Team:Washington/Magnetosomes/Background

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<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>
<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>
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As with the expansion of the iGEM competition, many iGEM teams have started to investigate the possibility of working with large-scale genomes. Large-scale gene manipulation often requires the use of tools which allow multiple gene inserts as to bring the cloning project from single gene level to a multiple gene level. However, the current BioBrick standard vectors available through iGEM are not designed for multiple-insert cloning. Therefore, the UW iGEM team decided to research methods to improve cloning efficiency and as a result, two "toolkits" were submitted to the registry.
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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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= '''Gibson Assembly Toolkit''' =
= '''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" vectors comprise the [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly Toolkit]. These vectors have much higher cloning efficiencies than the equivalent pSB vector and are fully compliant with BioBrick [http://www.synbio.org.uk/gibson/downloads/files/RFC57.pdf RFC 57] developed by the 2010 [https://2010.igem.org/Team:Cambridge Cambridge] iGEM team.
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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.
[[File:Igem2011 GibsonToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors]]  
[[File:Igem2011 GibsonToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors]]  
;'''What's in the Gibson Assembly Toolkit?'''
;'''What's in the Gibson Assembly Toolkit?'''
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* Five plasmid backbones
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Five plasmid backbones for high-efficiency, multiple-fragment assemblies.
* 2 high copy vectors for gene extraction and cloning: '''pGA1A3, pGA1C3'''
* 2 high copy vectors for gene extraction and cloning: '''pGA1A3, pGA1C3'''
* 1 medium copy expression vector: '''pGA3K3'''
* 1 medium copy expression vector: '''pGA3K3'''
* 2 low copy expression vectors: '''pGA4A5, pGA4C5'''
* 2 low copy expression vectors: '''pGA4A5, pGA4C5'''
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='''Magnetosome Toolkit'''=
='''Magnetosome Toolkit'''=
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To demonstrate the utility of pGA vectorsIn addition, we were also ambitious about assembling a large gene-construct of over 16 kb. Therefore, utilizing the pGA vectors and Gibson cloning methods, we assembled the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] with the goal to generate magnetic ''E. coli''; a novel characteristic observed in magnetotactic bacteria such as ''Magnetospirillum magneticum''.
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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]]
[[File:Igem2011 MagnetToolkit.png|left|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit]]
'''What’s in the Magnetosome Toolkit?'''
'''What’s in the Magnetosome Toolkit?'''
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*A set of the 18 essential genes for the various steps of magnetosome formation.
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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 in pGA vectors
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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
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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 [http://dspace.mit.edu/bitstream/handle/1721.1/46747/BBFRFC21.pdf?sequence=1 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