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Team:Trieste/parts/8
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<h2>Assembly</h2> | <h2>Assembly</h2> | ||
| - | The BioBrick BBa_K314200 coding for the Tse2 toxin was assembled with the weak BBa_B0031 RBS and with the BBa_B0015 terminator. This composite was then cloned under the inducible promoter T5 LacO ( that we submitted as BioBrick BBa_K875002 ) in order to be able to follow the toxin activity when expressed in E.coli. | + | The BioBrick BBa_K314200 coding for the Tse2 toxin was assembled with the weak BBa_B0031 RBS and with the BBa_B0015 terminator. This composite was then cloned under the inducible promoter T5 LacO ( that we submitted as BioBrick BBa_K875002 ) in order to be able to follow the toxin activity when expressed in <i>E.coli</i>. |
<center><img src="https://static.igem.org/mediawiki/2012/7/7f/Tse2ts.gif" width="500px"/></center> | <center><img src="https://static.igem.org/mediawiki/2012/7/7f/Tse2ts.gif" width="500px"/></center> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
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<p>M15 bacterial cells (containing the pREP4 plasmid coding for the Lac repressor) are transformed with this Biobrick that resides on pSB1C3 plasmid.</br> | <p>M15 bacterial cells (containing the pREP4 plasmid coding for the Lac repressor) are transformed with this Biobrick that resides on pSB1C3 plasmid.</br> | ||
</br> | </br> | ||
| - | After an over night liquid culture (LB broth) the O.D.<sub>600</sub> absorbance was recorded and the culture was diluted to O.D.=0,1 and splitted into 6 flask – 20ml each. Three of these were induced with IPTG 1mM and the others were not induced.</br> | + | After an over night liquid culture (LB broth) the O.D.<sub>600</sub> absorbance was recorded and the culture was diluted to O.D.<sub> 600</sub>=0,1 and splitted into 6 flask – 20ml each. Three of these were induced with IPTG 1mM and the others were not induced.</br> |
</br> | </br> | ||
| - | The O.D.<sub> 600</sub> was then recorded every hour.</br> | + | The O.D.<sub>600</sub> was then recorded every hour.</br> |
</br> | </br> | ||
| - | It is possible to observe that the not induced cells grew up to O.D.<sub> 600</sub>=1, while the induced ones did not grow, demonstrating the efficiency of both the inducible promoter and the toxin.</br> | + | It is possible to observe that the not induced cells grew up to O.D.<sub>600</sub> = 1, while the induced ones did not grow, demonstrating the efficiency of both the inducible promoter and the toxin.</br> |
</p> | </p> | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
<h2>Looking forward</h2> | <h2>Looking forward</h2> | ||
| - | <p> In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001). | + | <p> In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001). You can find the results on <a href="https://2012.igem.org/Team:Trieste/project/mainres"> Main Results page</a>, in the suicide system section. |
</p> | </p> | ||
</br> | </br> | ||
Latest revision as of 18:02, 26 October 2012
BBa_K875008
More
Description
The Tse2 protein is a toxin component of a toxin-immunity system and arrests the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 has no effect on eukaryotic cell.
Assembly
The BioBrick BBa_K314200 coding for the Tse2 toxin was assembled with the weak BBa_B0031 RBS and with the BBa_B0015 terminator. This composite was then cloned under the inducible promoter T5 LacO ( that we submitted as BioBrick BBa_K875002 ) in order to be able to follow the toxin activity when expressed in E.coli.
Results
M15 bacterial cells (containing the pREP4 plasmid coding for the Lac repressor) are transformed with this Biobrick that resides on pSB1C3 plasmid. After an over night liquid culture (LB broth) the O.D.600 absorbance was recorded and the culture was diluted to O.D. 600=0,1 and splitted into 6 flask – 20ml each. Three of these were induced with IPTG 1mM and the others were not induced. The O.D.600 was then recorded every hour. It is possible to observe that the not induced cells grew up to O.D.600 = 1, while the induced ones did not grow, demonstrating the efficiency of both the inducible promoter and the toxin.
Looking forward
In the final system, the toxin should be under the control of the T5 Cumate Operator (BBa_K875001). You can find the results on Main Results page, in the suicide system section.
Link to the Registry
