50 mL Culture Flasks
In order to investigate the best time to infect E. coli with a recombinant λ-bacteriophage containing a detectable signal, the growth kinetics of E. coli in LB media must be characterized. In this set of experiments, the main aim is to gage when cultures are entering particular stages of the growth curve (refer to figure 1). In conjunction with measuring the optical density (OD) of cultures at a wavelength of 600 nm (OD600), the level of background noise of potential signal molecules was also investigated. For instance GFP & RFP have excitation peaks at 395/475/501 nm and 555 nm respectively. We investigated if we could detect the expression of gfp by absorption at low culture densities.
Figure 1: Schematic of Microbial Growth Curve. After inoculation, microbial cells adapt to the new environment (lag phase). After ample time to adapt, the microbe starts to divide rapidly with the shortest doubling time (log phase). As nutrients become depleted, cells are unable to divide and hence the culture remains "stationary" with regards to growth.
The data from the growth curves also gives scope for when to induce the “payload” circuit. At different time points in the growth curve, anhydrotetracycline can be added in order to relieve repression of mrfp expression. Interesting time points for investigating signal intensity & detection limits are; the lag, mid log & stationary phase. The time point which yields the greatest signal & lowest detection limit will be considered for infection of E. coli cultures to engineered recombinant λ-bacteriophage.
The glycerol stock of E. coli cells was streaked out on a LB agar plate. Glycerol stock cells that bear a plasmid had to be selected for by supplementing the LB agar plates with the appropriate antibiotic at the MIC. Plates were incubated overnight at 37oC overnight.
Three single colony were picked from each plate and used to inoculate three 10 mL of LB broth. LB broth was supplemented with antibiotics if required. Cultures were grown overnight shaking at a rate of 180 rpm at 37oC.
Cultures were diluted back to an OD600 of ~ 0.4 in sterile and preheated LB broth. The diluted culture was used as a seed inoculum. A 1% inoculum was carried out in 50 mL of preheated LB broth. The initial OD600 was taken after inoculation to represent the starting point of the growth curve.
The cultures were left to grow for 1 hour with the OD600, OD475, OD395 & OD555. After 1 hour, culture ODs were measured at 20 minute intervals. At time points 60 minutes & 175 minutes, a viable count was taken. A dilution series ranging from 10-1 – 10-7 was carried out. 3 Χ 20 μL of each dilution was spotted on separate quadrants of the LB agar plate.
The OD of the cultures was monitored for 5 hours. A final reading of the culture OD was carried out at 24 hours. If delays in growth of cells under certain treatments are observed, the phenomenon can be further investigated by ethanol fixing cells and assessing the cellular lengths. Results can be reviewed InterLab Study Additional Results.
96-well Microtitre Plate Growth Curve.
Exactly the same preparation was carried out for the first 2 days with exception to 9 colonies being picked instead of 3. When it came to inoculation, an overnight culture was used to inoculate 200 μL of LB broth (1% inoculum). The general set up of the microtitre plate is displayed in Fig. 2.
Fig. 2: 96-Well Microtitre Plate Set Up for Growth Curve. The "BLANK" in row D was averaged & subtracted from each culture at different time points throughout the growth curve. The contents of each well is labelled; DH1-9 - E. coli DH5α, Pos1-9 - E. coli DH5α containing interlab study positive control gfp expression device, P1-1-9 - E. coli DH5α containing P1-gfp expression device & E. coli DH5α containing P2-gfp expression device.
Microtitre plates were sealed with adhesive tabe and placed in a BMG-LabTech SPECTROstar. An initial reading was taken with readings being take at intervals of 20 minutes for 580 minutes. Shaking was set at 200 rpm with an incubation temperature of 37oC.