Difference between revisions of "Part:BBa K4169028"

(→‎Functional Parameters)
 
(12 intermediate revisions by the same user not shown)
Line 12: Line 12:
 
===Functional Parameters===
 
===Functional Parameters===
 
<p>
 
<p>
−
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>).
+
To verify the activity of promoter Pcyd, we transferred pUC57(+)-Pcyd-GFP into <i>E.coil</i> DH5α. After coating the transformed strains, the plates were placed in special anaerobic boxes and cultured at 37 ° C. After 12 hours of culture, the fluorescence intensity of GFP was qualitatively observed by irradiation under normal light and UV lamp. The results are shown below (<b>Figure 1</b>).
 
</p>
 
</p>
  
Line 21: Line 21:
 
</head>
 
</head>
 
<body>
 
<body>
−
<center><img src="https://static.igem.org/mediawiki/parts/0/08/T--HZAU-China-HepT-basic-1.png" style="width:793px;height:360px"></center>
+
<center><img src="https://static.igem.wiki/teams/4169/wiki/suicide-figure-1/suicide-pcyd-1.png" style="width:300px"></center>
−
<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>
+
<center><b>Figure 1. A.</b> Growth of E. oilDH5α after incubation at 37℃ for 15 hours. The left one is anaerobic and the right one is aerobic.Photoed under natural lights. <b>B.</b> Growth was observed under UV light. </center>
 
<br>
 
<br>
 
</body>
 
</body>
 
</html>
 
</html>
 +
 +
<p>
 +
In order to further verify that this difference was caused by oxygen, the plates that had been incubated in the anaerobic box were also incubated at room temperature. After incubating at 37℃ for 3 hours, all the plates showed the green fluorescent color of GFP. (<b>Figure 2</b>).
 +
</p>
 +
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
<title>无标题文档</title>
 +
</head>
 +
<body>
 +
<center><img src="https://static.igem.wiki/teams/4169/wiki/suicide-figure-1/suicide-pcyd-2.jpg" style="width:300px"></center>
 +
<center><b>Figure 2. A.</b> Growth of E. oilDH5α after transiting into 37℃ for 3 hours. The left one is anaerobic and the right one is aerobic. <b>B.</b> Growth was observed under UV light. </center>
 +
<br>
 +
</body>
 +
</html>
 +
 +
<p>However, after consulting the data, we found that GFP could emit light only under aerobic conditions. Therefore, the start-up conditions of this element still need to be further verified.</p>
  
 
===Sequence and Features===
 
===Sequence and Features===
Line 33: Line 51:
  
 
<h3>References</h3>
 
<h3>References</h3>
−
<p>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.<i>Mol Microbiol.</i> 1997 Aug;25(3):605-15.</p>
+
<p>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.[J]Molecular Microbiology.1997 Aug;25(3):605-15.</p>

Latest revision as of 17:03, 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 activity of promoter Pcyd, we transferred pUC57(+)-Pcyd-GFP into E.coil DH5α. After coating the transformed strains, the plates were placed in special anaerobic boxes and cultured at 37 ° C. After 12 hours of culture, the fluorescence intensity of GFP was qualitatively observed by irradiation under normal light and UV lamp. The results are shown below (Figure 1).

无标题文档

Figure 1. A. Growth of E. oilDH5α after incubation at 37℃ for 15 hours. The left one is anaerobic and the right one is aerobic.Photoed under natural lights. B. Growth was observed under UV light.

In order to further verify that this difference was caused by oxygen, the plates that had been incubated in the anaerobic box were also incubated at room temperature. After incubating at 37℃ for 3 hours, all the plates showed the green fluorescent color of GFP. (Figure 2).

无标题文档

Figure 2. A. Growth of E. oilDH5α after transiting into 37℃ for 3 hours. The left one is anaerobic and the right one is aerobic. B. Growth was observed under UV light.

However, after consulting the data, we found that GFP could emit light only under aerobic conditions. Therefore, the start-up conditions of this element still need to be further verified.

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.[J]Molecular Microbiology.1997 Aug;25(3):605-15.