Difference between revisions of "Part:BBa K314100"
 
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High constitutive protein expression insert includes f1 origin [https://parts.igem.org/Part:BBa_K314110 K314110], a high expression promoter [https://parts.igem.org/Part:BBa_J23100 J23100], and the  Elowitz standard RBS [https://parts.igem.org/Part:BBa_B0034 B0034].  When ligated with a protein coding sequence at the SpeI site the scar site created will create proper spacing for protein expression.  
 
High constitutive protein expression insert includes f1 origin [https://parts.igem.org/Part:BBa_K314110 K314110], a high expression promoter [https://parts.igem.org/Part:BBa_J23100 J23100], and the  Elowitz standard RBS [https://parts.igem.org/Part:BBa_B0034 B0034].  When ligated with a protein coding sequence at the SpeI site the scar site created will create proper spacing for protein expression.  
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===Usage and Biology===
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|[[Image:Washington2010_vector_assay_data.png | 650px]]
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|<partinfo>E0040</partinfo> (GFP) was inserted between S and P using standard BioBrick assembly.  The resulting constructs were grow in DH5a (constitutive promoters <partinfo>BBa_J23100</partinfo> or <partinfo>BBa_J23114</partinfo>, as well as inducible promoter <partinfo>R0011</partinfo>) or BL21(DE3)* (T7 promoter, <partinfo>BBa_K314104</partinfo>) cells overnight at 298K in either the presence or absence of 0.5mM IPTG.  The cells were harvested, washed in PBS, and then fluorescence for the whole cells was measured on a fluorometer (ex. 485, em. 525) and normalized to cell density (OD600).  Cells that did not contain GFP were used as a blank.  As observed, the addition of IPTG significantly increased the observed fluorescence of the cells.
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K314100 parameters</partinfo>
 
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GFP expression assay shows activity of K314100 as compaired to other protein expression cassettes.
 
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[[Image:Washington2010_vector_assay_data.png|thumb|left|650px|'''Figure 1.  GFP fluorescence was measured (ex. 485, em. 525) and normalized to cell density, read as the optical density at 600nm.''']]
 

Latest revision as of 04:32, 23 October 2010

High constitutive expression cassette

High constitutive protein expression insert includes f1 origin K314110, a high expression promoter J23100, and the Elowitz standard RBS B0034. When ligated with a protein coding sequence at the SpeI site the scar site created will create proper spacing for protein expression.

Usage and Biology

Washington2010 vector assay data.png BBa_E0040 (GFP) was inserted between S and P using standard BioBrick assembly. The resulting constructs were grow in DH5a (constitutive promoters BBa_J23100 or BBa_J23114, as well as inducible promoter BBa_R0011) or BL21(DE3)* (T7 promoter, BBa_K314104) cells overnight at 298K in either the presence or absence of 0.5mM IPTG. The cells were harvested, washed in PBS, and then fluorescence for the whole cells was measured on a fluorometer (ex. 485, em. 525) and normalized to cell density (OD600). Cells that did not contain GFP were used as a blank. As observed, the addition of IPTG significantly increased the observed fluorescence of the cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 470
    Illegal NheI site found at 493
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 126
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters