Difference between revisions of "Team:Birkbeck/InterLab Study"
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Revision as of 13:24, 2 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!
Location: Birkbeck, University of London Malet Street London WC1E 7HX Contact us: |
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Tweets by @BBKiGEM |
InterLab Study
1. Introduction
The InterLab Study is aimed at investigating the expression levels of three promoters (J23101, J23106 & J23117). Grren fluorescent protein (gfp-mut3b – E0040) was used as a reporter for expression and the level of gene expression was determined by measuring the fluorescence of each E. coli DH5α strain generated. The promoters under investigation were designed by John Anderson and submitted by the Berkeley iGEM 2006 group (with reference to Part description & UC Berkeley/2006).
The previous InterLab Study (2014) was composed of 45 teams representing 18 countries globally. As part of our project in designing a phage-based diagnostic tool, detection of signals is fundamental to our end goal. Studying the expression levels of different promoters in a standardized fashion maybe one way in which an output can be achieved with our product.
2. Cloning
In order to investigate the promoter expression, gfp had to be expressed by each of the promoters. The BioBrick BBa_I3504 contains a ribosome binding site (RBS – B0034), gfp-mut3b (E0040) and two terminators (B0010 & B0012). Cloning this BioBrick into different promoters will result in the expression of gfp and therefore detectable fluorescence from cells. The level of fluorescence detected from each of the promoters can be used to assess promoter activity.
3 A assembly was carried out to make all 3 devices. The BBa_I3504 BioBrick was in a plasmid backbone containing an ampicillin resistance cassette with each of the promoter devices in a plasmid backbone containing a chloramphenicol resistance cassette (P1 [ BBa_J23101], P2 [ BBa_J23106] & P3 [ BBa_J23117]).
The BBa_I3504 plasmid was digested with XbaI & PstI in order to liberate the BBa_I3504 BioBrick. Each of the promoters were restricted with SpeI & PstI in order to open up the plasmid and allow digested BBa_I3504 to anneal and be ligated into each of the promoters. The cloning process is diagrammatically shown in Fig. 1.
Fig. 1: Cloning Strategy for Device Construction.
Three colonies from each transformation were picked and grown in 5 mL LB broth (supplemented with 35 μg/mL chloramphenicol). 500 μL of overnight culture was used to make glycerol stocks with the remaining cultures used for miniprepping & restriction diagnostics (EcoRI & PstI).
3. Experimental Procedure
After screening plasmids for the BBa_I3504 insert into the three promoter devices, successful transformants were streaked from glycerol stocks. LB agar (1.5%) plates were supplemented with chloramphenicol to a final concentration of 35 μg/mL chloramphenicol. Plates were incubated at 37oC overnight in a static incubator.
3 discrete single colonies were used to inoculate 10 mL of LB broth supplemented with 35 μg/mL of chloramphenicol. Cultures were grown at 37oC shaking at 250 rpm for 18 hours.
After 18 hours of incubation, the optical density (at a wavelength of 600 nm – OD600) of each culture was taken by aliquoting 200 μL of overnight culture into a 96-well plate and measuring the absorbance of each culture at OD600. Each culture was diluted back to an OD600 = 0.5 by calculating the required volume of culture to be diluted in sterile LB broth; Volume used in inoculation = (Desired OD/Actual OD) × Volume to be inoculated. The diluted OD600 was measured and adjusted where required. When an OD600 range between 0.475-0.525 was achieved, fluorescence of each culture was measured.
4. Results
The variances of each triplicate cultures were analysed. Fig. 2 displays the mean of each of the standardize OD600 with error bars representing 95% confidence intervals. All the statistical analysis tables can be found in the supplementary information page. All data was analysed using IBM SPSS statistics software package.
Fig. 2: Means of Standardized Triplicate Devices OD600.
A one-way ANOVA conducted between each of the triplicates showed there was no statistical differences between the triplicates of each expression device showed that there was no significant difference (P=0.669). Further investigation using independent samples T-test showed a significant difference in the variance between triplicates in P3 (triplicates 1 & 2 [P=0.022] and triplicates 2 & 3 [P=0.036]). There was however no significant difference between the means of P3 triplicates 1 & 2 (P=0.085) and triplicates 2 & 3 (P=0.193). No other statistical significance was observed between any other conditions within the standardization dilutions.
There was a statistically significant difference between the means of the dilution back to an OD600 between the E. coli DH5α containing the negative control plasmid (B0012) triplicates 2 & 3 (P=0.023). Considering the range between the 2 triplicates, each of the data points were within the 0.475-0.525 range. No significant difference between the variances was observed between the second and third triplicate of the B0012 negative control (P=0.305).
Fig. 3: Means of Standardized Devices OD600.
Each of the OD600 = 0.5 dilutions were grouped and averaged. The data is displayed in Fig. 3. A one-way ANOVA between the different expression devices showed that there was no statistical differences between each expression device (P=0.284).
Fig. 4: Means of Standardized Triplicate Devices Fluorescence.
The mean measurement of fluorescence for each triplicate is displayed in Fig. 4. There was a statistically significant difference in the variance observed between triplicate 1 & 3 of P2 expression device (P=0.039). There was no significant difference between the means of triplicate 1 & 3 of the P2 expression device (P=0.549).
Fig. 5: Means of Standardized Devices Fluorescence (au).
The data from Fig. 4 was grouped according to expression device and is displayed in Fig. 5. Conducting a one-way ANOVA on the data P1, P2 & positive control expression devices shows a very highly significant difference between all other expression devices (P<0.001). Comparing the mean of P3 device with P1, P2 & positive control, there appears to be a very highly significant difference (P<0.001) with P3 showing the lowest level of fluorescence. The P3 device shows no statistical significant difference between the B0012 & E. coli DH5α negative controls (P=0.704 & P=0.243 respectively). There is no statistically significant difference observed between the two negative controls (P=0.967).
5. Discussion
6. Conclusion
Results from this study indicate that the P1-gfp expression device yields the highest expression levels after 18 hours of growth. P2-gfp expression device has a higher expression level than the positive control but a lower level of expression when compared to the P1-gfp expression device. The P3-gfp expression device has a fluorescence level which is comparible to the negative controls used in this study.
7. Participants
Sean Ross Craig (data analysis, cloning, restriction diagnostics & measurements), Elliott Parris (measurements & restriction diagnostics), Rachel Wellman (restriction diagnostics & measurements) & Ariana Mirzarafie-Ahi (cloning & measurements).
With thanks to Dr. Vitor Pinheiro, Dr. Jane Nicklin & Bilkis Kazi.