Difference between revisions of "Part:BBa K3408000"
 
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We add the promoter “P<sub>nar</sub>nar”, <b>in response to the RES-FNR oxygen regulator system</b> of <i>Bacillus Subtilis</i>, to express <b>phy(ycD) phytase</b> efficiently in the absence of oxygen, and meanwhile express <b>CICI repressor protein</b> to inhibit toehold switch-Dpnc downstream.
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According to the paper, they have designed three kinds of strength of the promoter P<sub>nar</sub>nar and applied them in <i>E.coli</i> successfully. The expression level of GFP in the control of high-strength promoter was 19.7 higher than the wild-type promoter.
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<partinfo>BBa_k3408000 short</partinfo>
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<html>
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<head>
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</head>
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<body>
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    <p><strong>Contribution By Team:</strong> WestCentral-HS</p>
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    <p><strong>Group:</strong> WestCentral-HS iGEM 2023</p>
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    <h2>Summary</h2>
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    <p>The original part (BBa K3408000 Part:BBa K3408000 - parts.igem.org) modified the -35 and -15 regions on the gene sequence, whereas our protocol was designed and modified for the RBS locus. So, the sequence of the nar promoter we uploaded (BBa_K4842001 Part:BBa K4842001 - parts.igem.org) is different from the old part.</p>
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    <p>This study aimed to obtain nar promoter libraries with different translation levels by modulating the anaerobically induced nar promoter ribosome binding site (RBS) sequences.</p>
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    <p>Subsequently, the optimal promoters obtained from the screening were then applied to engineered Escherichia coli for the expression of the enzyme of the biosynthetic pathway for the conversion of soybean glycosides to (S)-estragenol.</p>
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    <h2>New part: Pnar</h2>
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    <p><strong>Name:</strong> Pnar</p>
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    <p><strong>Part:</strong> BBa K4842001 - parts.igem.org</p>
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    <p><strong>Base Pairs:</strong> 237 bp</p>
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    <p><strong>Origin:</strong> Escherichia coli str. K-12 substr. MG1655</p>
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    <p><strong>Properties:</strong> Promoter</p>
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    <h2>Usage and Biology</h2>
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    <p>Despite some exciting achievements in the heterologous biosynthesis of (S)-estragenol, heterologous expression of this pathway in E. coli currently relies on isopropyl-β-d-thiogalactopyranoside (IPTG) induction, and the high price and toxicity of IPTG to E. coli cells have limited the application of this induced expression on an industrial scale[1]. The dissolved oxygen (DO)-dependent nar promoter is considered an alternative to the above promoters because of its relatively simple and cost-effective induction mechanism and function at any stage of cell growth[2]. Therefore, our hypothesis was whether this oxygen-responsive promoter could be used to control our (S)-estradiol biosynthesis pathway in aerobic anaerobic two-stage cultures to achieve access to green-produced (S)-estradiol in a cost-effective manner. In addition, our nar promoter is derived from a 1992 reference [3] with the sequence name pMW618. It should be noted that the promoter ontology is 197 bp in length, and to facilitate the study, our sequence also contains enzymatic and RBS sites totaling 237 bp.</p>
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    <h2>Experimental Approach</h2>
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    <h3>1. Construction of plasmid pETM6-pnar</h3>
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    <p>To construct the plasmid pETM6-pnar, we first double-digested the synthetic pCDM4-pnar and pETM6 with NdeI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the nar fragment with the pETM6 vector using T4 DNA ligase to obtain the complete recombinant plasmid.</p>
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    <img src="https://static.igem.wiki/teams/4842/wiki/bba-k3408000/1.jpg" alt="Figure 1: Construction result of the plasmid pETM6-pnar." width="500">
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    <p>Figure 1: Construction result of the plasmid pETM6-pnar.</p>
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    <h3>2. Construction of plasmid pETM6-pnar-mCherry</h3>
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    <p>To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the mCherry fragment with the pETM6-pnar vector using T4 DNA ligase to obtain the complete recombinant plasmid.</p>
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    <img src="https://static.igem.wiki/teams/4842/wiki/bba-k3408000/2.jpg" alt="Figure 2: Construction result of the plasmid pETM6-pnar-mCherry." width="500">
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    <p>Figure 2: Construction result of the plasmid pETM6-pnar-mCherry.</p>
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    <h2>Characterization/Measurement</h2>
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    <h3>Mutation library construction for the plasmids pETM6-pnar-RBSx-mCherry</h3>
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    <p>We designed eight RBS sequences downstream of the nar promoter using the RBS Calculator (https://salislab.net/software/), as shown in the table below.</p>
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    <img src="https://static.igem.wiki/teams/4842/wiki/bba-k3408000/table-1-1.jpg" alt="Table 1: RBS sequences designed by RBS Calculator" width="00">
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    <p>Table 1: RBS sequences designed by RBS Calculator.</p>
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    <p>We then introduced the RBS sequence mutations by whole plasmid PCR. We successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.</p>
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    <img src="https://static.igem.wiki/teams/4842/wiki/bba-k3408000/3.jpg" alt="Figure 3: Electrophoresis results of whole plasmid PCR." width="500">
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    <p>Figure 3: Electrophoresis results of whole plasmid PCR.</p>
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    <p>We transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent E. coli BL21(DE3) cells, respectively. After overnight culture, positive transformants grew on LB plates. Colony PCR identification showed the correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while no bands were observed for pETM6-pnar-RBS (6-8)-mCherry. This may be due to mistakes in picking colonies or omissions in adding templates. Nevertheless, we inoculated all eight transformants for subsequent fluorescence intensity testing.</p>
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    <img src="https://static.igem.wiki/teams/4842/wiki/bba-k3408000/4.jpg" alt="Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry." width="500">
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    <p>Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.</p>
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    <h2>Reference</h2>
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    <ol>
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        <li>Lee P G, Kim J, Kim E J, et al. P212A mutant of dihydrodaidzein reductase enhances (S)-equol production and enantioselectivity in a recombinant Escherichia coli whole-cell reaction system[J]. Applied and Environmental Microbiology, 2016, 82(7): 1992-2002.</li>
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        <li>Clomburg J M, Blankschien M D, Vick J E, et al. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for synthesizing medium chain ω-functionalized carboxylic acids[J]. Metab Eng, 2015, 28(202-212).</li>
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        <li>Walker, M.S., DeMoss, J.A., 1992. Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. J Bacteriol 174(4), 1119-1123.</li>
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    </ol>
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</body>
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</html>
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P<sub>nar</sub> is an oxygen-free inducible promoter which can response to the ResD/E two-component regulatory system and the global regulator Nitrate Reductase Regulator (FNR) of <i>Bacillus subtilis</i>.
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In our project, we use it to express <b>Phy(ycD)</b> efficiently in the absence of oxygen, and meanwhile express <b>CⅠ repressor protein</b> to inhibit toehold switch-mazF downstream.
 +
 
 +
According to the paper, P<sub>nar</sub> with three levels of strength were designed and applied in <i>E.coli</i> successfully. The expression level of GFP in the control of high-strength promoter was 19.7 higher than the wild-type promoter.
 +
 
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https://2020.igem.org/wiki/images/9/92/T--NAU-CHINA--basic-parts1.png
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<span style="text-align:center; display:inline-block;">Fig.1. The spacer sequence of 15 bp between the -35 and -10 elements of the upstream region of the wild-type nar promoter was randomized(Hwang Hee Jin, et al., <i>BIOTECHNOL BIOFUELS</i>. 2018)</span>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3408000 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3408000 parameters</partinfo>
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Latest revision as of 09:30, 11 October 2023

promoter nar(fnr regulated)

Contribution By Team: WestCentral-HS

Group: WestCentral-HS iGEM 2023

Summary

The original part (BBa K3408000 Part:BBa K3408000 - parts.igem.org) modified the -35 and -15 regions on the gene sequence, whereas our protocol was designed and modified for the RBS locus. So, the sequence of the nar promoter we uploaded (BBa_K4842001 Part:BBa K4842001 - parts.igem.org) is different from the old part.

This study aimed to obtain nar promoter libraries with different translation levels by modulating the anaerobically induced nar promoter ribosome binding site (RBS) sequences.

Subsequently, the optimal promoters obtained from the screening were then applied to engineered Escherichia coli for the expression of the enzyme of the biosynthetic pathway for the conversion of soybean glycosides to (S)-estragenol.

New part: Pnar

Name: Pnar

Part: BBa K4842001 - parts.igem.org

Base Pairs: 237 bp

Origin: Escherichia coli str. K-12 substr. MG1655

Properties: Promoter

Usage and Biology

Despite some exciting achievements in the heterologous biosynthesis of (S)-estragenol, heterologous expression of this pathway in E. coli currently relies on isopropyl-β-d-thiogalactopyranoside (IPTG) induction, and the high price and toxicity of IPTG to E. coli cells have limited the application of this induced expression on an industrial scale[1]. The dissolved oxygen (DO)-dependent nar promoter is considered an alternative to the above promoters because of its relatively simple and cost-effective induction mechanism and function at any stage of cell growth[2]. Therefore, our hypothesis was whether this oxygen-responsive promoter could be used to control our (S)-estradiol biosynthesis pathway in aerobic anaerobic two-stage cultures to achieve access to green-produced (S)-estradiol in a cost-effective manner. In addition, our nar promoter is derived from a 1992 reference [3] with the sequence name pMW618. It should be noted that the promoter ontology is 197 bp in length, and to facilitate the study, our sequence also contains enzymatic and RBS sites totaling 237 bp.

Experimental Approach

1. Construction of plasmid pETM6-pnar

To construct the plasmid pETM6-pnar, we first double-digested the synthetic pCDM4-pnar and pETM6 with NdeI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the nar fragment with the pETM6 vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 1: Construction result of the plasmid pETM6-pnar.

Figure 1: Construction result of the plasmid pETM6-pnar.

2. Construction of plasmid pETM6-pnar-mCherry

To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the mCherry fragment with the pETM6-pnar vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 2: Construction result of the plasmid pETM6-pnar-mCherry.

Figure 2: Construction result of the plasmid pETM6-pnar-mCherry.

Characterization/Measurement

Mutation library construction for the plasmids pETM6-pnar-RBSx-mCherry

We designed eight RBS sequences downstream of the nar promoter using the RBS Calculator (https://salislab.net/software/), as shown in the table below.

Table 1: RBS sequences designed by RBS Calculator

Table 1: RBS sequences designed by RBS Calculator.

We then introduced the RBS sequence mutations by whole plasmid PCR. We successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.

Figure 3: Electrophoresis results of whole plasmid PCR.

Figure 3: Electrophoresis results of whole plasmid PCR.

We transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent E. coli BL21(DE3) cells, respectively. After overnight culture, positive transformants grew on LB plates. Colony PCR identification showed the correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while no bands were observed for pETM6-pnar-RBS (6-8)-mCherry. This may be due to mistakes in picking colonies or omissions in adding templates. Nevertheless, we inoculated all eight transformants for subsequent fluorescence intensity testing.

Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.

Figure 4: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.

Reference

  1. Lee P G, Kim J, Kim E J, et al. P212A mutant of dihydrodaidzein reductase enhances (S)-equol production and enantioselectivity in a recombinant Escherichia coli whole-cell reaction system[J]. Applied and Environmental Microbiology, 2016, 82(7): 1992-2002.
  2. Clomburg J M, Blankschien M D, Vick J E, et al. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for synthesizing medium chain ω-functionalized carboxylic acids[J]. Metab Eng, 2015, 28(202-212).
  3. Walker, M.S., DeMoss, J.A., 1992. Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. J Bacteriol 174(4), 1119-1123.


Pnar is an oxygen-free inducible promoter which can response to the ResD/E two-component regulatory system and the global regulator Nitrate Reductase Regulator (FNR) of Bacillus subtilis.

In our project, we use it to express Phy(ycD) efficiently in the absence of oxygen, and meanwhile express CⅠ repressor protein to inhibit toehold switch-mazF downstream.

According to the paper, Pnar with three levels of strength were designed and applied in E.coli successfully. The expression level of GFP in the control of high-strength promoter was 19.7 higher than the wild-type promoter.

T--NAU-CHINA--basic-parts1.png

Fig.1. The spacer sequence of 15 bp between the -35 and -10 elements of the upstream region of the wild-type nar promoter was randomized(Hwang Hee Jin, et al., BIOTECHNOL BIOFUELS. 2018)


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]