Click here to see the results from testing the constructs ORF-314, stf gene, tfa construct, P(Cat)-TetR construct, TetR-controlled tfa circuit and cI-Cro circuit.
To confirm the presence and correct size of the BioBricks we are submitting, all samples were run on an agarose gel. The constructs tested were:
ORF-314
the stf gene
the tfa construct
the P(Cat)-TetR construct
the TetR-controlled tfa circuit
the cI-Cro circuit
All samples were initially digested with two of the four standard iGEM restriction enzymes (XbaI, EcoRI, SpeI and PstI) and expected fragment sizes calculated (Fig 1a and 2a). Success of the cloning was then determined by comparing actual band sizes to the predicted values (Fig 1b and 2b).
Comparison of predicted and actual band sizes on gel 1.
Comparison of predicted and actual band sizes on gel 2.
Click here to see the results of our contribution to iGEM's InterLab Study, and some additional experiments that support our research.
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.
Descriptive Statistics of 1 hour Viable Count of E. coli DH5α.
N
Minimum
Maximum
Mean
Std. Deviation
Viable Count
9
1600000
3750000
2566666.67
627495.020
Valid N (listwise)
9
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.
Descriptive Statistics of 175 Minutes Viable Count of E. coli DH5α.
N
Minimum
Maximum
Mean
Std. Deviation
Viable Count
9
90000000
175000000
133333333.33
29154759.474
Valid N (listwise)
9
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 gfpE. 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 gfpE. 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 gfpE. 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 gfpE. 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. Comparing the P1-gfp expression device with E. coli DH5α, at 0 minutes there is a highly significant difference between the two cultures fluorescence (P=0.005). This significance is observed until 100 minutes (P=0.427). There is an insignificant difference between the two cultures until 160 minutes (P<0.001). At 220 minutes into the growth curve, the fluorescence between P1gfp expression device is insignificant (P=0.277) with significance being observed at 240 minutes (P=0.001). The fluorescence of P1-gfp expression device remains higher than the E. coli DH5α culture.
Comparing the positive control gfp expression device with E. coli DH5α, at no point in the growth curve does the fluorescence of the positive control gfp expression device increase above that of the E. coli DH5α culture. Even at 580 minutes of culturing the positive control does not increase in fluorescence above the E. coliE. coli DH5α (P=0.677).
Comparing the P2-gfp expression device with E. coli DH5α, Initially there is no significant difference between the two cultures in fluorescence (P=1). There is a highly significant Increase in fluorescence from P2 at 180 minutes (P=0.007). At 200 minutes, there is no significant difference in fluorescence observed between P2 + E. coli DH5α (P=0.146). Insignificance of fluorescence between the two cultures is observed until 500 minutes of culturing (P=0.038). The fluorescence from the P2-gfp expression device remains significantly higher than E. coli DH5α throughout the rest of the growth curve.
Comparing the P1-gfp expression device with the positive control gfp expression device there is a significantly higher fluorescence from 0 minutes (P=0.034) & 20 minutes (P=0.038). At 40 minutes of culturing there is no significant difference in fluorescence (P=0.164). At 60 minutes the P1-gfp expression device has a significantly higher fluorescence than the positive control (P=0.017). The significance is not observed at 80 minutes (P=0.849). There is no significant fluorescence from P1 compared to the positive control until 180 minutes (P=0.009). No significance is observed between the two promoters until 340 minutes where P1 has a higher fluorescence (P=0.019). The P1-gfp expression device has a higher fluorescence compared to the positive control at 360 minutes (P=0.024) and becomes insignificant at 380 minutes (P=0.245) where it remains insignificant for the result of the culturing time.
Comparing P2-gfp expression device with the positive control, there is no time point where the P2-gfp expression device has a statistically higher fluorescence than the positive control gfp expression device.
When comparing the data sets for the P1 & P2 gfp expression devices, there is no significant difference until 60 minutes (P=0.021). At 60 minutes, the P1-gfp expression device has a higher fluorescence. There is no significance between P1 & P2 gfp expression devices at 80 minutes (P=0.646) & no other time points show significance between these two cultures.
Results discussion & credit for the work are shown in the Discussion section of this website.
