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Team:TU Delft-Leiden/Project/Life science/EET/cloning/GoldenGate
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<h2> Extracellular Electron Transport Module - Cloning - Golden Gate Assembly</h2> | <h2> Extracellular Electron Transport Module - Cloning - Golden Gate Assembly</h2> | ||
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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, 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 our BioBrick. | + | 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, 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 our BioBrick (see figure 1). |
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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> | <center><img style="margin-left: -10%;" src="https://static.igem.org/mediawiki/2014/archive/8/86/20141011133410%21TUDELFT2014_goldengateET.PNG" width="120%"></center> | ||
<figcaption> | <figcaption> | ||
| - | Figure 1. <b>Golden Gate Assembly of the <i>mtrCAB</i> operon.</b> | + | Figure 1. <b>Golden Gate Assembly of the <i>mtrCAB</i> operon.</b> The BioBrick encodes an adjusted T7 lac promoter (red rectangle) and the coding sequences of <i>mtrC</i>, <i>mtrA</i> and <i>mtrB</i>, indicated by the grey arrows. The beam beneath the arrows visualizes the relative size and coding regions of the 5 DNA pieces that were ligated into pSB1C3. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
Revision as of 10:53, 12 October 2014
Extracellular Electron Transport Module - Cloning - Golden Gate Assembly
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, 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 our BioBrick (see figure 1).
References
1. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli. ACS Synth. Biol. 2 (3), pp 150–159 (2013)
