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Team:Missouri Miners/Project
From 2010.igem.org
Project Center
Abstract
The growing need for alternative fuel sources has sparked interest and research across many scientific and engineering disciplines. The fledgling field of microbial fuel cell development has previously relied on anaerobic metal reducing organisms such as Geobacter sulfurreduccens. This project sought to isolate genes from the electron shuttling pathway in Geobacter and transform them into the more manageable aerobic Escherichia coli. The Missouri University of Science and Technology iGEM team isolated four outer membrane cytochrome (omc) genes from Geobacter, vital to the extracellular transportation of electrons. The four genes; omcB, omcE, omcS and omcT, were cloned into individual plasmids. The eventual goal is to combine all four genes into one plasmid to transform into E. coli to create an aerobic, electron transporting microbial system.
Background
What exactly is a microbial fuel cell? What are current research initiatives focused on in this area? How is the S&T iGEM team's approach different?
Process in Detail
The isolated genes omcB, omcE, omcS, and omcT were first obtained as nonfunctional sequences in plasmids from the iGEM standard registry of parts. These sequences were digested from the plasmids using restriction enzymes XbaI and PstI. The band lengths for omcB, omcE, omcS, and omcT, were 483, 1298, 698, and 1293 bases, respectively. Ribosome binding site (RBS) plasmid with an Ampicillin and Kanamycin resistance genes from the iGEM standard registry of parts was digested in the middle of the Kanamycin resistance gene using SpeI and PstI restriction enzymes, and dephosphorylated to produce higher ligation yields. The now isolated genes of omcB, omcE, omcS, and omcT genes were then ligated into the cut RBS plasmids, creating a functional RBS+omc gene complex. The new plasmids were then transformed into electrocompetent Top10 E. coli and grown up in broth cultures of Circlegrow, which is a unique broth designed to enhance the growth of E. coli with higher yields of plasmid DNA. Ampicillin was used as a selective pressure to prevent undesirable microbial growth. The cultures were then grown up over night in a 37oC incubator and then smeared onto Lysogeny Broth plates with Ampicillin (LB Amp).
Individual colonies were replica plated on LB Amp and LB Kanamycin plates and grown over night in a 37oC incubator. Kanamycin was used as a form of selection because the Kanamycin resistance gene was interrupted in the RBS plasmid with the insertion of omcB, omcE, omcS, and omcT. From these plates, 5 mL cultures of Circlegrow with Ampicillin were started. These cultures were used to perform mini preps to isolate the plasmids from the cells. The pomcE RBS and pomcT RBS plamsids were digested with XbaI and PstI to entirely remove just the gene and RBS, while the pomcB RBS and pomcS RBS plasmids were cut open with SpeI and PstI and dephosphorylated. The resulting products were separated using gel electrophoresis and the DNA was removed using a gel/PCR extraction kit. The pomcB RBS plasmid was dephosphorylated and ligated to omcE, while the pomcS RBS plasmid was also dephosphorylated and ligated to omcT. PomcBE RBS and pomcST RBS plasmids were transformed into Top10 E. coli and grown, plated on LB Amp plates, and incubated at 37oC overnight.
Problems Encountered
There were many problems and downfalls experienced through this process. Transformations posed a big problem at the beginning of the project. The chemically competent cells orginially used were not working properly; the solution was to use electroporation as an alternative method for transformation. Additionally, some of the restriction enzymes used were old and the specific activity of the enzyme was low, giving low digestion yields. To fix this problem, new enzymes were obtained. The mini preps were not pelleting properly, possibly due to problems with the detergent in the cell lysis solution. This problem was dealt with by fishing the cell debris out of the solution instead of removing the supernatant from atop the pelleted debris. Other problems encountered were due to human error in different processes and were resolved with more careful lab technique and redoing of the processes.
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