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Team:ETH Zurich/Mutant
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| - | < | + | <h1>Triple knockout strain</h1> |
| - | < | + | <p align="justify">In order to use the orthogonal [https://2013.igem.org/Team:ETH_Zurich/Experiments_7 hydrolases] as our reporter system in the sender-receiver set-up, we needed to use a strain of ''E. coli'' where the expression of native hydrolase genes was knocked out. This is to prevent background hydrolysis of our colorimetric substrates. <br> |
| + | In our sender-receiver circuit, we use three hydrolases'' gusA'' , ''aes'' and ''nagZ''. Hence, three hydrolase genes were knocked out of ''E. coli'' strain MG1655. The ''gusA'' hydrolase was knocked out by a scarless deletion scheme that left no scar sequences after the deletion method (1). The subsequent deletions of ''aes'' and ''nagZ'' were carried out by the P1 phage transduction by using a deletion strain from the Keio collection (2). Thus we were able to use this strain MG1655△''gusA''△''aes''△''nagZ'' knocked out of expression of the three hydrolase genes in our project.<br> </p> | ||
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| + | The triple mutant is used for the sender cells which are made to constitutively express ''nagZ''. In the receiver cells, the hydrolases ''aes'' and ''gusA'' are expressed under the control of OHHL induced P<sub>luxR</sub> promoters which serve as [https://2013.igem.org/Team:ETH_Zurich/Experiments_5 highpass filters]. | ||
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| + | <h1>References</h1> | ||
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| + | (1) Martinez-Garcia .E, de Lorenzo .V. 2012. Transposon based and plasmid based genetic tools for editing genomes of gram-negative bacteria. Methods in Molecular Biology. 813:267-283.doi: 10.1007/978-1-61779-412-4_16. <br> | ||
| + | (2) Thomason L.C., Costantino .N, Court .D .L, 2007. ''E. coli'' Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology.1.17.1-1.17.8. | ||
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Latest revision as of 03:32, 5 October 2013
Triple knockout strain
In order to use the orthogonal hydrolases as our reporter system in the sender-receiver set-up, we needed to use a strain of E. coli where the expression of native hydrolase genes was knocked out. This is to prevent background hydrolysis of our colorimetric substrates.
In our sender-receiver circuit, we use three hydrolases gusA , aes and nagZ. Hence, three hydrolase genes were knocked out of E. coli strain MG1655. The gusA hydrolase was knocked out by a scarless deletion scheme that left no scar sequences after the deletion method (1). The subsequent deletions of aes and nagZ were carried out by the P1 phage transduction by using a deletion strain from the Keio collection (2). Thus we were able to use this strain MG1655△gusA△aes△nagZ knocked out of expression of the three hydrolase genes in our project.
The triple mutant is used for the sender cells which are made to constitutively express nagZ. In the receiver cells, the hydrolases aes and gusA are expressed under the control of OHHL induced PluxR promoters which serve as highpass filters.
References
(1) Martinez-Garcia .E, de Lorenzo .V. 2012. Transposon based and plasmid based genetic tools for editing genomes of gram-negative bacteria. Methods in Molecular Biology. 813:267-283.doi: 10.1007/978-1-61779-412-4_16.
(2) Thomason L.C., Costantino .N, Court .D .L, 2007. E. coli Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology.1.17.1-1.17.8.
