Technical Reference #1839

1839.

Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products Henrike Niederholtmeyer; Bernd T. Wolfstaedter; David F. Savage; Pamela A. Silver; and Jeffrey C. Way, Harvard Medical School, Applied and Environmental Microbiology, 76(1839), (2010)
Link To Paper

Materials & Methods:
Synechococcus-E. coli coculture. To test whether the sugar-secreting Synechococcus strain could support growth of E. coli cultures of the Synechococcus glf invA strain and a non-sugar-secreting control strain were grown to an OD750 of 0.1. Sugar secretion was induced with 200 mM NaCl and 100 M IPTG. E. coli cells were washed in phosphate-buffered saline (PBS) three times and incubated with shaking in PBS containing 100 M IPTG and antibiotics for 1 h at 37°C after which 106 cells/ml were added. The medium used was BG-11 medium with 100 M IPTG 2.5 g/ml kanamycin 2 g/ml spectinomycin and 1 mg/ml NH4Cl. Growth of E. coli was monitored by plating serial dilutions on LB agar and measuring the yellow fluorescent protein (YFP) fluorescence of culture samples with a Victor 3V plate reader. For microscopic quantitation of Synechococcus and E. coli bacteria in samples of the liquid coculture were visualized by red chlorophyll autofluorescence and YFP fluorescence respectively. To observe microcolony formation 100 l of the initial coculture was plated on BG-11 agar containing the compounds listed above. After 4 days pieces of agar were cut out and placed onto a MatTek glass bottom dish for microscopy.

Microscopic Technique
Red chlorophyll autofluorescence and YFP fluorescence

Cell Type(s)
Synechococcus-E. coli coculture.