Difference between revisions of "Team:TU Darmstadt/Measurement"

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<div class="abstract">
 
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<p><b>ABSTRACT</b><br/></p>
 
<p><b>ABSTRACT</b><br/></p>
<p>Artificial plasmids are a significant burden to the host. The design of our pathways, for example the combination of a promoter and RBS, results in different amounts of product. The measurement of the metabolic burden is the key for a quantitative optimization  
+
<p>Artificial plasmids are a significant burden to the host. The design of pathways, e.g. the combination of different promoter and RBS systems, results in different amounts of product. Measurement of the metabolic burden is a key for quantitative optimization of metabolic engineering approaches. We want to establish a new approach to iGEM by providing a measurement strain to the community. As described by F.&nbsp;Ceroni <i>et al.</i>, we integrated one copy of the <i>gfp</i> gene into the genome of <i>E. coli</i>, which offers us a highly accurate and instantaneous measurement of the impact of our plasmids on the host. Metabolic burden measurement is of economical interest, because it enables academic and industrial research testing several different pathways at once in a short period of time by using microplate reader.
in metabolic engineering. We want to establish a new approach to iGEM by providing a measurement strain to the community. As described by F.&nbsp;Ceroni et al., we genomically integrated one copy of GFP into <i>E.&nbsp;coli</i>, which offers us a highly accurate and instantaneous measurement of the impact of our plasmids on the host. This is of economical interest because it enables academic and industrial researchers to test a lot of different pathways at once in a short time just by using a microplate reader. For the integration we used the &lambda;&#8209;Integrase site&#8209;specific recombination pathway, described by A.&nbsp;Landy in 2015 [9]. Therefore, we designed two plasmids (BBa_K1976000 and BBa_K1976001) and measured them using single cell measurement and via microplate reader.
+
For the integration we used the &lambda;&#8209;Integrase site&#8209;specific recombination pathway, described by A.&nbsp;Landy in 2015 [9]. Therefore, we designed two plasmids (<a href="http://parts.igem.org/Part:BBa_K1976000">BBa_K1976000</a> and <a href="http://parts.igem.org/Part:BBa_K1976001">BBa_K1976001</a>) and measured them using single cell measurement via microplate reader.
  
 
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    <p><h5>Metabolic burden</h5></p>
+
    <p><h5>Metabolic Burden</h5></p>
  
  
<p>In synthetic biology, the term &quot;metabolic&nbsp;burden&quot; describes the influence of heterologously expressed genes on the distribution availability of resources in the host&#8209;cell. Thereby the extent of metabolic burden is not only dependent on the consumption of energy and reaction equivalents, but is also influenced by a number of different factors. Next to general factors, like for example size and copy number of the used plasmid [1], especially the specific properties of the heterologous proteins play an important role. Proteins that interfere with the host's metabolism, influence the proton gradient or are toxic in a different way can already at low levels of expression pose a strong burden for the cell [1, 2]. I.&nbsp;Shachrai et al. could furthermore show that ribosome availability, as a limiting factor, represents a big portion of metabolic burden [3].<br>
+
<p>In synthetic biology, the term &quot;metabolic&nbsp;burden&quot; describes the influence of heterologously expressed genes on the distribution availability of resources in the host cell. Thereby the extent of metabolic burden is not only dependent on the consumption of energy and reaction equivalents but is also influenced by a number of different factors. In addition to size and copy number of the used plasmid [1], the specific properties of the heterologous proteins play an important role. Proteins that interfere with the host's metabolism, influence the proton gradient or are toxic in a different way can pose a strong burden for the cell already at low expression levels [1, 2]. I.&nbsp;Shachrai <i>et al</i>. could show that ribosome availability represents a highly limiting factor for metabolic performance [3].<br>
If the metabolic burden on the host cell is too high, the physiology and biochemistry of the cell is drastically altered. In this case, for example the viability or proliferation of the cell could be disturbed. In addition there is a higher error rate in the translation which heightens the immunogenicity of the incorporated proteins and can lead to a reduced protein activity and stability [1]. Exactly for this reason the metabolic burden poses a big problem, especially in the industrial sector [4]. That is because it has a big impact on expression efficiency and thus affects the amount of obtained product. <br>
+
If the metabolic burden on the host cell is too high, the physiology and biochemistry of the cell will be drastically altered, <i>e.g.</i> the viability or proliferation of the cell could be disturbed. In addition, the error rate in translation increases which heightens the immunogenicity of the incorporated proteins and can lead to a reduced protein activity and stability [1]. Metabolic burden is a big issue especially in the industrial sector [4], because lowered expression efficiency decreases product yield. <br>
Examples for often used methods to reduce the metabolic burden include the separation of the biosynthetic pathway into multiple organisms through co&#8209;cultivating them or the identification and deletion of expendable genes as a part of strain optimization [6]. Another approach is the regulation of protein expression with <i>Dynamic Sensor and Regulatory Systems</i> (DSRSs). Basis of such systems is the usage of transcription factors that can detect certain key&#8209;metabolites and regulate the transcription simultaneously [7]. <br>
+
Often used methods to reduce the metabolic burden include for example the separation of the biosynthetic pathway into multiple organisms through co&#8209;cultivating them or the identification and deletion of expendable genes as a part of strain optimization [6]. Another approach is the regulation of protein expression with <i>Dynamic Sensor and Regulatory Systems</i> (DSRSs). Such systems are based on the usage of transcription factors which detect certain key&#8209;metabolites and regulate the transcription simultaneously [7]. <br>
One possibility to quantify the metabolic burden <i>in&nbsp;vivo</i> was described by F.&nbsp;Ceroni et al. In this method, a GFP reporter gene is integrated in the genome of <i> E.&nbsp;coli </i> using the <i>&lambda;</i>&#8209;integrase [8]. Through measuring the fluorescence it could be shown that the constitutive expression of GFP after transformation with expression&#8209;plasmids drastically lowers in comparison to not transformed cells. </p>
+
One possibility to quantify the metabolic burden <i>in&nbsp;vivo</i> was described by F.&nbsp;Ceroni <i>et al.</i> In this method a GFP reporter gene is integrated in the genome of <i> E.&nbsp;coli </i> using the <i>&lambda;</i>&#8209;integrase [8]. Through measuring the fluorescence it could be shown that the constitutive expression of GFP after transformation with expression&#8209;plasmids drastically decreases in comparison to not transformed cells. </p>
  
  
<h6>Measurement of the metabolic burden via microplate reader</h6>
+
<h6>Measurement of the Metabolic Burden via Microplate Reader</h6>
<p> The total amount of formed GFP is, next to the stress forced onto the cell, also significantly influenced by the cell amount. Because of that reason the measurement of GFP with a microplate reader is done under continuous observation of the cell density. This kind of measurement seems feasible and with a lot of samples, like in <i>96&#8209;well&#8209;plates</i>, easily doable. It is possible, with the measurement of different combinations of plasmid and cell&#8209;type, to determine the artificially caused stress on the cells proportional to the decrease in GFP expression.  Due to this reason this kind of measurement is a more economical approach than the single cell measurement described in the next paragraph.</p>
+
<p> In addition to the stress forced onto the cell, the total amount of formed GFP is significantly influenced by the cell amount. For this reason the measurement of GFP with a microplate reader was performed under continuous observation of the cell density. With this kind of measurement many samples, like in <i>96&#8209;well&#8209;plates</i>, can be analyzed. Since the measurement of different combinations of plasmid and cell&#8209;type, would enable to determine the artificially caused stress on the cells proportional to the decrease in GFP expression, this kind of measurement is assumed to be a more economical approach than the single cell measurement described in the next paragraph.</p>
<h6>Single-cell-measurement</h6>
+
<h6>Single-cell Measurement</h6>
<p>While measuring every single cell individually, the cell density can be neglected, which concludes in a smaller error in the analysis of the fluorescence. This method shows the direct influence of the metabolic burden on the GFP production and is therefore a more exact method than the previously described method using a microplate reader.</p>
+
<p>While measuring every single cell individually, the cell density can be neglected which leads to a smaller error in the fluorescence measurement. This method enables the detection of the influence of the metabolic burden based on the GFP production. Therefore, it is assumed to be a more exact method than the previously described method using a microplate reader.</p>
  
  
<p><h5>Genomic integration</h5></p>
+
<p><h5>Genomic Integration</h5></p>
  
<p>The <i>&lambda;</i>&#8209;integrase, originally derived from the <i>&lambda;</i>&#8209;Phage, catalyzes in combination with several assisting proteins the excessive and integrative recombination of the phage's genome with the chromosomal genome of a host. For this, two attachment sites are needed: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>&lambda;</i>&#8209;genome (<i>attP</i>), which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB'&nbsp;Region (<i>attB</i>) and COC'&nbsp;Region (<i>attP</i>). These can be connected by the <i>&lambda;</i>&#8209;integrase and the bacterial <i>integration host factor</i> (IHF) via <i>Holliday junction</i> forming an intasome, a DNA&#8209;protein&#8209;complex, producing hybrid attachment sites <i>attL</i> and <i>attR</i>. <br>
+
<p>The <i>&lambda;</i>&#8209;integrase, originally derived from the <i>&lambda;</i>&#8209;phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>&lambda;</i>&#8209;genome (<i>attP</i>), which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB'&nbsp;region (<i>attB</i>) and COC'&nbsp;region (<i>attP</i>). These regions can be connected by the <i>&lambda;</i>&#8209;integrase and the bacterial <i>integration host factor</i> (IHF) via <i>Holliday junction</i> forming an intasome, a DNA&#8209;protein&#8209;complex, producing hybrid attachment sites <i>attL</i> and <i>attR</i>. <br>
<p><h6>Integration plasmid and helper plasmid</h6></p>
+
 
<p>For the integration of a gene of interest (GOI) into the chromosomal genome of <i>E.&nbsp;coli</i> there are two plasmids needed.  
+
<p><h6>Integration Plasmid and Helper Plasmid</h6></p>
The integration plasmid contains the constitutively expressed GOI GFP, which, as previously mentioned, is also the reporter that is necessary for the measurement of the metabolic burden and should be integrated into the <i>E.&nbsp;coli</i> genome. To measure only the temporary fluorescence a LVA degradation tag is added to the GFP. The plasmid also contains the <i>attP&nbsp;</i>site that enables the integration. Additionally, two bidirectional terminators are located on each side of the <i>attP</i> to protect the GFP&nbsp;operon from the transcription of the other neighbouring genes.  
+
<p>For the integration of the gene of interest (GOI) into the chromosomal genome of <i>E.&nbsp;coli</i> two plasmids are needed. The integration plasmid contains the constitutive generator of GFP, which is also the necessary reporter for the measurement of the metabolic burden and should be integrated into the genome of <i>E.&nbsp;coli</i>. To ensure that only temporary fluorescence are measured, a LVA degradation tag was added to the GFP. The plasmid also contains the <i>attP</i> that enables the integration. Additionally, two bidirectional terminators are located on each side of the <i>attP</i> to protect the GFP&nbsp;operon from the transcription during biosynthesis of neighbouring genes. To create the integration plasmid E0240 (RBS(BB0032+GFP)) was cloned in a J61002 backbone to locate the GFP gene under the control of the promoter J23101. The construct J23101+E0240 was mutated via mutagenesis PCR for optimizing BBa_I11023 and afterwards cloned in the high&#8209;copy vector pSB1C3 to increase the yield of the plasmid preparation. The mutagenesis PCR aimed to mutate <i>attp2</i> to <i>&lambda&;&#8209;attP</i>. To finalize the construct, we changed the backbone to pSB4A5 which possesses a <i>low-copy ori</i> to facilitate the later performed plasmid curing.
To create the integration plasmid E0240 (RBS(BB0032+GFP)) was put on J61002 to locate the GFP behind the promoter J23101. The construct J23101+E0240 was then transformed on the high copy vector pSB1C3 to increase the yield of the prep after a Quick Change PCR, which was necessary for the optimization of  BBa_I11023, mutating attp2 to <i>&lambda;</i>&#8209;attP. The final construct was then transformed on the backbone pSB4A5, which possesses a <i>low copy ori</i> and eases the later performed plasmid curing.
+
The second plasmid is a helper plasmid which is necessary for transposing the <i>gfp</i> gene into the chromosomal genome as it contains the <i>&lambda;</i>&#8209;integrase generator. For inscription in the registry the construct was cloned in a pSB1C3 backbone and , additionally, to facilitate additional performed plasmid curing via sustained lack of selection pressure, we again chose the <i>low&#8209;copy vector</i> pSB4K5 .<br>
The second plasmid is a helper plasmid, that is necessary for transposing the GFP into the chromosomal genome as it contains the synthesized protein <i>&lambda;</i>&#8209;integrase with a ribosomal binding site (RBS). For the registry the construct was transformed on a pSB1C3, then we again chose a <i>low copy vector</i> pSB4K5 to ease the later transformed plasmid curing via sustained lack of selection pressure.<br>
+
To verify the success of the recombination, PCR was performed, using specific primers to the <i>attB</i> site of the <i>E.&nbsp;coli</i> and the VR&nbsp;primer binding on every BioBrick standard vectors. Since the first primer binds to the genome and the other is complimentary to the plasmid, a PCR&nbsp;amplicon could be optained only if the integration has succeeded. </p>
To verify whether the recombination was successful one can perform a PCR with primers binding to the <i>attB</i> site of the <i>E.&nbsp;coli</i> and the VR&nbsp;Primer, which binds on every BioBrick compliant plasmid. As the one primer binds on the genome and the other on a plasmid, there can only be a PCR&nbsp;amplicon if the integration has succeeded. </p>
+
  
 
<h5>Plasmid Curing</h5>
 
<h5>Plasmid Curing</h5>
 
<p>.....</p>
 
<p>.....</p>
  
<h5>Integration strains</h5>
+
<h5>Integration Strains</h5>
<p>A suitable genomic integration strain needs to carry the <i>attB</i> sequence needed for <i>&lambda;</i>&#8209;integrase mediated recombination, which can be troublesome because many commonly used <i>E.&nbsp;Coli</i> strains already have the <i>&lambda;</i>&#8209;phage integrated into their genome. Also, the <i>attB site</i> needed for the integration is blocked in <i>&lambda;</i> (DE3) phages.<br>
+
<p>A suitable genomic integration strain needs to carry the <i>attB</i> sequence which is necessary for the <i>&lambda;</i>&#8209;integrase mediated homologous recombination. Many commonly used <i>E.&nbsp;coli</i> strains carry the <i>&lambda;</i>&#8209;phage integrated in their genome. But since the <i>attB</i> site was often used to integrate genes via <i>&lambda;</i> recombination, <i>e.g.</i> T7 polymerase in BL21 (DE3), in many strains the attachment site is not accessible anymore. For our integration we chose <i>E.&nbsp;coli</i> JM109 because it matches all our demands and is also freely and easily available to our lab.</p>
For our integration strain we chose the <i>E.&nbsp;Coli</i> JM109 strain because it matched all our demands and was also freely and easily available to us.</p>
+
 
 +
<h5>Results</h5>
 +
 
 +
<h6>1. Integration Plasmid - GFP&#8209;construct</h6>
 +
<p>
 +
The first major step in assembling the integration plasmid was to choose a useful promoter for an expression of GFP, which should be strong enough to be measured but low enough to keep the metabolic burden as low as possible.<br>
 +
<center><div class="bild" id="figure_1"><img src="https://static.igem.org/mediawiki/2016/4/4e/T--TU_Darmstadt--promotorentest_complete.png" width="60%"></div>
 +
<div><br><b>Figure&nbsp;1: </b> <i>E.coli</i> transformed with GFP behind three different promoters. A: GFP and JM23109, B: GFP and JM23115, C: GFP and JM23101</center> <br>
 +
Three different promoters and GFP were transformed into E. Coli to test their strenght.
 +
In dish A the GFP was combined with the JM23109 promoter, in dish B with the JM23115 promoter and in dish C with the JM23101 promoter. As it can be seen above in the comparison, GFP is best transcripted with the JM23101 promoter, so we decided to use it in our integration plasmid. With the other two promoters the fluorescence would not be strong enough to be measured after plasmid curing, as there would be only one GFP copy left in the cells.
 +
</p>
 +
<p>
 +
The next major step was to mutate the synthesized <i>attp2</i>&#8209;site to the needed <i>attp</i>&#8209;site of the &lambda;&#8209;integrase.<br>
 +
 
 +
This sequence was <b>improved</b> to the following sequence:<br><center>
 +
<div class="bild" id="figure_2"><center><img src="https://static.igem.org/mediawiki/2016/8/8b/T--TU_Darmstadt--attpalignment.png" width="60%"></div><br><div><b>Figure&nbsp;2:</b> Compared alignment of the <i>attp2</i> gene and the <i>attp</i> gene. The red marked spots show the mutations that were corrected
 +
</p></div></center><br>
 +
<p>
 +
The third major step in the assembly of the integration plasmid was the adding of a LVA&#8209;Tag to the GFP sequence to ensure a fast degradation. (This was done to further decrease the metabolic burden caused by the GFP and to make a fast answer in the fluorescence possible so accurate measurements can be made.)<br>
 +
//Sequenz mit LVA-Tag hier einfügen//<br>
 +
The sequence shows that the mutagenesis PCR for adding the LVA&#8209;Tag was succesful.
 +
</p>
 +
<p>
 +
The last major step of the assembly was to ligate the &lambda;&#8209;<i>attp</i>&#43;GFP&#8209;LVA construct into a pSB1C3 vector to submit the part to the iGEM registry.<br>
 +
To determine if the ligation was succesful a gelelectrophoresis was made using VR&#8209;&nbsp;and&nbsp;VF2&#8209;primers.<br><center><div class="bild" id="figure_3"><img src="https://static.igem.org/mediawiki/2016/f/fa/T--TU_Darmstadt--GFP_Nachweil_Gel_complete.jpg" width="30%"></div><br><div>Figure&nbsp;3: Gelelectrophoresis of the &lambda;&#8209;<i>attp</i>&#43;GFP&#8209;LVA construct in pSB1C3. The marked band shows the wanted product</div></center> <br>
 +
The band coming from the sixth batch of the gelelectrophoresis of the cPCR shows that the ligation was succesful.
 +
</p>
 +
<h6>2. Helping Plasmid - Integrase </h6>
 +
<p>
 +
Due to a mistake in ordering the integrase there was a LVA&#8209;tag at the end of the sequence. <br>
 +
Therefore the LVA&#8209;tag was deleted with a mutagenesis PCR.
 +
The sequencing shows that the deletion of the LVA&#8209;Tag was succesful.<br>
 +
Click the following link to get a .zip file with further informations on our <a href="https://2016.igem.org/File:T--TU_Darmstadt--Sequencing_genomic_integration_TUD.zip">sequences</a>.
 +
 
 +
</p>
 +
<p>
 +
Next was the ligation of the integrase on a pSB1C3 for part submission and to measure if the integrase is expressed.<br><center><img src="https://static.igem.org/mediawiki/2016/9/94/T--TU_Darmstadt--helferplasmid_nachweis_gel_complete.jpg" width="30%"><br>Figure&nbsp;4: Gelelectrophoresis of the &lambda;&#8209;integrase in pSB1C3. The marked band shows the wanted product</center><br>
 +
The lanes three and five show a succesful ligation in pSB1C3 and are therefore used for further experiments. To determine if the integrase is expressed a SDS&#8209;Page was done.<br><center><img src="https://static.igem.org/mediawiki/2016/4/47/T--TU_Darmstadt--integrase_nachweis_page_complete.jpg" width="30%"><br>Figure&nbsp;5: SDS&#8209;Page of the &lambda;&#8209;integrase<br>
 +
The marked spot on the SDS&#8209;Page shows a sufficient expression of the wanted integrase</center>
 +
</p>
 +
<p>
 +
For the detemination if our integrase works as expected and therefore have a proof of concept we tried to genomically integrate our integration plasmid into the genome of <i>E. coli</i> with the help of the integrase. But before doing that we put both constructs on midi&#8209;copy vectors (pSB1K3 and JM23101). To test if this was succesful a cPCR which only yields product if the genomic integration was succesful was performed.<br>
 +
<center><img src="https://static.igem.org/mediawiki/2016/c/cc/T--TU_Darmstadt--genomic_integration_nachweis.png" width="30%"><br>Figure&nbsp;6: Proof for the genomic integration
 +
The gelelectrophoresis shows that in colonies three and four the genomic integration were successful and therefore <b>our Integrase works as expected</b>.
 +
</p></center>
 +
<h6>3. Genomic Integration and Measurement</h6>
 +
<p>
 +
As previously described the genomic integration using the integration&nbsp;plasmid and the helping&nbsp;plasmid was succesful.<br>
 +
To test if our measurement system works as expected we transformed the cells which carry the GFP with the Naringenin Biosensor BBa_K1497021 of the 2014 TU Darmstdt iGEM Team.
 +
<img src="//Grafiken Messung 2.0//" align=center>Graphen fehlen noch<br>
 +
The first graph shows that the optical density is decreasing during the measurement. The second graph shows that
 +
</p>
 +
<a name="improvedpart">
 +
<h6>4. Improved Part </h6> </a>
 +
 
 +
<p> For genomic integration via &lambda-phage (attP/attB) recombination, a functional phage-attachment site (attP) is essential. Here, we initally used the attachment sequence from BBa_I111023 that we combined with two bidirectional terminators (BB1001) and had it synthesized by iDT. But the attachment sequence used in BBa_I111023 is not the &lambda;-attP-site, it corresponds to the attachment site attp2, that is used in context of the gateway &reg; cloning system. Compared to the &lambda;-phage attP-sequence it bears three significant mutations within it’s for the recombination process highly relevant O-site.
 +
 
 
</div>
 
</div>
 
<div class="references"><h6>References</h6>
 
<div class="references"><h6>References</h6>
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                             <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/OrthogonalPair">Incorporation of OMT</a><br/>
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                             <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/OrthogonalPair">Incorporation of a nnAA</a><br/>
 
<a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/Reporter">Reporter</a><br/>
 
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ABSTRACT

Artificial plasmids are a significant burden to the host. The design of pathways, e.g. the combination of different promoter and RBS systems, results in different amounts of product. Measurement of the metabolic burden is a key for quantitative optimization of metabolic engineering approaches. We want to establish a new approach to iGEM by providing a measurement strain to the community. As described by F. Ceroni et al., we integrated one copy of the gfp gene into the genome of E. coli, which offers us a highly accurate and instantaneous measurement of the impact of our plasmids on the host. Metabolic burden measurement is of economical interest, because it enables academic and industrial research testing several different pathways at once in a short period of time by using microplate reader. For the integration we used the λ‑Integrase site‑specific recombination pathway, described by A. Landy in 2015 [9]. Therefore, we designed two plasmids (BBa_K1976000 and BBa_K1976001) and measured them using single cell measurement via microplate reader.

Metabolic Burden

In synthetic biology, the term "metabolic burden" describes the influence of heterologously expressed genes on the distribution availability of resources in the host cell. Thereby the extent of metabolic burden is not only dependent on the consumption of energy and reaction equivalents but is also influenced by a number of different factors. In addition to size and copy number of the used plasmid [1], the specific properties of the heterologous proteins play an important role. Proteins that interfere with the host's metabolism, influence the proton gradient or are toxic in a different way can pose a strong burden for the cell already at low expression levels [1, 2]. I. Shachrai et al. could show that ribosome availability represents a highly limiting factor for metabolic performance [3].
If the metabolic burden on the host cell is too high, the physiology and biochemistry of the cell will be drastically altered, e.g. the viability or proliferation of the cell could be disturbed. In addition, the error rate in translation increases which heightens the immunogenicity of the incorporated proteins and can lead to a reduced protein activity and stability [1]. Metabolic burden is a big issue especially in the industrial sector [4], because lowered expression efficiency decreases product yield.
Often used methods to reduce the metabolic burden include for example the separation of the biosynthetic pathway into multiple organisms through co‑cultivating them or the identification and deletion of expendable genes as a part of strain optimization [6]. Another approach is the regulation of protein expression with Dynamic Sensor and Regulatory Systems (DSRSs). Such systems are based on the usage of transcription factors which detect certain key‑metabolites and regulate the transcription simultaneously [7].
One possibility to quantify the metabolic burden in vivo was described by F. Ceroni et al. In this method a GFP reporter gene is integrated in the genome of E. coli using the λ‑integrase [8]. Through measuring the fluorescence it could be shown that the constitutive expression of GFP after transformation with expression‑plasmids drastically decreases in comparison to not transformed cells.

Measurement of the Metabolic Burden via Microplate Reader

In addition to the stress forced onto the cell, the total amount of formed GFP is significantly influenced by the cell amount. For this reason the measurement of GFP with a microplate reader was performed under continuous observation of the cell density. With this kind of measurement many samples, like in 96‑well‑plates, can be analyzed. Since the measurement of different combinations of plasmid and cell‑type, would enable to determine the artificially caused stress on the cells proportional to the decrease in GFP expression, this kind of measurement is assumed to be a more economical approach than the single cell measurement described in the next paragraph.

Single-cell Measurement

While measuring every single cell individually, the cell density can be neglected which leads to a smaller error in the fluorescence measurement. This method enables the detection of the influence of the metabolic burden based on the GFP production. Therefore, it is assumed to be a more exact method than the previously described method using a microplate reader.

Genomic Integration

The λ‑integrase, originally derived from the λ‑phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (attB) and the other located on the λ‑genome (attP), which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB' region (attB) and COC' region (attP). These regions can be connected by the λ‑integrase and the bacterial integration host factor (IHF) via Holliday junction forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites attL and attR.

Integration Plasmid and Helper Plasmid

For the integration of the gene of interest (GOI) into the chromosomal genome of E. coli two plasmids are needed. The integration plasmid contains the constitutive generator of GFP, which is also the necessary reporter for the measurement of the metabolic burden and should be integrated into the genome of E. coli. To ensure that only temporary fluorescence are measured, a LVA degradation tag was added to the GFP. The plasmid also contains the attP that enables the integration. Additionally, two bidirectional terminators are located on each side of the attP to protect the GFP operon from the transcription during biosynthesis of neighbouring genes. To create the integration plasmid E0240 (RBS(BB0032+GFP)) was cloned in a J61002 backbone to locate the GFP gene under the control of the promoter J23101. The construct J23101+E0240 was mutated via mutagenesis PCR for optimizing BBa_I11023 and afterwards cloned in the high‑copy vector pSB1C3 to increase the yield of the plasmid preparation. The mutagenesis PCR aimed to mutate attp2 to &lambda&;‑attP. To finalize the construct, we changed the backbone to pSB4A5 which possesses a low-copy ori to facilitate the later performed plasmid curing. The second plasmid is a helper plasmid which is necessary for transposing the gfp gene into the chromosomal genome as it contains the λ‑integrase generator. For inscription in the registry the construct was cloned in a pSB1C3 backbone and , additionally, to facilitate additional performed plasmid curing via sustained lack of selection pressure, we again chose the low‑copy vector pSB4K5 .
To verify the success of the recombination, PCR was performed, using specific primers to the attB site of the E. coli and the VR primer binding on every BioBrick standard vectors. Since the first primer binds to the genome and the other is complimentary to the plasmid, a PCR amplicon could be optained only if the integration has succeeded.

Plasmid Curing

.....

Integration Strains

A suitable genomic integration strain needs to carry the attB sequence which is necessary for the λ‑integrase mediated homologous recombination. Many commonly used E. coli strains carry the λ‑phage integrated in their genome. But since the attB site was often used to integrate genes via λ recombination, e.g. T7 polymerase in BL21 (DE3), in many strains the attachment site is not accessible anymore. For our integration we chose E. coli JM109 because it matches all our demands and is also freely and easily available to our lab.

Results
1. Integration Plasmid - GFP‑construct

The first major step in assembling the integration plasmid was to choose a useful promoter for an expression of GFP, which should be strong enough to be measured but low enough to keep the metabolic burden as low as possible.


Figure 1: E.coli transformed with GFP behind three different promoters. A: GFP and JM23109, B: GFP and JM23115, C: GFP and JM23101

Three different promoters and GFP were transformed into E. Coli to test their strenght. In dish A the GFP was combined with the JM23109 promoter, in dish B with the JM23115 promoter and in dish C with the JM23101 promoter. As it can be seen above in the comparison, GFP is best transcripted with the JM23101 promoter, so we decided to use it in our integration plasmid. With the other two promoters the fluorescence would not be strong enough to be measured after plasmid curing, as there would be only one GFP copy left in the cells.

The next major step was to mutate the synthesized attp2‑site to the needed attp‑site of the λ‑integrase.
This sequence was improved to the following sequence:


Figure 2: Compared alignment of the attp2 gene and the attp gene. The red marked spots show the mutations that were corrected


The third major step in the assembly of the integration plasmid was the adding of a LVA‑Tag to the GFP sequence to ensure a fast degradation. (This was done to further decrease the metabolic burden caused by the GFP and to make a fast answer in the fluorescence possible so accurate measurements can be made.)
//Sequenz mit LVA-Tag hier einfügen//
The sequence shows that the mutagenesis PCR for adding the LVA‑Tag was succesful.

The last major step of the assembly was to ligate the λ‑attp+GFP‑LVA construct into a pSB1C3 vector to submit the part to the iGEM registry.
To determine if the ligation was succesful a gelelectrophoresis was made using VR‑ and VF2‑primers.


Figure 3: Gelelectrophoresis of the λ‑attp+GFP‑LVA construct in pSB1C3. The marked band shows the wanted product

The band coming from the sixth batch of the gelelectrophoresis of the cPCR shows that the ligation was succesful.

2. Helping Plasmid - Integrase

Due to a mistake in ordering the integrase there was a LVA‑tag at the end of the sequence.
Therefore the LVA‑tag was deleted with a mutagenesis PCR. The sequencing shows that the deletion of the LVA‑Tag was succesful.
Click the following link to get a .zip file with further informations on our sequences.

Next was the ligation of the integrase on a pSB1C3 for part submission and to measure if the integrase is expressed.


Figure 4: Gelelectrophoresis of the λ‑integrase in pSB1C3. The marked band shows the wanted product

The lanes three and five show a succesful ligation in pSB1C3 and are therefore used for further experiments. To determine if the integrase is expressed a SDS‑Page was done.

Figure 5: SDS‑Page of the λ‑integrase
The marked spot on the SDS‑Page shows a sufficient expression of the wanted integrase

For the detemination if our integrase works as expected and therefore have a proof of concept we tried to genomically integrate our integration plasmid into the genome of E. coli with the help of the integrase. But before doing that we put both constructs on midi‑copy vectors (pSB1K3 and JM23101). To test if this was succesful a cPCR which only yields product if the genomic integration was succesful was performed.


Figure 6: Proof for the genomic integration The gelelectrophoresis shows that in colonies three and four the genomic integration were successful and therefore our Integrase works as expected.

3. Genomic Integration and Measurement

As previously described the genomic integration using the integration plasmid and the helping plasmid was succesful.
To test if our measurement system works as expected we transformed the cells which carry the GFP with the Naringenin Biosensor BBa_K1497021 of the 2014 TU Darmstdt iGEM Team. Graphen fehlen noch
The first graph shows that the optical density is decreasing during the measurement. The second graph shows that

4. Improved Part

For genomic integration via &lambda-phage (attP/attB) recombination, a functional phage-attachment site (attP) is essential. Here, we initally used the attachment sequence from BBa_I111023 that we combined with two bidirectional terminators (BB1001) and had it synthesized by iDT. But the attachment sequence used in BBa_I111023 is not the λ-attP-site, it corresponds to the attachment site attp2, that is used in context of the gateway ® cloning system. Compared to the λ-phage attP-sequence it bears three significant mutations within it’s for the recombination process highly relevant O-site.

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