Team:Washington/Magnetosomes/Background

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<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>
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Magnetosomes and Gibson Vectors
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As the iGEM competition grows, iGEM teams are looking to build increasingly complex systems with many gene inserts. In some cases, we want to transfer whole organelle-level systems from host organisms that may be difficult to culture or have other undesirable traits to model organisms that are easy to manipulate. This type of large-scale gene manipulation has been accelerated by new DNA assembly techniques, perhaps most notably the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning Gibson Assembly] method. These new tools allow for multiple inserts and allow precise design down to the nucleotide level without ligation scars which can degrade the performance of gene circuits. Since 2010 the UW iGEM team has been researching methods to improve cloning efficiency for Gibson Assembly and have submitted to the Parts Registry two "toolkits"': the Gibson Assembly Toolkit and the Magnetosome Toolkit.
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<h2>Project goal: </h2>
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<p> The goal of this project was to use gibson cloning to transfer the genes involved in magnetosome synthesis, into E.Coli.  </p>
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= '''Gibson Assembly Toolkit''' =
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What are magnetosomes?
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To expand on work started by the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW IGEM team], this year we developed and submitted a set of plasmid backbones for BioBricks that are optimized for Gibson assembly. Based on the BglBrick standard [http://dspace.mit.edu/bitstream/handle/1721.1/46747/BBFRFC21.pdf?sequence=1 RFC 21], these pGA (plasmids for Gibson Assembly) vectors comprise the [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly Toolkit]. These vectors have [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults much higher] cloning efficiencies than the equivalent pSB vector and are fully compliant with BioBrick [https://2010.igem.org/Team:Cambridge/Gibson/RFC RFC 57] developed by the 2010 Cambridge iGEM team.
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[[File:Igem2011 GibsonToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors]]
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Magnetosomes are small invaginations of the bacterial cell membrane that contain magnetite nanoparticles. These particles range in size between 20 and several hundred nanometers and are aligned in one or several chains along the long axis of the bacteria. <br />
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;'''What's in the Gibson Assembly Toolkit?'''
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Five plasmid backbones for high-efficiency, multiple-fragment assemblies.
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These particles act together to form a magnetic dipole across the bacteria, allowing it to perceive the earths magnetic field. In the northern hemisphere, magnetotactic bacteria (bacteria with magnetosomes) swim north along the earths magnetic field lines in search of a micro-environement with a specific oxygen content. It is believed that the magnetosomes help bacteria turn the search for this perfect oxygen level from a three dimensional one (in all directions) to a one dimensional one along a single path. <br />
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* 2 high copy vectors for gene extraction and cloning: '''pGA1A3, pGA1C3'''
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* 1 medium copy expression vector: '''pGA3K3'''
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* 2 low copy expression vectors: '''pGA4A5, pGA4C5'''
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='''Magnetosome Toolkit'''=
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One amazing thing some bacteria do is to fabricate nano-scale magnets from soluble iron. They can use these magnetic particles, aligned in chains, to sense and navigate along the earth's magnetic field. In order to bring these fabrication and sensing capabilities to future iGEM teams, we created the  [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit]: a set of 18 genes from the essential <i>mamAB</i> operon of <i>Magnetospirillum magneticum</i> strain AMB-1. We extracted these genes from AMB-1, and used our pGA vectors to efficiently BioBrick and sequence verify constructs of all 18 genes. We have also begun to characterize these genes, with some exciting [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results results]. The ultimate goal of this toolkit is to enable future iGEM teams to generate '''magnetoColi''': ''E. coli'' with magnetic properties.
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[[File:Igem2011 MagnetToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit]]
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'''What’s in the Magnetosome Toolkit?'''
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* A set of gene clusters from the essential mamAB operon from strain AMB-1
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* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors
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* A table compiling individual gene functions from our literature search

Latest revision as of 23:57, 28 September 2011


iGEM Toolkits: Background


As the iGEM competition grows, iGEM teams are looking to build increasingly complex systems with many gene inserts. In some cases, we want to transfer whole organelle-level systems from host organisms that may be difficult to culture or have other undesirable traits to model organisms that are easy to manipulate. This type of large-scale gene manipulation has been accelerated by new DNA assembly techniques, perhaps most notably the Gibson Assembly method. These new tools allow for multiple inserts and allow precise design down to the nucleotide level without ligation scars which can degrade the performance of gene circuits. Since 2010 the UW iGEM team has been researching methods to improve cloning efficiency for Gibson Assembly and have submitted to the Parts Registry two "toolkits"': the Gibson Assembly Toolkit and the Magnetosome Toolkit.


Gibson Assembly Toolkit

To expand on work started by the 2010 UW IGEM team, this year we developed and submitted a set of plasmid backbones for BioBricks that are optimized for Gibson assembly. Based on the BglBrick standard RFC 21, these pGA (plasmids for Gibson Assembly) vectors comprise the Gibson Assembly Toolkit. These vectors have much higher cloning efficiencies than the equivalent pSB vector and are fully compliant with BioBrick RFC 57 developed by the 2010 Cambridge iGEM team.

Igem2011 GibsonToolkit.png
What's in the Gibson Assembly Toolkit?

Five plasmid backbones for high-efficiency, multiple-fragment assemblies.

  • 2 high copy vectors for gene extraction and cloning: pGA1A3, pGA1C3
  • 1 medium copy expression vector: pGA3K3
  • 2 low copy expression vectors: pGA4A5, pGA4C5











Magnetosome Toolkit

One amazing thing some bacteria do is to fabricate nano-scale magnets from soluble iron. They can use these magnetic particles, aligned in chains, to sense and navigate along the earth's magnetic field. In order to bring these fabrication and sensing capabilities to future iGEM teams, we created the Magnetosome Toolkit: a set of 18 genes from the essential mamAB operon of Magnetospirillum magneticum strain AMB-1. We extracted these genes from AMB-1, and used our pGA vectors to efficiently BioBrick and sequence verify constructs of all 18 genes. We have also begun to characterize these genes, with some exciting results. The ultimate goal of this toolkit is to enable future iGEM teams to generate magnetoColi: E. coli with magnetic properties.

Igem2011 MagnetToolkit.png

What’s in the Magnetosome Toolkit?

  • A set of gene clusters from the essential mamAB operon from strain AMB-1
  • Our favorite genes as translational fusions with superfolder gfp in pGA vectors
  • A table compiling individual gene functions from our literature search
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