Difference between revisions of "Part:BBa K590010"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for [https://parts.igem.org/wiki/index.php?title=Part:BBa_I7107 LacI-repressible GFP]. | + | This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for [https://parts.igem.org/wiki/index.php?title=Part:BBa_I7107 LacI-repressible GFP]. This plasmid has been altered to conform to [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] standards, with arbitrary, Gibson-friendly sequences placed in between the restriction enzyme sites EcoRI-BglII, and BamHI-PstI. |
===Characterization=== | ===Characterization=== | ||
This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of just the pGA backbone. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements. | This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of just the pGA backbone. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements. | ||
| − | <center>https://static.igem.org/mediawiki/2011/d/d1/Washington_pGAefficiency_summary.jpg</center> | + | <center>https://static.igem.org/mediawiki/2011/d/d1/Washington_pGAefficiency_summary.jpg</center> |
| + | To understand how the copy number varies from plasmid to plasmid, the pGA vectors were transformed into a <i>lacI</i> knockout (strain [http://cgsc.biology.yale.edu/Strain.php?ID=16959 2.320]) and the fluorescence levels were measured by flow cytometry. It was found that the fluorescence level is highest in the high copy plasmids: | ||
| + | |||
| + | <center> [[Image:Washington 2011 pGA vector fluorescence means v2.pdf|400px]] </center> | ||
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Latest revision as of 00:00, 3 November 2011
pGA1A3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
This is a Gibson Cloning friendly 1A3 plasmid backbone that was made by [http://2011.igem.org/Team:Washington UW iGEM Team 2011] as part of the [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly toolkit]. Using this plasmid increases the efficiency for anyone doing Gibson cloning into a 1A3 vector.
Usage and Biology
This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for LacI-repressible GFP. This plasmid has been altered to conform to [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] standards, with arbitrary, Gibson-friendly sequences placed in between the restriction enzyme sites EcoRI-BglII, and BamHI-PstI.
Characterization
This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of just the pGA backbone. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements.
To understand how the copy number varies from plasmid to plasmid, the pGA vectors were transformed into a lacI knockout (strain [http://cgsc.biology.yale.edu/Strain.php?ID=16959 2.320]) and the fluorescence levels were measured by flow cytometry. It was found that the fluorescence level is highest in the high copy plasmids:
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2136 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2136 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2136
Illegal BglII site found at 2151
Illegal BamHI site found at 1
Illegal XhoI site found at 16 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2136 - 25INCOMPATIBLE WITH RFC[25]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2136 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI.rc site found at 1175