Prha Characterization with GFP

The purposes of this protocol is to determine how glucose as opposed rhamnose influences expression of genes under control of the rhamnose promoter. To this end, we exposed cultures of E. coli which contained P_rha—GFP to media with varying concentrations of glucose and rhamnose. Prepare 7mL LB broth overnight cultures of the following:

• P_rha—RBS—K082003 of interest
• TetR—RBS—K0082003 for a positive control
• Negative control, IE some Top ten E. coli not expressing GFP

Reagents and materials:

• 5x concentrate version of M9 minimal media (link)--do NOT add glucose
• 10x concentrate of casamino acids
• 2% w/v rhamnose
• 2% w/v glucose
• ddH_2O

The media for the assay is as follows:

1. You will dilute the M9 and casamino acids to a 1x working solution. Additionally, add glucose or rhamnose to desired testing concentrations. In our test of the rhamnose promoter, we used the results of Jeske and Altenbuchner (2010) as initial concentrations for each sugar (ie, 0.2% w/v of glucose or rhamnose). We also conducted the test with 0.5% of each sugar, as well as samples which contained no sugar and only minimal media.
2. Aliquot 1mL portions of the cultures into 2mL microfuge tubes for each sugar condition to be tested. Spin down the cells and discard supernatant. Resuspend in 1mL of the desired minimal media. Spin down and discard supernatant. Finally resuspend the cells another sample of the minimal media and add the appropriate selective antibiotic agent: in our 100 ug/mL of ampicillin and 30 ug/mL of chloramphenocol.
3. Pipet out 200uL of culture per well into a black-sided, clear-bottomed 96 well plate. Incubate and shake the plate at 37 degrees Celsius. Read fluorescence (475nm and 515nm for K082003 (LINK)) and OD at 600__ at intervals of every hour.