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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===Usage and Biology===
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The results showed that PT5lac has a RPU of 0.4 when repressed by natural occuring (no IPTG added) LacI and a RPU of 0.6 when LacI expression was repressed with IPTG.
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[[Image:Promoter characterization.png]]
 
[[Image:Promoter characterization.png]]
  
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'''iGEM11_Uppsala-Sweden:''' Promoter characterization. We characterised the promotor in a TOP10 ''E. coli'' strain together with PcpcG2 (<partinfo>BBa_K592003</partinfo>), PLlacO (<partinfo>BBa_R0011</partinfo>), <partinfo>BBa_J23113</partinfo>, PfixK (<partinfo>BBa_K592006</partinfo>) and as reference promoter we used <partinfo>J23101</partinfo>. The backbone pSB3K3, the RBS <partinfo>BBa_B0032</partinfo> and the reporter BFP (<partinfo>BBa_K592100</partinfo>) 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 grew the cells in LB medium for 10 hours and we used flow cytometry to quantify the output. The flow cytometer used was a BD FACSAria II, using a 407 nm violet laser and a DAPI filter (band pass 450/40).
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The results showed that PT5lac has a strength of 0.4 RPU in TOP10 when repressed by LacI (not lacIq) and a strength of 0.6 RPU when induced with IPTG.
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===References===
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[http://www.ncbi.nlm.nih.gov/pubmed/8045263] Oehler S, Amouyal M, Kolkhof P, von Wilcken-Bergmann B, Muller-Hill B (1994) Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J 13: 3348-3355.
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[http://www.ncbi.nlm.nih.gov/pubmed/3900050] Gentz R, Bujard H (1985) Promoters Recognized by Escherichia-Coli Rna-Polymerase Selected by Function - Highly Efficient Promoters from Bacteriophage-T5. J Bacteriol 164: 70-77.
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[http://www.ncbi.nlm.nih.gov/pubmed/3057497] Lanzer M, Bujard H (1988) Promoters Largely Determine the Efficiency of Repressor Action. P Natl Acad Sci USA 85: 8973-8977.
  
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===Usage and Biology===
 
  
 
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Latest revision as of 13:49, 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.

Usage and Biology

Promoter characterization.png

iGEM11_Uppsala-Sweden: Promoter characterization. We characterised the promotor in a TOP10 E. coli strain together with PcpcG2 (BBa_K592003), PLlacO (BBa_R0011), BBa_J23113, PfixK (BBa_K592006) and as reference promoter we used BBa_J23101. The backbone pSB3K3, the RBS BBa_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 grew the cells in LB medium for 10 hours and we used flow cytometry to quantify the output. The flow cytometer used was a BD FACSAria II, using a 407 nm violet laser and a DAPI filter (band pass 450/40).

The results showed that PT5lac has a strength of 0.4 RPU in TOP10 when repressed by LacI (not lacIq) and a strength of 0.6 RPU when induced with IPTG.

References

[http://www.ncbi.nlm.nih.gov/pubmed/8045263] Oehler S, Amouyal M, Kolkhof P, von Wilcken-Bergmann B, Muller-Hill B (1994) Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J 13: 3348-3355.

[http://www.ncbi.nlm.nih.gov/pubmed/3900050] Gentz R, Bujard H (1985) Promoters Recognized by Escherichia-Coli Rna-Polymerase Selected by Function - Highly Efficient Promoters from Bacteriophage-T5. J Bacteriol 164: 70-77.

[http://www.ncbi.nlm.nih.gov/pubmed/3057497] Lanzer M, Bujard H (1988) Promoters Largely Determine the Efficiency of Repressor Action. P Natl Acad Sci USA 85: 8973-8977.


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]