Difference between revisions of "Team:Birkbeck/Measurement"

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. 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. coli E. 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.

Discussion of these results & credit for the work are shown in the tab below.

Signal Detection

Considering our results, at time 0 minutes, there is a significant signal for P1-gfp expression device compared to the E. coli DH5α (P=0.005, with reference to Fig. 9 in results section). This result cannot be regarded as this is probably carry over fluorescence from sub-culturing. Photo-bleaching prior to the first reading may yield more accurate readings when considering the expression of gfp from promoters. Also the error bars in each of the expression devices do appear excessive. A means of reducing this may be to sub-culture the cells & grow to an OD₆₀₀ of 0.2 and sub-culturing into experimental cultures in order to minimise the error bars.

One technique which has been developed is transcription-reverse transcription concerted reaction (TCR) (Ishiguro et al., 1996). TCR monitors transcription in a sequence-dependant manner in in-vitro systems (Ishiguro et al., 1996). The TCR protocol was adapted in order to improve sensitivity for detecting M. tuberculosis from clinical sputum samples (Drouillon et al., 2009 [TCR-2]). Considering P1-gfp culture at 20 minutes, there is a significant fluorescent signal (P=0.002) with no difference between the OD₆₀₀ growth curves at this point (P=1). This is significantly less than the hours taken for results to be generated by TCR-2 (Drouillon et al., 2009). Cells cannot be quantified at the 20 minutes time point in our study as no viable count was carried out, therefore the detection limit of the E. coli DH5α with regards to fluorescence cannot be determined.

With regards to Decontamination Kits, samples would take just under one hour to process. Of note, some bacteria (such as Staphylococcus aureus) can be resistant to decontamination (refer to the kit previously mentioned). A key point to note here is that the decontamination step must not be carried out for longer than 15 minutes due to the reduction in cell viability of Mycobacterium cells.

As transduction was not characterised in this study, it is difficult to assess exactly how long this step may take. Considering the application of D29 mycobacteriophage could be spotted directly onto soft agar plates giving rise to plaques on both M. smegmatis & M. tuberculosis (Sampson et al., 2009). A mycobacteriophage assay for detection of live Mycobacterium cells has previously been carried out using a plaque assay (Alcaide et al., 2003). A 1 mL decontaminated sample was washed and grown overnight in the standard Mycobacterium media Middlebrook 7H9 supplemented with 10% (vol/vol) oleic acid-albumin-dextrose catalase (OADC) (Alcaide et al., 2003). 100 μL of mycobacteriophage was added to the overnight cultures and incubated at 37^oC for 1 hour before adding 100 μL of viricidal solution (Alcaide et al., 2003). A soft agar lawn was poured and incubated for 24 hours before counting plaques. The use of a fluorescent protein as a tag would perhaps remove the need for overnight incubation (in fact a 48 hour protocol).

Alcaide & co-workers did demonstrate that smear positive samples were extremely sensitive to the phage assay and claim that the detection limit was 10 cfu/mL with smear negative samples showing much higher detection limits. Another study using mycobacteriophages as a diagnostic tool used the cheaper medium Mueller–Hinton instead of Middlebrook 7H9 (Hemvani et al., 2012). The Hemvani study showed similar results to the Alcaide study. The change in media did however result in 3.5% of sputum samples being un-interpretable due to high levels of contamination even after treatment with vancomycin & polymyxin B (Hemvani et al., 2012).

Considering the presorption phase of the assay developed by Alcaide et al, this would add an hour onto the protocol. Given that transduction is 100% efficient (unlikely) and that the kinetics of gfp expression behaves the same in E. coli DH5α as does a Mycobacterium sp. cell (another wild assumption), we are looking at from sputum sample collection until signal detection at approximately 2-4 hours (given that decontaminated sputum samples could be directly exposed to recombinant bacteriophages). It must be noted that in our approach, the signal is produced by transduction of genetic material as opposed to amplification of phage particles being the signal. The use of transduction hass been successfully applied in genetic engineering of Mycobacterium sp. (Jain et al., 2014; Tufariello et al., 2014) which is promising for the development of a diagnostic tool in introducing a detectable "payload" for diagnostic purposes.

Considering our results, at 1 hour of culturing E. coli DH5α, there is a significant signal observed from the culture expressing the P1-gfp expression device (P<0.001). Considering the growth, there was no significant difference between the OD₆₀₀ of the P1-gfp expression device & the E. coli without plasmid (P=1 for both 50 mL cultures & 200 μL, with reference to Fig. 1 & Fig. 5 respectively). At the 1 hour time point, there is a significant difference between the 50 mL & 200 μL cultures (P=0.017) with the 200 μL cultures showing a higher OD₆₀₀. This entails that the fluorescence of the 200 μL cultures at 1 hour corresponds to a higher culture density than 2.57×10⁶ cfu/mL.

Considering the TCR-2 method of detection, 2.57×10⁶ cfu/mL is an increase in detection limit by a magnitude of 10⁵ (30 cfu of M. tuberculosis/mL of sputum [Drouillon et al., 2009]) which is clearly an undesirable due to the slow growth of M. tuberculosis. There appears to be a reduction in fluorescence from the P1-gfp expression device after 20 minutes which might correspond to the depletion of mRNA/GFP. The increase in fluorescence when comparing the 20 & 40 minute time points may be the expression of gfp from each of the promoter increasing to a detectable level.

An interesting observation was made on the growth kinetics of the E. coli DH5α containing P1-gfp expression device & E. coli DH5α not containing any plasmid. Between 100-400 minutes, the E. coli DH5α containing P1-gfp expression device has a significantly lower OD₆₀₁ & higher fluorescence when compared to E. coli DH5α. The significant difference is not observed in the positive control or P2-gfp expression device with limited detection of fluorescence. This data suggests that compromising cellular growth with regard to expression of a signal may yield a quicker detection of a signal with the product.

It is obvious that the P1 promoter will not be able to drive gene expression in all kinds of bacteria. The proposed T7-RNAP-driven rfp expression was not characterised as the circuit was not constructed due to cloning issues. However, the growth kinetics would be expected to be slower than that of the P1-gfp expression device. Fig. 1 is a diagramatic representation of the proposed genetic circuit for the characterization of a signal.

Fig. 1: TetR rfp Expression device.

The idea behind the circuit displayed in Fig. 1 is to have the amplification of rfp expression inducible. The induced expression of the amplification step in this circuit would mimic the infection of the E. coli cells by a recombinant λ-bacteriophage. This would allow different time points in the growth curve to be stimulated and signal intensities could be compared relative to the cell dose & physiological state of cells.

In the first part of the circuit displayed in Fig. 1, a strong constituitive promoter (such as pCAT) will drive the expression of tetR. The TetR will bind to tetO & repress the expression of T7-RNAP. The T7-RNAP would then drive the exmpreesion of rfp via T7 promoter.

The highly mutated rfp was chosen as a marker due to the ability of the protein to be observed on a plate as well as liquid culture without the use of fluorescence. The absorption of GFP could not be detected accurately (see Fig. 2 & Fig. 6-8 in the additional Interlab Results. This approach can't be applied to detecting an early signal however, left on a plate can yield very suggestive & qualitative results.

Another proposed way in which to characterize this circuit was to clone into a gt 11 λ-bacteriophage vector. with reference to Fig. 1, the right hand side of the line down the circuit (i.e. from the tetO) would be cloned into the gt 11 vector. There could be many possible time points to assess what part of the growth curve would yield the highest signal.

References

Sean Ross Craig (data analysis, cloning, restriction diagnostics, measurements & uploading content to the wiki), Elliott Parris (measurements & restriction diagnostics), Rachel Wellman (restriction diagnostics & measurements) & Ariana Mirzarafie-Ahi (cloning).

With thanks to Dr. Vitor Pinheiro, Dr. Jane Nicklin, Bilkis Kazi, Barbara Steijl, Luba Prout.