Difference between revisions of "Part:BBa K3257001"

Latest revision as of 00:16, 9 October 2022

T5 Promoter

Bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator.

Biology

Figure 1. The relative fluorescence intensity of EGFP with T5 promoter measured by 96-well plate reader EGFP with T5 promoter represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding EGFP protein. The Negative represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding nonsense mutated EGFP protein. Significant difference between them shows that EGFP can be expressed and function well inside E.coli cells.

Contribution: New documentation from Evry_Paris-Saclay 2022

This part is a LacI regulated hybrid promoter derived from the P_N25 promoter of the bacteriophage T5 [1–3].

P_N25 is an early phase strong promoter of the bacteriophage T5 which is recognized by the E. coli RNA polymerase [1], contrary to the well known T7 promoter which requires its own specific bacteriophage T7 RNA polymerase.

This hybrid promoter (Figure 1) was created to achieve high protein expression in any E. coli strain [2,3]. It contains a LacI binding site embedded between the -35 and the -10 boxes.

Figure 1. Sequence comparaison between the P_N25 and P_T5 promoters.

As for all other LacI controlled promoters, this promoter is repressed in E. coli strains overexpressing the LacI transcriptional regulator and is inducible by IPTG. However, with only a single LacI binding site, the efficiency of repression is reduced [4].

References

[1] Gentz R, Bujard H. Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. Journal of Bacteriology (1985) 164: 70–77.

[2] Bujard H, Gentz R, Lanzer M, Stueber D, Mueller M, Ibrahimi I, Haeuptle M-T, Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins in Vitro and in Vivo. Methods in Enzymology. Vol155. Academic Press (1987) 416–433.

[3] Hu MC. Efficient protein expression system. US7528245B2 (2009).

[4] Oehler S, Amouyal M, Kolkhof P, von Wilcken-Bergmann B, Müller-Hill B. Quality and position of the three lac operators of E. coli define efficiency of repression. The EMBO journal (1994) 13: 3348–3355.

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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3257001 short</partinfo>
-This part is T5 promoter and it comes from T5 bacteriophage. It is a rather strong promoter and it has a lacO site inside its sequence so that it can be used in lac operon.
+Bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator.
-<!-- Add more about the biology of this part here
+'''Biology'''
-===Usage and Biology===
+[[File:EGFP with T5.jpeg|center|400px|thumb|'''Figure 1. The relative fluorescence intensity of EGFP with T5 promoter measured by 96-well plate reader''' EGFP with T5 promoter represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding EGFP protein. The Negative represents the relative fluorescence intensity of BL21(DE3) transformed with plasmid encoding nonsense mutated EGFP protein. Significant difference between them shows that EGFP can be expressed and function well inside E.coli cells.]]
+===Contribution: New documentation from Evry_Paris-Saclay 2022===
+This part is a LacI regulated hybrid promoter derived from the P<sub>N25</sub> promoter of the bacteriophage T5 [1–3].
+P<sub>N25</sub> is an early phase strong promoter of the bacteriophage T5 which is recognized by the ''E. coli'' RNA polymerase [1], contrary to the well known T7 promoter which requires its own specific bacteriophage T7 RNA polymerase.
+This hybrid promoter (Figure 1) was created to achieve high protein expression in any ''E. coli'' strain [2,3]. It contains a LacI binding site embedded between the -35 and the -10 boxes.
+[[File:T--Evry_Paris-Saclay--PT5core-PN25.png|900px|thumb|left| Figure 1. Sequence comparaison between the P<sub>N25</sub> and P<sub>T5</sub> promoters.]]
+As for all other LacI controlled promoters, this promoter is repressed in ''E. coli'' strains overexpressing the LacI transcriptional regulator and is inducible by IPTG. However, with only a single LacI binding site, the efficiency of repression is reduced [4].
+=====References=====
+[1] Gentz R, Bujard H. Promoters recognized by ''Escherichia coli'' RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. Journal of Bacteriology (1985) 164: 70–77.
+[2] Bujard H, Gentz R, Lanzer M, Stueber D, Mueller M, Ibrahimi I, Haeuptle M-T, Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins ''in Vitro'' and ''in Vivo''. Methods in Enzymology. Vol155. Academic Press (1987) 416–433.
+[3] Hu MC. Efficient protein expression system. US7528245B2 (2009).
+[4] Oehler S, Amouyal M, Kolkhof P, von Wilcken-Bergmann B, Müller-Hill B. Quality and position of the three lac operators of ''E. coli'' define efficiency of repression. The EMBO journal (1994) 13: 3348–3355.
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
+<span class='h3bb'>'''Sequence and Features'''</span>
 <partinfo>BBa_K3257001 SequenceAndFeatures</partinfo>