Team:TU Delft-Leiden/Project/Life science/EET/cloning

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<h2>Module Electron Transport &ndash; Cloning</h2>
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<h2> Module Electron Transport - Cloning</h2>
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<p>In the wet lab we integrated the Electron Transport pathway of <i>Shewanella oneidensis</i> into <i>Escherichia coli</i>. Here you can find information with respect to cloning of the BioBricks for the Electron Transport pathway. </p>  
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click to return to the&nbsp; <a href="https://2014.igem.org/Team:TU_Delft-Leiden/Project/Life_science/EET"> <b> Module Electron Transport </b> </a>
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         <ul>
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            <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET">Module Electron Transport</a>
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<h3> Final constructs </h3>
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The final BioBricks constructed for this module were:
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<li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning#mtrCAB"><p>mtrCAB and mtrCAB-His </p>
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<li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning#ccmAH"><p>ccmAH</p>
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        <ul>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/theory">Context</a></li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/integration">Integration of Departments</a></li>
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            <li>Cloning</li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning#mtrCAB"><i>mtrCAB</i> and <i>mtrCAB:HIS</i></a></li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning#ccmAH"><i>ccmAH</i></a></li>
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        </ul>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/characterisation">Characterization</a></li>
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<a name="mtrCAB"></a>  
<a name="mtrCAB"></a>  
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<h3> mtrCAB  and mtrCAB-His </h3>
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<h3> <i>mtrCAB</i> and <i>mtrCAB-HIS</i> </h3>
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<p>
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<h4> Final constructs </h4>
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Our BioBricks <a href="http://parts.igem.org/Part:BBa_K1316012" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316012</font></a> and <a href="http://parts.igem.org/Part:BBa_K1316017" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316017</font></a> encode the <i>mtrCAB</i> genes under control of a weakened T7 promoter with the lac operator (T7 lacO). In addtion, BBa_K1316017 carries a histidine (HIS) tag at the 5' end of the coding sequence which makes it easier to purify MtrB during characterization experiments. The end constructs are visualized in figure 1 and 2.
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The two BioBricks constructed using the mtrCAB genes were
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<a href="http://parts.igem.org/Part:BBa_K1316012" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316012</font></a>
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and  
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<a href="http://parts.igem.org/Part:BBa_K1316017" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316017</font></a> (figures 1 and 2).
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<img src="https://static.igem.org/mediawiki/2014/0/02/TUDelft_2014_BBa_K1316012_Map.png" width="100%" height="100%">
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<img src="https://static.igem.org/mediawiki/2014/0/02/TUDelft_2014_BBa_K1316012_Map.png" width="60%" height="60%">
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Figure 1: Plasmid carrying the BBa_K1316012 BioBrick.
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Figure 1: <b>BBa_K1316012</b>. Schematic overview of our BioBrick encoding the <i>mtrCAB</i> genes under control of a T7 lacO promoter.
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<img src="https://static.igem.org/mediawiki/2014/d/d3/TUDelft_2014_BBa_K1316017_Map.png" width="100%" height="100%">
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<img src="https://static.igem.org/mediawiki/2014/d/d3/TUDelft_2014_BBa_K1316017_Map.png" width="60%" height="60%">
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Figure 2: Plasmid carrying the BBa_K1316017 BioBrick.
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Figure 2: <b>BBa_K1316017</b>. Schematic overview of our BioBrick encoding the <i>mtrCAB</i> genes and <i>HIS</i> tag under control of a T7 lacO promoter.
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<h4> Cloning Scheme </h4>
<h4> Cloning Scheme </h4>
<p>
<p>
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The template used to obtain the mtrCAB genes was the Ajo-Franklin plasmid [1], from which 5 different pairs of primers were designed to eliminate the main iGEM illegal restriction sites by means of the  
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Making a BioBrick of the <i>mtrCAB</i> operon is quite challenging. First of all, the coding sequence of <i>mtrCAB</i> contains several illegal restrictions sites. Secondly, we need to have the operon under the regulation of a weakened T7 lacO promoter[1]. To get rid of the illegal restriction sites as well as implementing the T7 lacO promoter in our BioBricks <a href="http://parts.igem.org/Part:BBa_K1316012">BBa_K1316012 </a> and <a href="http://parts.igem.org/Part:BBa_K1316017">BBa_K1316017 </a>, we ended up having 5 pieces of DNA to be ligated into pSB1C3. To work efficiently, we used the <a href="https://2012.igem.org/Team:Freiburg/Project/Golden#GGC">Golden Gate Assembly </a> to clone these BioBricks  (see figure 3).  
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<a href="https://2014.igem.org/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning/GoldenGate" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">Golden Gate Assembly method</font></a>.  
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<center><img style="margin-left: -10%;" src="https://static.igem.org/mediawiki/2014/archive/8/86/20141011133410%21TUDELFT2014_goldengateET.PNG" width="120%"></center>
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<figcaption>
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Figure 3. <b>Golden Gate Assembly of the <i>mtrCAB</i> operon.</b> The BioBricks <a href="http://parts.igem.org/Part:BBa_K1316012">BBa_K1316012</a> and <a href="http://parts.igem.org/Part:BBa_K1316017">BBa_K1316017</a> encode a weakened T7 lacO promoter (red rectangle) and the coding sequences of <i>mtrC</i>, <i>mtrA</i> and <i>mtrB</i>, indicated with grey arrows. The beam beneath the arrows visualizes the regions and relative sizes of the 5 DNA pieces that were ligated into pSB1C3.
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<p>
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To generate BBa_K1316017 (construct with mtrCAB-His), the last reverse primer was designed in a way that a 6 x His tag was introduced at the C-terminus of the MtrB protein. This tag aims to be able to easily detect and purify this protein.
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Once the 5 fragments were PCRed, they were assembled together via the Golden Gate Assembly method. The Golden Gate primers were designed so that, due to the used BsaI enzyme,  at the end of this assembly the 5' end of the assembled construct was compatible with a fragment restricted with EcoRI and the 3' end with a fragment restricted with SpeI. Consequently, the Assembled fragment was compatible with an iGEM backbone restricted with the enzymes EcoRI and SpeI. This way, the assembled construct was introduced into the pSB1C3 iGEM backbone.
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<p>The Golden Gate primers were designed in a way that restriction with the BsaI enzyme resulted in a EcoRI overhang at the 3' end of the DNA and a SpeI overhang at the 5' of the DNA. In this way we were able to clone the DNA fragment into pSB1C3 by using EcoRI and SpeI.
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To construct the <i>mtrCAB-HIS</i>, the reverse primer of the last DNA part was designed in a way that a HIS tag was introduced at the C-terminus of the MtrB protein. However, there was no additional stop codon included in this primer. Therefore we do not characterize this BioBrick.  
<p>
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Later on, the mtrCAB BioBricks (BBa_K1316012 and BBa_K1316017) were introduced into pSB3K3, because it is a backbone with a much lower copy number than pSB1C3, and in the literature the mtrCAB genes were found to be quite toxic for Escherichia coli.
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For characterization the <i>mtrCAB</i> BioBrick was introduced into pSB3K3. This backbone has a lower copy number than pSB1C3 and is therefore more consistent with the literature [1].
</p>
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<p> A more graphical representation of the cloning strategy can be found on figure 3.
 
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<h6>Important notes </h6>
 
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<li>It is important to notice that the actual assembly used the 5 fragments PCRed from the original Ajo-Franklin plasmid containing the mtrCAB genes. In the
 
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<a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning#mtrCAB Fig">mtrCAB
 
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figure it might seem that this happened in two steps, but that is due to the Software used to check the assembly "in silico" (SnapGene), which was limited to an assembly of four fragments.</li>
 
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<li>The construct with MtrB carrying the 6xHisTag (BBa_K1316017) was made the same way as BBa_K1316012 but the reverse primer amplifying mtrB was designed in order to incorporate the Tag right before the STOP codon of this gene. It must be pointed out that there was a mistake on the design of this construct and the STOP codon is not right after the HisTag but few baseparis afterwards.</li>
 
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<a name="mtrCAB Fig"></a>
 
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<img src="https://static.igem.org/mediawiki/2014/1/1f/TUDelft_2014_Mtr_in_pSB3k3_GOOD_History.png" width="100%" height="100%">
 
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Figure 3: Cloning scheme of the BBa_K1316012 BioBrick. BioBrick BBa_K1316017 was designed the same was made the same way as BBa_K1316012 but the reverse primer amplifying mtrB was designed in order to incorporate a 6xHisTag on its C-terminus end.
 
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<a name="ccmAH"></a>  
<a name="ccmAH"></a>  
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<h3> ccmAH </h3>
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<h3> <i>ccmAH</i> </h3>
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<h4> Final constructs </h4>
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Our BioBrick <a href="http://parts.igem.org/Part:BBa_K1316011" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316011</font></a> encodes the <i>cytochrome C maturation</i>  genes (<i>ccm</i>) under control of the pFAB640 promoter [1]. The Ccm proteins help to mature the MtrCAB conduit and are therefore needed to facilitate Electron Transport. The end construct is visualized figure 4.  
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The BioBrick constructed using the ccmAH gene cluster is
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<a href="http://parts.igem.org/Part:BBa_K1316011" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K1316011</font></a> (figure 4).  
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<img src="https://static.igem.org/mediawiki/2014/a/a0/TUDelft_Pfab_promoter_%2B_NdeI_%2B_ccmAH_Map.png" width="100%" height="100%">
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<img src="https://static.igem.org/mediawiki/2014/c/ca/TUDELFT2014ccmAH_Map.png" width="60%" height="60%">
<figcaption>
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Figure 4: Plasmid carrying the BBa_K1316011 BioBrick.
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Figure 4: <b>BBa_K1316011</b>. Schematic overview of the <i>ccm</i> genes under control of the pFAB640 promoter.
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<h4> Cloning Scheme </h4>
<h4> Cloning Scheme </h4>
<p>
<p>
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The BioBrick BBa_K1316011 was built up by amplifying the best performing promoter according to Goldbeck [1] and introducing it upstream de coding region of the ccmAH cluster of the Biobrick
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In order obtain our <i>ccm</i> BioBrick we inserted the pFAB640 promoter in 
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<a href="http://parts.igem.org/Part:BBa_K917006" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K917006</font></a>
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constructed by the iGEM team Edinburgh 2012, which was already BioBrick compatible.
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<a href="http://parts.igem.org/Part:BBa_K917006" style="text-decoration: none"" target="_blank"><font color="#0080FF" size="3">BBa_K917006</font></a>, a BioBrick that encodes the <i>ccm</i> gene cluster,
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constructed by the Edinburgh 2012 iGEM team.
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A schematic representation of the cloning strategy can be found on figure 5.
</p>
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<p> A more graphical representation of the cloning strategy can be found on figure 5.
 
<p>
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<figure>
<figure>
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<img src="https://static.igem.org/mediawiki/2014/1/10/TUDelft_2014_Pfab_promoter_%2B_NdeI_%2B_ccmAH_History.png" width="100%" height="100%">
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<a href="https://static.igem.org/mediawiki/2014/1/10/TUDelft_2014_Pfab_promoter_%2B_NdeI_%2B_ccmAH_History.png" class="zfgallery2" style="display: block; width:100%; height:100%;">
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        <img src="https://static.igem.org/mediawiki/2014/1/10/TUDelft_2014_Pfab_promoter_%2B_NdeI_%2B_ccmAH_History.png" style="display: block; width:60%; height:120%;">
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    </a>
<figcaption>
<figcaption>
Figure 5: Cloning scheme of BBa_K1316011 BioBrick.
Figure 5: Cloning scheme of BBa_K1316011 BioBrick.
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<h3> References </h3>
<h3> References </h3>
<p>[1] C.P. Goldbeck, H.M. Jensen et al., “Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli”, ACS Synth. Biol. 2, 150-159, 2013. </p>
<p>[1] C.P. Goldbeck, H.M. Jensen et al., “Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli”, ACS Synth. Biol. 2, 150-159, 2013. </p>

Latest revision as of 16:40, 17 October 2014

Module Electron Transport – Cloning

In the wet lab we integrated the Electron Transport pathway of Shewanella oneidensis into Escherichia coli. Here you can find information with respect to cloning of the BioBricks for the Electron Transport pathway.

mtrCAB and mtrCAB-HIS

Our BioBricks BBa_K1316012 and BBa_K1316017 encode the mtrCAB genes under control of a weakened T7 promoter with the lac operator (T7 lacO). In addtion, BBa_K1316017 carries a histidine (HIS) tag at the 5' end of the coding sequence which makes it easier to purify MtrB during characterization experiments. The end constructs are visualized in figure 1 and 2.

Figure 1: BBa_K1316012. Schematic overview of our BioBrick encoding the mtrCAB genes under control of a T7 lacO promoter.
Figure 2: BBa_K1316017. Schematic overview of our BioBrick encoding the mtrCAB genes and HIS tag under control of a T7 lacO promoter.


Cloning Scheme

Making a BioBrick of the mtrCAB operon is quite challenging. First of all, the coding sequence of mtrCAB contains several illegal restrictions sites. Secondly, we need to have the operon under the regulation of a weakened T7 lacO promoter[1]. To get rid of the illegal restriction sites as well as implementing the T7 lacO promoter in our BioBricks BBa_K1316012 and BBa_K1316017 , we ended up having 5 pieces of DNA to be ligated into pSB1C3. To work efficiently, we used the Golden Gate Assembly to clone these BioBricks (see figure 3).

Figure 3. Golden Gate Assembly of the mtrCAB operon. The BioBricks BBa_K1316012 and BBa_K1316017 encode a weakened T7 lacO promoter (red rectangle) and the coding sequences of mtrC, mtrA and mtrB, indicated with grey arrows. The beam beneath the arrows visualizes the regions and relative sizes of the 5 DNA pieces that were ligated into pSB1C3.


The Golden Gate primers were designed in a way that restriction with the BsaI enzyme resulted in a EcoRI overhang at the 3' end of the DNA and a SpeI overhang at the 5' of the DNA. In this way we were able to clone the DNA fragment into pSB1C3 by using EcoRI and SpeI. To construct the mtrCAB-HIS, the reverse primer of the last DNA part was designed in a way that a HIS tag was introduced at the C-terminus of the MtrB protein. However, there was no additional stop codon included in this primer. Therefore we do not characterize this BioBrick.

For characterization the mtrCAB BioBrick was introduced into pSB3K3. This backbone has a lower copy number than pSB1C3 and is therefore more consistent with the literature [1].


ccmAH

Our BioBrick BBa_K1316011 encodes the cytochrome C maturation genes (ccm) under control of the pFAB640 promoter [1]. The Ccm proteins help to mature the MtrCAB conduit and are therefore needed to facilitate Electron Transport. The end construct is visualized figure 4.

Figure 4: BBa_K1316011. Schematic overview of the ccm genes under control of the pFAB640 promoter.


Cloning Scheme

In order obtain our ccm BioBrick we inserted the pFAB640 promoter in BBa_K917006, a BioBrick that encodes the ccm gene cluster, constructed by the Edinburgh 2012 iGEM team. A schematic representation of the cloning strategy can be found on figure 5.

Figure 5: Cloning scheme of BBa_K1316011 BioBrick.

References

[1] C.P. Goldbeck, H.M. Jensen et al., “Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli”, ACS Synth. Biol. 2, 150-159, 2013.

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