Team:Birkbeck/InterLab Study

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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), described by Dr. Jake Beal (iGEM Foundation) at the 2014 Jamboree in the following link, 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.

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.

The positive control in this study was; pSB1C3-Bba_I20270. Two negative controls were used; pSB1C3-BBa_R0040 & E. coli DH5α containing no plasmid

2 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 culture used for miniprepping & restriction diagnostics with EcoRI & PstI. The expected restriction pattern is displayed in Fig. 2 with the restriction diagnostic results being displayed in Fig. 3. Plasmid maps can be visualised in Fig. S1-S5.

Fig. 2: Expected Restriction Pattern. In the lane MW is a 2Log ladder. All substrate plasmids were digested with EcoRI & PstI. Lanes 1-3 are restriction diagnostics of P1, P2 & P3 gfp Expression devices respectively. Lane 4 is the restriction diagnostic of pSB1C3-BBa_I3504. Lane 5 is the restriction digest of the positive control used (Bba_I20270). Lane 6 is the negative control plasmid restriction (pSB1C3-BBa_R0040)

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Fig. 3: Restriction Diagnostic (EcoRI & PstI Digestion) of Interlab gfp Expression Devices. In Lane 1 is 5 μL of 2-Log NEB ladder. In lanes 2-4 are 5 μL of P1-3-gfp expression device digests respectively. Lane 5 contains 5 μL of the gfp part source digest. Lane 6 contains 5 μL of digested gfp-positive control. Lane 7 contains 5 μL of digested gfp-negative control expression device.

Experimental Procedure

After screening plasmids for the BBa_I3504 insert into the three promoter devices, successful transformants were streaked from glycerol stocks on LB agar (1.5%) plates were supplemented with chloramphenicol to a final concentration of 35 μg/mL chloramphenicol. Plates were incubated at 37^oC 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 37^oC 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 then measured after standardization. Fluorescence was measured using a BMG-LabTech FLUOstar omega plate reader. The excitation was 485 nm with fluorescence being measured at 520 nm

Results

The differences in means of each triplicate cultures were analysed. Fig. 4 displays the mean of each of the standardize OD₆₀₀ with error bars representing 95% confidence intervals. All data was analysed using IBM SPSS statistics software package.

Fig. 4: 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=0.123). Inspecting the multiple comparison table, there is no significant difference between the means of the E. coli DH5α strains standardized. This data shows that each culture was successfully standardized by dilution to an OD₆₀₀ range between 0.475-0.525.

Fig. 5: Means of Standardized Devices OD₆₀₀.

Each of the OD₆₀₀ = 0.5 dilutions were grouped and averaged. The data is displayed in Fig. 4. A one-way ANOVA between the different expression devices showed that there was no statistical differences between each expression device with regard to OD₆₀₀ standardization (P=0.284).

Fig. 6: Means of Standardized Triplicate Devices Fluorescence.

The mean measurement of fluorescence for each triplicate is displayed in Fig. 5. There was very highly statistically significant differences the between all the triplicates (P<0.001). However when comparing the same E. coli DH5α strain, there was no significant difference between the triplicates. This data shows that there is a difference between the different promoters under investigation & the control E. coli DH5α strains with no difference between each of the triplicates.

Fig. 7: Means of Standardized Devices Fluorescence (au).

The data from Fig. 5 was grouped according to expression device and is displayed in Fig. 6. 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).

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. Te P3-gfp expression device has a fluorescence level which is comparible to the negative controls used in this study. Each of the gfp expression devices P1, P2 & positive control from this study were further characterised in our InterLab Study Additional Results Fig. 9: Growth Curves of Different Strains of E. coli DH5α Following Culture Fluorescence, which is in the tab below. The data from this experiment also shows the same pattern of expression from each of the promoters under investigation.

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, Barbara Steijl, aunty Luba Prout.