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Team:Yale/Our Project
From 2010.igem.org
our project
Project Overview
What would it take to...
Build a circuit using microbially catalyzed metal sulfide deposition?
Background: SRB, H2S, and Copper Sulfides
The inspiration for this idea came from an ecological observation made of copper biomineralization by a species of Sulfate Reducing Bacteria. Sulfate Reducing Bacteria (SRB) comprise a family of chemolithotrophic bacteria that use sulfate as the terminal electron acceptor in anaerobic metabolism. As a result of sulfate reduction, these bacteria produce gaseous H2S. Subsequently, microbially generated H2S reduces copper in solution and precipitates soluble copper ions in the form of insoluble copper sulfide (CuS). It was discovered that by this method, some strains of SRB formed a CuS compound nearly identical to covellite - a natural superconductor (Weber, 2009). If this activity could be enhanced under spatial and temporal control, bacteria could be harnessed to deposit metal sulfide in specified geometries for manufacturing and engineering applications.
Building H2S Production Activity into E. coli
Investigation of H2S production in bacteria has been well documented in E. coli. Work by Dr. Jay Keasling at University of California, Berkeley, has shown that a gene encoding Thiosulfate Reductase from Salmonella enterica serovar Typhimurium has previously been expressed in E. coli to overproduce hydrogen sulfide from thiosulfate.
The gene phsABC functionally encodes Thiosulfate reductase. The gene itself contains an open reading frame for each of the three genes phsA, phsB, and phsC. The resulting complex of three transmembrane proteins constitutes Thiosulfate reductase and catalyzes the reduction of inorganic thiosulfate to hydrogen sulfide.
This activity can be monitored by growing bacteria on
Triple Sugar Iron (TSI) Agar. Evolution of H2S as a gas, results in the reduction of Iron Sulfate to an Iron Sulfide. Iron sulfide precipitates as a black solid and can be easily identified visually on the macroscopic scale. Bacteria that are actively producing H2S will reduce the Iron in the medium, and turn the agar black.
Growing Bacteria In Copper Medium
Bacteria expressing Thiosulfate Reductase can then be grown in media containing both a source of sulfur and free copper in solution. Some strains of E. coli exhibit robust growth in copper containing media, despite toxicity of high levels of copper in solution. By measuring cell growth and viability at varying concentrations of copper, we can maximize the reaction of H2S resulting from bacterial activity.
Correlating Bacterial Growth to Copper Deposition
In this experimental system, the formation of the copper sulfide should be directly related to the disappearance of Cu2+ with time as the insoluble metal sulfide formed is a complex of copper and sulfur. As H2S production is turned on, there should be a subsequent decrease in soluble copper concentrations as copper is bound in the precipitated copper sulfide.
Localizing Copper Deposition
With bacteria able to affect CuS deposition, the nest logical step is to control this activity spatially and temporally. Inspired by the work of Christine Jacobs-Wagner (Yale, MCDB), we built a mold for constructing agar channels in which to grow and constrain the active bacteria. A master mold was designed and constructed out of the polymer PDMS. This could then be used to iteratively create indented agar channels in our solid growth medium.
When bacteria are washed over these channels, they become trapped in the indentation. By imposing a physical restraint prior to the induction of metal deposition activity, it is possible to control where the metal sulfide is deposited. This is a necessary requirement in the construction of a circuit.
Modeling the Construction of Metal Sulfides
Project
In an effort to achieve our goal, we have designed a bacterial model using synthetic biology. A gene encoding the protein Thisulfate Reductase has been inserted Our initial research indicated that several observations have been made of natural mechanisms for metal deposition and precipitation in a family of microorganisms called Sulfate Reducing Bacteria (SRB). These SRB comprise a class of chemolithotrophic microbes that couple anaerobic electron transport to ATP synthesis using sulfate as the terminal electron acceptor. Importantly, as a consequence of their metabolism, SRB produce hydrogen sulfide: a gas that can be used to reduce metals in solution.These H2S Producing bacteria were found to form significant concentrations of solid metals on and around their cell surface. The H2S gas reduces free metal ions in solution to form a metal sulfide, which subsequently precipitates out of solution. The metal sulfide can self-associate to form clusters of had the capability to reduce could be used to catalyze the precipitation of metal solids in solutions containing metal in the appropriate ionic state. Biochemistry of Sulfate Reducing Bacteria
Papers on Sulfate Reducing Bacteria
Sulfate Reducing Bacteria (SRB) comprise a class of chemolithotrophic microorganisms that couple anaerobic electron transport to ATP synthesis, using sulfate as a terminal electron acceptor.
