Difference between revisions of "Part:BBa K590014"

Latest revision as of 00:16, 3 November 2011

pGA3K3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 3K3 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 3K3 vector.

Usage and Biology

This is a medium copy plasmid backbone that confers kanamycin 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.

Below is the gel image of the plasmid amplified with universal pGA backbone primers pGAsuffix_fwd and pGAprefix_rev. The band is near the expected length of 2750 bp.

1kb Ladder (left), pGA3k3 (right)

Characterization

A sister plasmid (pGA1A3) with the same BglBrick prefix and suffix regions 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. As expected, we found the medium copy pGA3K3 plasmid to express GFP at levels between the low and high copy plasmids.

Washington 2011 pGA vector fluorescence means v2.pdf

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2729
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2729
Illegal NheI site found at 1384
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2729
Illegal BglII site found at 2744
Illegal BamHI site found at 1
Illegal XhoI site found at 16
Illegal XhoI site found at 177
Illegal XhoI site found at 1020
23
INCOMPATIBLE WITH RFC[23]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2729
25
INCOMPATIBLE WITH RFC[25]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2729
Illegal AgeI site found at 1470
Illegal AgeI site found at 1793
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI.rc site found at 2498

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K590014 short</partinfo>
-This is a Gibson Cloning friendly 3K3 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].
+This is a Gibson Cloning friendly 3K3 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 3K3 vector.
 ===Usage and Biology===
-This is a medium copy plasmid backbone that confers kanamycin 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 medium copy plasmid backbone that confers kanamycin 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.
-[Image:https://static.igem.org/mediawiki/2011/d/d1/Washington_pGAefficiency_summary.jpg]
 Below is the gel image of the plasmid amplified with universal pGA backbone primers [https://parts.igem.org/Part:BBa_K590086 pGAsuffix_fwd] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590084 pGAprefix_rev]. The band is near the expected length of 2750 bp.
 [[Image:Igem2011 3k3 placGFP gel.png|thumb|center|1kb Ladder (left), pGA3k3 (right)]]
+===Characterization===
+A sister plasmid ([https://parts.igem.org/Part:BBa_K590010 pGA1A3]) with the same BglBrick prefix and suffix regions 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>
+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. As expected, we found the medium copy pGA3K3 plasmid to express GFP at levels between the low and high copy plasmids.
+<center> [[Image:Washington 2011 pGA vector fluorescence means v2.pdf|400px]] </center>
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