Team:CMUQ/Contribution
Characterization
Effect of glucose on arabinose-induced expression:
Group: (Team iGEM CMUQ 2019)
Author: CMUQ iGEM Bio Team
Summary: The below experiments provide additional data about the araC promoter described on this page; specifically, the effect of glucose on the activity level of the promoter.
We investigated the effect of glucose on the levels of expression of araC controlled GFP by measuring fluorescence levels. The strain of E. coli used was E. coli BL21 (DE3). The bacteria were transformed with pLysSBL-21 (DE3) from Promega (catalog number L1198 ) plasmid, to which we integrated the inducible araC-pBad promoter with GFPmut3b, a strong ribosome binding site (Bba_K731250).
FIGURE 4. Concentration of E. coli cells across various conditions.
The concentration of bacteria remained relatively constant across the different concentrations of arabinose. Hence, arabinose does not affect the growth rate of cells and changes in levels of GFP shown in Figure 5 may be attributable to promoter activity. However, we note that LB+chl+IPTG had a slower growth compared to the other permutation of the media.
FIGURE 5. Expression levels of GFP agains E. coli media composition
Expression levels of GFP in various different conditions of E. coli environment was measured. We observe that an increase in arabinose levels leads to an increase in the levels of GFP expressed. We also observe that the presence of glucose in the media significantly reduces the levels of GFP expressed. Thus we conclude that araC promoter activity is positively regulated by arabinose but negatively regulated by glucose.
Characterization Protocol
pLysSBL-21 (DE3) from Promega (catalog number L1198) was transformed with Bba_K731250 which is an inducible araC-pBad promoter with GFPmut3b, strong RBS
1ul of plasmid was added to 50ul of cells. Pipette up and down to mix. Kept on ice for 30min
Heat-shock at 42°C for 45s using heat block
Incubate on ice for 5min
950ul SOC media was added
Transformed mixture was incubated at 37°C for 1h at 240rpm
100ul was spread plate onto LB+chl plate
Growth and Fluorescence:
Colonies were inoculated in 5ml of Luria Broth +25ug/ml Chloramphenicol, grown overnight until saturation.
Saturated cultures were inoculated into tryptone (lambda) + 25ug/ml Chloramphenicol or Luria Broth +25ug/ml Chloramphenicol to an OD of 30 using a Klett colorimeter. Cultures were grown in an air shaker at 37°C until OD reached 80.
100ul of culture was added into 96-well plate (clear) according to the well-plate plan
Arabinose concentrations (0, 0.01, 0.02, 0.05, 0.1 and 0.53mM) was added with and without IPTG. IPTG induces the T7 RNA polymerase.
At t=0, absorbance at 600nm (growth) and fluorescence of GFP at Ex/Em 485nm/526nm was measured using the multimode microplate reader (Varioskan Multimode Microplate Reader, Thermofisher).
Plate was kept in an air shaker at 37°C for 5h following which OD at 600nm and fluorescence of GFP was measured.
Data was presented as change in absorbance (to indicate growth) and change in fluorescence (to indicate expression of GFP)