Difference between revisions of "Team:Birkbeck/Results"

Revision as of 21:36, 8 September 2015

Birkbeck iGEM

The Owligos are the first-ever team entered into the international Genetically Engineered Machine (iGEM) Competition by Birkbeck, University of London. We’re a varied group of students who reflect the diversity and unique character of our institution: many of us have chosen science as a second career, having already spent some time in full-time work. For most of us, this has meant making our way through a degree while continuing to work full-time. Hopefully this kind of dedication will help us successfully navigate our way through our iGEM project.

Project Aim

Our project aims to create a new diagnostic solution that will be low-tech and cost-effective enough to allow its usage in deprived and remote communities. We’re attempting to engineer a bacteriophage lambda chassis to change its host affinity, while simultaneously adding a marker that will facilitate easy detection of a target bacterial pathogen in patient samples.

To demonstrate this approach as a proof of concept for the competition, we plan to change this affinity between different strains of E.coli; however, ultimately we hope to demonstrate that this principle could also be applied to alter the phage’s host range to other bacterial species. We could then provide a modular system capable of diagnosing a range of diseases. Of course, we haven’t chosen a simple goal. But as Birkbeck pioneers, we are determined to prove ourselves by making our project a success. We can’t wait to present the results of our work at the Giant Jamboree in September!

Under Construction

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.

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

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

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

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

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 <center><img src="https://static.igem.org/mediawiki/2015/9/9a/Fluorescence_growth_curve_of_multiple_strains_of_E._coli_DH5_alpha_team_Birkbeck_iGEM_2015.jpg "></center>
 <p><b><u>Fig. 9: Growth Curves of Different Strains of <i>E. coli</i> DH5α Following Culture Fluorescence</u></b>.</p>
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