Difference between revisions of "Part:BBa K314100"
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<partinfo>BBa_K314100 short</partinfo> | <partinfo>BBa_K314100 short</partinfo> | ||
| − | High constitutive protein expression insert includes f1 origin and RBS B0034 | + | 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=== | ||
| + | {| cellpadding="1" cellspacing="8" | ||
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| + | |[[Image:Washington2010_vector_assay_data.png | 650px]] | ||
| + | |<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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| + | |} | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
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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
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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:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 470
Illegal NheI site found at 493 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 126
- 1000COMPATIBLE WITH RFC[1000]