentration used during system characterization steps was 20uM.
Construct Assembly
Cloning of pSB-AraC-pBAD
The AraC and pBAD elements were obtained by PCR, using the Biobricks BBa_K325108, BBa_K325218 and BBa_K325219 as DNA templates, all of which follow the standard structure showed on the image below (left hand side, add legend, add figure number). The primers used during this procedure were as follows:
- Forward: TACTAGTAGCGGCCGCTGCAG
- Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
The amplified elements were DpnI-treated following a standard procedure (New England Biolabs), and were subsequently submitted to a purification step (Qiaquick PCR clean up, Qiagen) prior to self-ligation. Three different template concentrations were tested during ligation (specifically 3ng, 7.5ng and 15 ng), and were incubated at room temperature for 1h+. The ligation mix was then transformed into chemically competent DH10B E.coli using a standard chemical transformation protocol and plated out on Chloramphenicol-specific LB plates.
Engineering Sub-Section 4
Cras dictum. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia mauris vel est.
Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.
Modelling
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etia
Testing
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.
Results
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim.
References
