Team:Yale/Our Project/Methods/cu localization

• home
• our project
• our team

our project

• introduction
• applications
• methods
• plasmid
• H2S production
• cu growth assay
• cu localization
• parts
• lab notebook
• protocols
• safety
• future work

experimental methods

After the bacteria begin to deposit copper sulfide through the active expression of the transformed plasmid, the next logical step is to localize this copper deposition in a specified geometry. Thus, by placing the active bacteria in a physical constraint copper would be deposited locally at only select areas. The deposition could be tested by simply applying a current and measuring the conductance of the construct.

To create such a local constraint for bacterial copper deposition, our team employed PDMS microchannels. Originally, the Yale iGEM team voyaged to, faulty member, Christine Jacob's lab to discover how bacteria were grown in localized environments. There we observed bacterial "molds," or templates, where channels were imprinted into agar and other growth media and then the bacteria were inserted and allowed to grow due to specific resistances within the mold. For our project, we desired different dimensions and more specific structure than these molds, so we proceeded to make our own.

Agar Channels from PDMS and Silicon Molds

Using standard metal lithography and etching techniques, we created a master mold for our channel in etched silicon wafers. The template for our channels range from 3 to 10 microns in depth and diameter and include multiple conductive pathways from a source and drain commonly used for microfludics. In these loosely defined "source" and "drain" wells, we later inserted electrodes when transfered to agar to allow for the creation of electric current within our deposited layer of copper. Over the metal master mold (with the elevated microchannels) we molded PDMS at approximately 1 inch. The PDMS was made with the use of 5% curing agent, and "cooked" for 3-4 hours at 60 degrees C, then hardened for another 5 hours. The resulting PDMS, when separated from the master mold, contained a microchannel perfect for our bacterial localization. PDMS was then placed on the channel again, to create an inverse, for use of agar. Our ultimate agar channels for actual bacterial growth resulted from two methods (1) either direct use of the silcion master mold to create the agar channel (inverted) in the same way as the PDMS channel was created or (2) double inversion of the PDMS channel through two rounds of PDMS mold creation (with foil in between) to create the indented agar channel (rather than elevated, using the PDMS first round alone). Generally, we found the first method was better to create an agar channel.
Circuit Completion

Upon the creation of the agar channel, holes could be created at the wells on either side of the channel to insert electrodes for current conduction.When expressing bacteria are restricted within the channel, this would allow for copper localization and subsequent conduction using CuS.