Difference between revisions of "Part:BBa K4169028"
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<p>This is a promoter sequence that initiates transcription of subsequent gene sequences under aerobic conditions and is part of the operon structure of cydAB. Under anaerobic conditions, the Fnr protein will bind to a part of the promoter sequence, impeding RNA polymerase binding, playing a competitive role, and the downstream gene sequence cannot be transcribed. However, under microoxygen conditions, AcrA protein binds to this promoter and recruits RNA polymerase to bind to the promoter, thereby normally initiating downstream transcription. Under aerobic conditions, RNA polymerase normally binds to the promoter. So this is a promoter sequence that can be transcribed normally under aerobic conditions.
 
<p>This is a promoter sequence that initiates transcription of subsequent gene sequences under aerobic conditions and is part of the operon structure of cydAB. Under anaerobic conditions, the Fnr protein will bind to a part of the promoter sequence, impeding RNA polymerase binding, playing a competitive role, and the downstream gene sequence cannot be transcribed. However, under microoxygen conditions, AcrA protein binds to this promoter and recruits RNA polymerase to bind to the promoter, thereby normally initiating downstream transcription. Under aerobic conditions, RNA polymerase normally binds to the promoter. So this is a promoter sequence that can be transcribed normally under aerobic conditions.
 
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===Functional Parameters===
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To verify the cytotoxicity of HepT, we transferred pET-28a(+)-HepT into <i>E.coil</i> BL21(DE3). The <i>E.coil</i> strain was cultured to OD<sub>600</sub> = 0.4 ~ 0.6, induced with or without 0.5 mM IPTG, and was allowed to grow overnight at 37℃. In the 96-well plates, the Synergy H1 microplate reader was used to measure the cytotoxicity by comparing the OD<sub>600</sub> between experimental group and control group. The results obtained from triple independent experiments are shown below (<b>Figure 1</b>).
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<center><img src="https://static.igem.org/mediawiki/parts/0/08/T--HZAU-China-HepT-basic-1.png" style="width:793px;height:360px"></center>
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<center><b>Figure 1. A.</b> Turbidity change of the experimental group (induced by IPTG) and control group (without IPTG). <b>B.</b> The result of OD<sub>600</sub> (-IPTG and +IPTG) presents the cytotoxicity of HepT. The error bars are obtained from three independent experiments. </center>
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===Sequence and Features===
 
===Sequence and Features===

Revision as of 03:28, 11 October 2022


BBa_K4169028

Pcyd: A promoter open in aerobic conditions

This is an aerobically initiated promoter that initiates transcription of subsequent sequences under microaerobic and aerobic conditions. The mechanism is based on the fact that Fnr takes over the promoter sequence under anaerobic conditions, competes with RNA polymerase for binding sites, and thus prevents the transcription of subsequent sequences. However, under microaerobic and aerobic conditions, Fnr protein does not bind to the sequence, and thus can continue the subsequent transcription.

Usage and Biology

This is a promoter sequence that initiates transcription of subsequent gene sequences under aerobic conditions and is part of the operon structure of cydAB. Under anaerobic conditions, the Fnr protein will bind to a part of the promoter sequence, impeding RNA polymerase binding, playing a competitive role, and the downstream gene sequence cannot be transcribed. However, under microoxygen conditions, AcrA protein binds to this promoter and recruits RNA polymerase to bind to the promoter, thereby normally initiating downstream transcription. Under aerobic conditions, RNA polymerase normally binds to the promoter. So this is a promoter sequence that can be transcribed normally under aerobic conditions.

Functional Parameters

To verify the cytotoxicity of HepT, we transferred pET-28a(+)-HepT into E.coil BL21(DE3). The E.coil strain was cultured to OD600 = 0.4 ~ 0.6, induced with or without 0.5 mM IPTG, and was allowed to grow overnight at 37℃. In the 96-well plates, the Synergy H1 microplate reader was used to measure the cytotoxicity by comparing the OD600 between experimental group and control group. The results obtained from triple independent experiments are shown below (Figure 1).

无标题文档

Figure 1. A. Turbidity change of the experimental group (induced by IPTG) and control group (without IPTG). B. The result of OD600 (-IPTG and +IPTG) presents the cytotoxicity of HepT. The error bars are obtained from three independent experiments.


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]


References

Cotter PA, Melville SB, Albrecht JA, Gunsalus RP. Aerobic regulation of cytochrome d oxidase (cydAB) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation.Mol Microbiol. 1997 Aug;25(3):605-15.