Team:Birkbeck/Experiments

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Under construction

Growth Curve

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 (OD600), the level of background noise of potential signal molecules was also investigated. For instance gfp or rfp would have absorption peaks at 395/475 nm and 555 nm respectively. The signal from GFP or RFP can be detected using a spectrophotometer by using the absorption peaks.

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.

he 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.

Day 1

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 37^oC overnight.

Day 2

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 37^oC.

Day 3

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 & 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

here

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