P_rha characterization with GFP

The purpose of this protocol is to determine how glucose as opposed rhamnose influences expression of genes under control of the rhamnose promoter. We wanted to show how glucose can be used to repress this promoter for the purpose of regulating our kill switch. To this end, we exposed cultures of E. coli which contained P_rha—GFP to media with varying concentrations of glucose and rhamnose. We prepared 7mL LB broth overnight cultures of the following:

• P_rha—RBS—K082003
• P_TetR—RBS—K082003 for a positive control
• negative control, i.e. a sample of Top ten E. coli lacking GFP

Reagents and materials:

• 5x concentrate version of M9 minimal media--do NOT add glucose
• 10x concentrate of casamino acids
• 2% w/v rhamnose
• 2% w/v glucose
• ddH₂O

Here are the steps for preparing the media and analysing the results:

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 excitation and 515nm emission for K082003 as in our assays) and OD at 600nm at intervals of every hour.