Difference between revisions of "Part:BBa K4169028"
 
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===Pcyd: A promoter open in aerobic conditions===
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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.
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<p>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.
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===Usage and Biology===
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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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===Sequence and Features===
 
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===Functional Parameters===
 
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Use a constitutive promoter (BBa_J23110), RNA thermometer (BBa_K3733011), strong RBS (BBa_B0034), toxin HepT (BBa_K3733010), and a strong transcriptional terminator Lambda t1 transcriptional terminator (BBa_K864601). Will express the HepT toxin below 28 &#8451; to commit suicide.
 
  
 
===Usage and Biology===
 
===Usage and Biology===
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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.
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This composite part is one of temperature-based suicide schemes for the engineered bacteria functioning in mammal intestine. It was designed to lead bacteria to commit suicide as they are leaked into the environment (at low temperatures) but do not affect the growth of engineered bacteria in intestine (at high temperatures).
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===Functional Parameters===
 
===Functional Parameters===
 
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To verify the function of this composite part, we transferred it into <i>E.coli</i> DH5α. Meanwhile, we also transformed blank plasmid (only with <i>ori</i> and <i>cmR</i>) into DH5α as control group. We incubated engineered bacteria at 37 ℃ and 28 ℃, taking the bacteria with blank plasmid as control. As the <b>Figure 1</b> shows, medium of experimental group shaked at 28 ℃ is more limpid than ones shaked at 37 ℃; however, in the control group, the medium shaked at 28 ℃ is almost as turbid as ones shaked at 37 ℃.
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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>).
 
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<center><img src="https://static.igem.org/mediawiki/parts/7/79/T--HZAU-China-HepT-comp-1.png" style="width:793px;height:360px"></center>
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<center><img src="https://static.igem.wiki/teams/4169/wiki/suicide-figure-1/suicide-pcyd-1.png" style="width:300px"></center>
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<center><b>Figure 1. A.</b> The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. Sch.1 means the experimental group (toxin system). Control means the control group. <b>B.</b> The specific OD<sub>600</sub> data of the experimental group and control group. </center>
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<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>
 
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We also plotted the quantitative growth curves at 28 ℃ in this suicide scheme. We got OD<sub>600</sub> data changing over time by culturing our engineered bacteria and control bacteria in an automatic microplate reader for 12 hours. Compared with controls, the growth of our engineered bacteria was inhibited obviously(<b>Figure 2</b>).
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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>).
 
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<center><b>Figure 2.</b> The quantitative growth curves in 12 hours at 28 ℃. </center>
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<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>
 
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<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>
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To further verify the temperature sensibility of this composite part, we reput bacteria cultured at 28 ℃ into an oribital shaker at 37 ℃ overnight. Compared with themselves, the medium becomes turbid observably at 37 ℃, which means this part could make engineered bacteria kill themselves at 28 ℃ and let them survive at 37 ℃, working as expected(<b>Figure 3</b>).
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<center><b>Figure 3.</b> The comparison photo of the bacteria transferred from 28 ℃ to 37 ℃ and the bacteria cultured at 37 ℃ all along. </center>
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===Sequence and Features===
 
===Sequence and Features===
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===Functional Parameters===
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<h3>References</h3>
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<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).

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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).

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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.