Difference between revisions of "Part:BBa K1976000"

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        <h1>Integration plasmid</h1>
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        Green fluorescent protein under control of a constitutive promoter, with attachment-site-plasmid (attP) of phage λ, flanked by bidirectional terminators. By genomic integration of this part into bacteria carrying the bacterial attachment site (attB), the metabolic burden on the microorganism can be quantified measuring the GFP concentration. The genomic integration is achieved by attP/attB recombination and catalysed by the λ-integrase.
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        <img style="width: 74%; height: 74%; margin-left: 15px; margin-right: 15px;" alt="" src="https://static.igem.org/mediawiki/2016/8/85/T--TU_Darmstadt--genomic_integration_complete.png">
  
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===Usage and Biology===
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            <b>Figure 1:</b> Scheme of the &lambda;-integration system. The &lambda;-Integrase cuts at the attB-; and attP-site, with the help of IHF (Integration Host Factor), which is naturally located in <i>E.&#8239;Coli</i> (shown in detail here:&#8239;<a href="https://parts.igem.org/Part:BBa_K1976001">BBa_K1976001</a>)
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<p> Green fluorescent protein with attachment-site-plasmid (attP) of phage &lambda;. By genomic integration of this part into bacteria carrying the bacterial attachment site (attB), the metabolic burden on the microorganism can be quantified measuring the GFP concentration. The genomic integration is achieved by attP/attB recombination and catalysed by the &lambda;-integrase. <b>Come back for additional information soon!</b></p>
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<span class='h3bb'>Sequence and Features</span>
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===<h2>Design and Usage</h2>===
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<p>The design of our plasmid follows its function. We want to have the lowest possible stress to the cell, this is why we flanked our attP-site with bidirectional promoters (<a href="https://parts.igem.org/Part:BBa_B1001">BBa_B1001</a>) to prevent transcription of the hole plasmid. The purpose of this Biobrick is to make a new measurement strain for high accurate quantitative measurement of the metabolic burden, produced by synthetic plasmids. To accomplish that, it is necessary to have just one copy of a reporter under the control of a constitutive promoter, in our case GFP(<a href="https://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) and (<a href="https://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) , in the cell. The reporter has a LVA-Tag added, for constitutive degradation of the fluorophore - otherwise it would not be possible to measure a decline of the signal of fluorescence. Genomic integration though, is not possible without <a href="https://parts.igem.org/Part:BBa_K1976001">BBa_K1976001</a>.
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It is to mention, that you have to be cautious with transformation of this Biobrick on low copy vectors. We showed, that under the stress of a heat shock a transposase can transpose into our plasmid. We described our observations in <a href="https://parts.igem.org/Part:BBa_K1976000:Experience">experience</a>.
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===<h2>Selection of the Promoter</h2>===
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<p>To get a expression, as gentle as possible, but as strong as needed, we tried three different <a href="https://parts.igem.org/Promoters/Catalog/Anderson">Anderson promoters</a>:&#8239;<a href="https://parts.igem.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>, <a href="https://parts.igem.org/wiki/index.php/Part:BBa_J23115">BBa_J23115</a>, <a href="https://parts.igem.org/wiki/index.php/Part:BBa_J23109">BBa_J23109</a> (from strong to weak). As seen in Figure 2, only our strongest used promoter (J23101) showed a detectable signal. Though we sure could measure the other reporters, it was important for us to have a practical solution, for efficient work in the lab.
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        <img style="width: 70%; height: 70%; margin-left: 15px; margin-right: 15px;" alt="" src="https://static.igem.org/mediawiki/parts/8/8c/T--TU_Darmstadt--J23109%2BJ23115%2BJ23101.png">
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            <b>Figure 2:</b> Determination of GFP fluorescence under control of different Anderson promoters on a UV-Transilluminator. The signal of GFP expression induced by our weak promoter J23109 (left side)and the middle promoter J23115 (center) are not detectable. Our strongest promoter J23101 (right side) shows a significant signal of GFP.
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===<h2>Improvement of &lambda;-attp</h2>===
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<p>It is important to mention, that <b>you have to use this Biobrick for a functional genomic integration</b>, because we detected mistakes in three bases in the <a href="https://parts.igem.org/Part:BBa_I11023">Biobrick</a> provided from iGEM. The problem is, that the bases are located in the center of the homologous region of the attP-site, which is significant for the recognition of the &lambda;-Integrase.
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===<h2>Plasmid Curing</h2>===
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<h2>Sequence and Features</h2>
 
<partinfo>BBa_K1976000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1976000 SequenceAndFeatures</partinfo>
  
  
 
<partinfo>BBa_K1976000 parameters</partinfo>
 
<partinfo>BBa_K1976000 parameters</partinfo>

Revision as of 00:03, 27 October 2016

Integration plasmid

Green fluorescent protein under control of a constitutive promoter, with attachment-site-plasmid (attP) of phage λ, flanked by bidirectional terminators. By genomic integration of this part into bacteria carrying the bacterial attachment site (attB), the metabolic burden on the microorganism can be quantified measuring the GFP concentration. The genomic integration is achieved by attP/attB recombination and catalysed by the λ-integrase.

Figure 1: Scheme of the λ-integration system. The λ-Integrase cuts at the attB-; and attP-site, with the help of IHF (Integration Host Factor), which is naturally located in E. Coli (shown in detail here: BBa_K1976001)

Design and Usage

The design of our plasmid follows its function. We want to have the lowest possible stress to the cell, this is why we flanked our attP-site with bidirectional promoters (BBa_B1001) to prevent transcription of the hole plasmid. The purpose of this Biobrick is to make a new measurement strain for high accurate quantitative measurement of the metabolic burden, produced by synthetic plasmids. To accomplish that, it is necessary to have just one copy of a reporter under the control of a constitutive promoter, in our case GFP(BBa_E0240) and (BBa_J23101) , in the cell. The reporter has a LVA-Tag added, for constitutive degradation of the fluorophore - otherwise it would not be possible to measure a decline of the signal of fluorescence. Genomic integration though, is not possible without BBa_K1976001.
It is to mention, that you have to be cautious with transformation of this Biobrick on low copy vectors. We showed, that under the stress of a heat shock a transposase can transpose into our plasmid. We described our observations in experience.

Selection of the Promoter

To get a expression, as gentle as possible, but as strong as needed, we tried three different Anderson promoters: BBa_J23101, BBa_J23115, BBa_J23109 (from strong to weak). As seen in Figure 2, only our strongest used promoter (J23101) showed a detectable signal. Though we sure could measure the other reporters, it was important for us to have a practical solution, for efficient work in the lab.

Figure 2: Determination of GFP fluorescence under control of different Anderson promoters on a UV-Transilluminator. The signal of GFP expression induced by our weak promoter J23109 (left side)and the middle promoter J23115 (center) are not detectable. Our strongest promoter J23101 (right side) shows a significant signal of GFP.

Improvement of λ-attp

It is important to mention, that you have to use this Biobrick for a functional genomic integration, because we detected mistakes in three bases in the Biobrick provided from iGEM. The problem is, that the bases are located in the center of the homologous region of the attP-site, which is significant for the recognition of the λ-Integrase.

Plasmid Curing

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 315
    Illegal NheI site found at 338
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1014