Team:UTK-Knoxville/procedures

Sucrose Study Protocol (Plate Reader)

• Grow cells in LB overnight
• Spin down and resuspend to OD=.1 in M9 4g/L glucose media, grow until OD=.5-.6
• Split cultures in half-one half will be with sucrose, one half will remain without
• Spin the cells that will have sucrose down again, resuspend in M9 4g/L glucose+20% sucrose media
• Do not dilute, OD should remain constant
• Aliquot (in triplicate) cells with and without sucrose media into a plate for the reader (carefully keep up with all well IDs)
• Plates need positive (RFP WT and ΔOmpR) and negative controls (WT and ΔOmpR, no plasmid), empty wells, wells with water, and wells measuring media alone
• Observe fluorescence changes via plate reader

Dilution Study Protocol

• Stocks were removed from -80̊C and streaked onto LB plates with respective antibiotics. Plates were put into 37̊C incubator and left overnight.
• Seed cultures:
• Seed cultures were started from a single colony in liquid LB medium with respective antibiotics. Seeds were grown overnight (about 8-10hr) in 37̊C shaker to OD 1.0-2.0.
• Main cultures:
• M9 Medium + glucose (either 2g/L or 4g/L) was put in the 37̊C water bath to warm up. 250mL flasks were prepared with 50mL M9 medium with respective antibiotics. Cultures were started from seed cultures at an OD of 0.01 in triplicate as well as a tube containing only medium in order to check for medium contamination. Time points were taken every 2-4 hours depending on how quickly cultures were growing. Once an OD of 0.6-0.8 was reached, samples were taken and cultures were diluted to an OD of 0.01 into 50mL of fresh M9 medium. This was repeated twice, resulting in three samples for each replicate of each plasmid in each strain (e.g.one plasmid would have 18 samples when done in triplicate, one plasmid in WT would have nine samples when done in triplicate). Samples were 50% (v/v) glycerol and 50% (v/v) culture. These were stored in the -80̊C freezer.
• Measurements for these studies were made using a microscope with a fluorescence filter and the image processing software ImageJ.