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Team:Cornell/project/wetlab/assembly
From 2012.igem.org
Summary of DNA Assembly
Genetic Parts Rely on Complementation Strategy To Sense Analyte
When MtrB is not present, it is as if the switch is open, and current cannot flow.
When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.
As described on our chassis page, the protein MtrB is required for
S. oneidensis to be able to shuttle electrons outside the cell. Therefore, when a strain of S. oneidensis lacking mtrB is inoculated in a bioelectrochemical system, current cannot be significantly produced.
We can think of this in terms of a simple switch analogy—visualized in the figures to the left: Without MtrB, no extracellular transfer of electrons is possible, and no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.
To sensitize Shewanella’s metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the Shewanella MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB—and therefore current—should only be produced in the presence of analyte.
One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist.
We can think of this in terms of a simple switch analogy—visualized in the figures to the left: Without MtrB, no extracellular transfer of electrons is possible, and no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.
To sensitize Shewanella’s metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the Shewanella MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB—and therefore current—should only be produced in the presence of analyte.
One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist.
