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Fig. 1: Growth Curve of E. coli DH5α Strains Following Culture Optical Density of 600 nm.

Growth kinetics was initially studied using 50 mL cultures. Fig. 1 shows the growth kinetics of E. coli DH5α & derivative strains containing plasmids from the InterLab study. The growth curve shows that the E. coli strain that contains the P1-gfp expression device grows at a slower rate than the other strains investigated. At 220 minutes the E. coli DH5α P1 strain has a significantly lower OD₆₀₀ than the E. coli DH5α (P=0.023). E. coli DH5α remains significantly higher in OD₆₀₀ than E. coli DH5α with the P1-gfp expression device (P=<0.001). The only difference between the E. coli DH5α & E. coli DH5α positive control device is observed at 280 minutes into the growth curve (P=0.016) where the positive control has a higher OD₆₀₀. The multiple comparison table showing P values can be viewed in Table S1.

Fig. 2: Growth Curve of E. coli DH5α Strains Following Culture Optical Density of 395 nm.

In order to investigate if there could be a point in the E. coli DH5α growth curve in which a signal from the GFP could be detected by absorbance, the growth curves were also conducted using the major absorption peak of GFP (wavelength 395 nm). The growth curve data for the culture optical density is displayed in Fig. 2.

When comparing the data point of E. coli DH5α strains, there appears to be a significant increase in culture OD₃₉₅ in the E. coli cells with the P1-gfp expression device (P<0.001). This apparent signal is only present between 60-100 minutes of growth. When comparing the positive control & E. coli DH5α1, there is no significant difference between the data sets at 60 or 100 minutes (P=1 for both time points). A small potential signal is observed from the oositive control gfp expression device at 280 minutes (P=0.012) & 300 minutes (P=0.006). This significance is lost after 300 minutes (P=0.262). See Table S2 for more details. In order to verify these results & to test for the feasibility of scaling down to a 96-well microtitre plate assay, a 10 hour growth curve was conducted in a 96-well microtitre plate (see Fig. 6-8).

Fig. 3: Viable Count of E. coli DH5α After 60 mins.

In order to assess how many viable cells correspond to different optical densities, a viable count was conducted on E. coli DH5α1 at 60 minutes (Fig. 3 & Table 1), 175 minutes (Fig. 5 & Table 2) & 225 minutes (data not shown due to high level of contamination).

Considering the OD₆₀₀ of the E. coli DH5α1 cultures at 60 mins, triplicate cultures were OD₆₀₀ = 0.029, 0.01 & 0.025. The viable count of each of the cultures gave a mean of 2.57×10⁶ cfu/mL (Table 1). It can therefore be concluded that an E. coli DH5α culture at an OD₆₀₀ = 0.021 corresponds to approximately 2.57×10⁶ cfu/mL.

Table 1: Descriptive Statistics of 60 minutes Viable Count of E. coli DH5α..

Fig. 4: Viable Count of E. coli DH5α After 175 mins.

At 175 minutes into growth, the E. coli DH5α1 cultures had OD₆₀₀ of; 0.255, 0.216 & 0.262. The viable count of each of the cultures gave a mean of 1.33×10⁸ cfu/mL (Table 2). It can therefore be concluded that an E. coli DH5α culture at an OD₆₀₀ = 0.244 corresponds to approximately 1.33×10⁸ cfu/mL. Considering the previous OD₆₀₀ (0.021), there is approximately a 10-fold increase in OD₆₀₀ which corresponds to nearly a 100-fold increase in viable cells.

Table 2: Descriptive Statistics of 175 minutes Viable Count of E. coli DH5α.

Fig. 5: Growth Curves of Different Strains of E. coli DH5α Following Culture Optical Density at 601 nm.

All data analysis tables for the OD₆₀₁ growth curves are in; Table S3, Table S5 & Table S6 for 0-200 minutes, 220-420 minutes & 440-580 minutes respectively. Comparing the growth of E. coli DH5α containing P1-gfp expression device with the E. coli DH5α, there is no significant difference between the two cultures at 80 minutes (P=0.943), however, at 100 minutes there is a very highly significant difference between the 2 strains of E. coli DH5α (P<0.001). The OD₆₀₁ of E. coli DH5α containg the P1-gfp expression device remains at a significantly lower that the E. coli DH5α untill 400 minutes (P=0.441).

Comparing the E. coli DH5α P1-gfp expression device with the positive control gfp E. coli DH5α cultures, the OD₆₀₁ remains insignificantly different at 100 minutes (P=0.848) with significance being observed at 120 minutes (P<0.001). The significance is observed until 280 minutes into the growth curves (P=0.065). Interesting;ly, this is a shorter window of significance compared to E. coli P1-gfp expression device & E. coli DH5α (160 minute Vs 300 minutes respectively).

Comparing P1 & P2-gfp expression devices in E. coli DH5α, no significance is observed at 100 minutes (P=0.132). P1-gfp expression device is very highly significantly lower (P<0.001) than P2-gfp expression device after 120 minutes of culturing. This significance is observed until 320 minutes of culturing (P=0.075).

When comparing the positive control, P2-gfp & E. coli DH5α, there is no significant difference throughout the growth curve. After 420 minutes, there is no significant difference between any of the E. coli DH5α cultures OD₆₀₁ (P=0.81).

Fig. 6: Growth Curves of Different Strains of E. coli DH5α Following Culture Optical Density at 501 nm.

All data analysis tables for the OD₅₀₁ growth curves are in; Table S7, Table S8 & Table S9 for 0-200 minutes, 220-420 & 440-580 minutes respectively. Comparing the growth of E. coli DH5α containing P1-gfp expression device with the E. coli DH5α, there is no significant difference between the two cultures at 80 minutes (P=0.987). After 100 minutes there is a highly significant difference between the two cultures (P=0.003). This difference remains significant until 400 minutes into culturing (P=0.093).

Comparing the E. coli DH5α P1-gfp expression device with the positive control gfp E. coli DH5α cultures, the OD₅₀₁ remains insignificantly different at 100 minutes (P=0.85) with significance being observed at 120 minutes (P<0.001). The P1-gfp expression device culture remains significantly lower than the positive control gfp expression device until 320 minutes (P=0.053).

Comparing P1 & P2-gfp expression devices in E. coli DH5α, no significance is observed at 100 minutes (P=0.403). P1-gfp expression device is very highly significantly lower (P<0.001) than P2-gfp expression device after 120 minutes of culturing. This significance is observed until 320 minutes of culturing (P=0.096).

When comparing the positive control, P2-gfp & E. coli DH5α, there is no significant difference throughout the growth curve. After 420 minutes, there is no significant difference between any of the E. coli DH5α cultures OD₅₀₁ (P=0.09). These results are identical to the results obtained in th OD₅₀₁ absorption of the E. coli DH5α cultures.

Fig. 7: Growth Curves of Different Strains of E. coli DH5α Following Culture Optical Density at 475 nm.

All data analysis tables for the OD₄₇₅ growth curves are in; Table S10, Table S11 & Table S12 for 0-200 minutes, 220-420 & 440-580 minutes respectively. Comparing the growth of E. coli DH5α containing P1-gfp expression device with the E. coli DH5α, there is no significant difference between the two cultures at 100 minutes (P=0.982). After 120 minutes there is a highly significant difference between the two cultures (P=0.003). This difference remains significant until 440 minutes into culturing (P=0.745).

Comparing the E. coli DH5α P1-gfp expression device with the positive control gfp E. coli DH5α cultures, the OD₄₇₅ remains insignificantly different at 100 minutes (P=0.844) with significance being observed at 120 minutes (P<0.001). The P1-gfp expression device culture remains significantly lower than the positive control gfp expression device until 340 minutes (P=0.19).

Comparing P1 & P2-gfp expression devices in E. coli DH5α, no significance is observed at 100 minutes (P=0.464). P1-gfp expression device is very highly significantly lower (P<0.001) than P2-gfp expression device after 120 minutes of culturing. This significance is observed until 340 minutes of culturing (P=0.163).

When comparing the positive control, P2-gfp & E. coli DH5α, there is no significant difference throughout the growth curve. Conducting a one-way ANOVA of the culture density at 440+ minutes shows there is a highly significant difference between the means (P=0.003). The multiple comparisons table shows no significant difference between any of the individual data point (data not shown).

Fig. 8: Growth Curves of Different Strains of E. coli DH5α Following Culture Optical Density at 395 nm.

All data analysis tables for the OD₃₉₅ growth curves are in; Table S13, Table S14 & Table S15 for 0-200 minutes, 220-420 & 440-580 minutes respectively. Comparing the growth of E. coli DH5α containing P1-gfp expression device with the E. coli DH5α, there is no significant difference between the two cultures at 80 minutes (P=0.937). After 100 minutes there is a highly significant difference between the two cultures (P<0.001). This difference remains significant until 440 minutes into culturing (P=0.083)

Comparing the E. coli DH5α P1-gfp expression device with the positive control gfp E. coli DH5α cultures, the OD₃₉₅ remains insignificantly different at 80 minutes (P=0.992) with significance being observed at 100 minutes (P=0.011). The P1-gfp expression device culture remains significantly lower than the positive control gfp expression device until 440 minutes (P=0.325).

Comparing P1 & P2-gfp expression devices in E. coli DH5α, no significance is observed at 100 minutes (P=0.358). P1-gfp expression device is very highly significantly lower (P<0.001) than P2-gfp expression device after 120 minutes of culturing. This significance is observed until 340 minutes of culturing (P=0.171).

When comparing the positive control, P2-gfp & E. coli DH5α, there is no significant difference throughout the growth curve. Conducting a one-way ANOVA of the culture density at 440+ minutes shows there is a highly significant difference between the means (P<0.001). The multiple comparisons table shows no significant difference between any of the individual data point (data not shown).

Fig. 9: Growth Curves of Different Strains of E. coli DH5α Following Culture Fluorescence.

All data analysis tables for the culture fluorescence growth curves are; Table S16, Table S17 & Table S18 for 0-200 minutes, 220-420 & 440-580 minutes respectively.

Results discussion & credit for the work are shown in the Discussion section of this website.