Team:Washington/Magnetosomes/Background

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Magnetosomes and Gibson Vectors
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<p> Magnetotactic Bacteria are prokaryotic organisms which possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome organelle. 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.  These particles act together to form a magnetic dipole across the bacteria, allowing it to perceive the earth’s magnetic field. In the northern hemisphere, magnetotactic bacteria (bacteria with magnetosomes) swim north along the earth’s magnetic field lines in search of a micro-environment with 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.
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<p>The formation of the magnetosome organelle is a highly regulated, step-wise process requiring a cascade of essential genes. Earlier gene products must be present for later gene products to be formed as shown in the diagram on the right: <sup>
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<a href="http://www.pnas.org/content/107/12/5593.full.pdf+html">[1] </a></sup>:
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<div style="text-align: right;"><img src="http://www.pnas.org/content/107/12/5593/F6.medium.gif">
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<p>It is thought that many of the essential genes associated with magnetosome formation are located within a well-conserved region known as the magnetosome island (MAI). The MAI consists of 14 gene clusters labeled R1-R14 (see diagram below).Our team focused on the genes of the mamAB gene cluster (R5)  as they were previously shown to be essential for magnetosome membrane biogenesis in AMB-1.<sup>
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<a href="http://www.pnas.org/content/107/12/5593/F1.expansion.html">[2] </a></sup>:
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<div style="text-align: center;"><img src="https://static.igem.org/mediawiki/2011/e/ee/MamAB.png">
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<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>
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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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<p>The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in E.coli. This was done in order to understand more about magnetosome formation and the magnet synthesis mechanism because many of the genes' functions are still unknown in the host species. Ultimately, we would like to show complete magnetosome formation within E.coli.</p>
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= '''Gibson Assembly Toolkit''' =
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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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;'''What's in the Gibson Assembly Toolkit?'''
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Five plasmid backbones for high-efficiency, multiple-fragment assemblies.
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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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