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

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<h4> Cloning Scheme </h4>
<h4> Cloning Scheme </h4>
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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 an adjusted T7 lac promoter, which was found to be the best promoter to express <i>mtrCAB</i> in <i> E. coli </i> [1]. To get rid of the illegal restriction sites as well as implementing the adjusted T7 lac promoter in our BioBrick <a href="http://parts.igem.org/Part:BBa_K1316012">BBa_K1316012 </a> , we ended up having 5 pieces of DNA to be ligated into pSB1C3. To work efficiently, we used the LINK Golden Gate Assembly to clone <a href="http://parts.igem.org/Part:BBa_K1316012">BBa_K1316012 </a>  (see figure 1).
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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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Figure 1. <b>Golden Gate Assembly of the <i>mtrCAB</i> operon.</b> The BioBrick <a href="http://parts.igem.org/Part:BBa_K1316012">BBa_K1316012 </a>  encodes an adjusted T7 lac 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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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.  
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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<p>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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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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Revision as of 22:39, 14 October 2014

Module Electron Transport - Cloning

click to return to the  Module Electron Transport


Final constructs

The final BioBricks constructed for this module were:

  • mtrCAB and mtrCAB-His

  • ccmAH

    • mtrCAB and mtrCAB-His

    Final constructs

    The two BioBricks constructed using the mtrCAB genes were BBa_K1316012 and BBa_K1316017 (figures 1 and 2).
    Figure 1: Plasmid carrying the BBa_K1316012 BioBrick.
    Figure 2: Plasmid carrying the BBa_K1316017 BioBrick.


    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 an adjusted T7 lac promoter, which was found to be the best promoter to express mtrCAB in E. coli [1]. To get rid of the illegal restriction sites as well as implementing the adjusted T7 lac promoter in our BioBrick BBa_K1316012 , we ended up having 5 pieces of DNA to be ligated into pSB1C3. To work efficiently, we used the LINK Golden Gate Assembly to clone BBa_K1316012 (see figure 1).

    Figure 1. Golden Gate Assembly of the mtrCAB operon. The BioBrick BBa_K1316012 encodes an adjusted T7 lac 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.


    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.

    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.

    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.

    A more graphical representation of the cloning strategy can be found on figure 3.

    Important notes
  • 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 mtrCAB 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.
  • 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.
    • 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.


    ccmAH

    Final constructs

    The BioBrick constructed using the ccmAH gene cluster is BBa_K1316011 (figure 4).

    Figure 4: Plasmid carrying the BBa_K1316011 BioBrick.


    Cloning Scheme

    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 BBa_K917006 constructed by the iGEM team Edinburgh 2012, which was already BioBrick compatible.

    A more graphical 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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