Difference between revisions of "Part:BBa K590013"

(→‎Usage and Biology)
(→‎Characterization)
 
Line 8: Line 8:
  
 
===Characterization===
 
===Characterization===
−
To understand how the copy number varies from plasmid to plasmid. The pGA vectors were transformed and the fluorescence levels were measured by flow cytometer. It was found that the fluorescence level is higher is high copy plasmid:
+
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 low copy pGA4A5 plasmid to express GFP at levels lower than medium and high copy plasmids.
  
 
<center> [[Image:Washington 2011 pGA vector fluorescence means v2.pdf|400px]] </center>
 
<center> [[Image:Washington 2011 pGA vector fluorescence means v2.pdf|400px]] </center>

Latest revision as of 00:21, 3 November 2011

pGA4A5, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 4A5 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].

Usage and Biology

This is a low copy plasmid backbone which has Ampicillin resistance, with pLac promoter and 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

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 low copy pGA4A5 plasmid to express GFP at levels lower than medium 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 3371
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3371
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3371
    Illegal BglII site found at 3386
    Illegal BamHI site found at 1
    Illegal XhoI site found at 16
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3371
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3371
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 3023