Difference between revisions of "Part:BBa K592008"
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T5-lac promoter is a hybrid promoter made from the phage T5 early promoter and the lac-operon. It contains three LacI binding sites and remains repressed in LacIq strains where LacI is expressed in high levels. It is inducible by IPTG, and recognizable by ''E coli'' RNA polymerase. Compared to the T7 promoters, T5-lac promoter doesn't require the co-expression of phage polymerase.  
 
T5-lac promoter is a hybrid promoter made from the phage T5 early promoter and the lac-operon. It contains three LacI binding sites and remains repressed in LacIq strains where LacI is expressed in high levels. It is inducible by IPTG, and recognizable by ''E coli'' RNA polymerase. Compared to the T7 promoters, T5-lac promoter doesn't require the co-expression of phage polymerase.  
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We characterised the promotor in a TOP10 E.coli strain together with PcpcG2, PLlacO, J23113, PfixK and as reference promoter we used J23101. The backbone pSB3K3, the RBS B0032 and the reporter BFP (BBa_K592100) were used in every construct and we calculated the relative promoter units, RPU, compared to J23101. The actual test was done in a slightly different way than that presented at the parts.igem. Here we use flow-cytometry which also results in that we did not use M9 media to grow the cells but used LB instead. Also when it comes to the time of incubation of the selected colonies which were 10 hours and no dilutions were made except for the settings requirements of the FACS AriaII machine.
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The results showed that PT5lac has a RPU of 0.4 when repressed by natural occuring LacI and a RPU of 0.6 when LacI expression was repressed with IPTG.
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[[Image:Promoter characterization.png]]
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Revision as of 01:44, 22 September 2011

T5-lac Promoter

T5-lac promoter is a hybrid promoter made from the phage T5 early promoter and the lac-operon. It contains three LacI binding sites and remains repressed in LacIq strains where LacI is expressed in high levels. It is inducible by IPTG, and recognizable by E coli RNA polymerase. Compared to the T7 promoters, T5-lac promoter doesn't require the co-expression of phage polymerase.

We characterised the promotor in a TOP10 E.coli strain together with PcpcG2, PLlacO, J23113, PfixK and as reference promoter we used J23101. The backbone pSB3K3, the RBS B0032 and the reporter BFP (BBa_K592100) were used in every construct and we calculated the relative promoter units, RPU, compared to J23101. The actual test was done in a slightly different way than that presented at the parts.igem. Here we use flow-cytometry which also results in that we did not use M9 media to grow the cells but used LB instead. Also when it comes to the time of incubation of the selected colonies which were 10 hours and no dilutions were made except for the settings requirements of the FACS AriaII machine.

The results showed that PT5lac has a RPU of 0.4 when repressed by natural occuring LacI and a RPU of 0.6 when LacI expression was repressed with IPTG.

Promoter characterization.png


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]