Team:Oxford/Interlab Study

Interlab Study

Introduction

Overview

One of the fundamental principles of synthetic biology is the characterization of standard biological parts. iGEM HQ's International InterLab Measurement Study is an endeavour at achieving the large-scale characterization of a set of biological devices which has been previously designed and catalogued in the BioBricks Registry. In this study, iGEM teams from across the world set out to measure the fluorescence of model organisms which have been transformed with devices comprising the same green fluorescent protein (GFP) gene expressed at different levels by being fused to promoters of different strengths. This would provide a large dataset from which a more accurate analysis that accounts for lab-to-lab variations can be drawn.

The promoters characterized in the 2015 InterLab Study are three constitutive promoters of different strengths found within the Anderson Promoter Collection, which were included in the 2015 iGEM Distribution Kit. We assembled each of these three promoters upstream of a given GFP-encoding BioBrick and transformed each of the composite parts into three different strains of E. coli to measure their respective fluorescence levels. We measured fluorescence levels using a 96-well microplate reader, a flow cytometer, as well as a confocal microscope.

Theoretically, we should obtain the same results from different teams since the devices used are the same but differences in lab protocol and equipment can often cause variation in results between labs. The use of multiple methods allows differences between different techniques to be quantified as well. Hence this year, we have introduced yet another method of using the MicrobeTracker software suite which is an easy-to-use data analysis method that measures fluorescence in absolute values of mean pixel intensity. We have also conducted the measurement in three different strains of E. coli for comparison of fluorescence between different strains too. Again, we believe this is an approach that can be adopted by other iGEM teams in the future to further explore possible variations in lab protocol and equipment.

Key Results and Findings

Through the data obtained from our microplate reader, we found that the strengths of the three promoters investigated are in the same order as Anderson et al's initial characterization across the three E. coli strains which we studied. The relative fluorescence magnitudes we measured however do not agree with the ratios presented in the Anderson study.

To eliminate the dependence on equipment parameters associated with arbitrary fluorescence units, we calibrated our fluorescence readings against different concentrations of a fluorescent chemical standard, sodium fluorescein, and expressed fluorescence intensity in units of its concentration.

Methods and Materials

Bacterial Strains and Growth Conditions

E. coli DH5α was used for all cloning purposes. E. coli DH5α, E. coli MG1655, and E. coli RP437 ∆FliC were used as expression hosts. Cultures for cloning were grown in Lysogeny Broth (LB) at 37°C, whereas for GFP expression cultures were grown in Knight's M9 Supplemented Media at 37°C.

Plasmid Constructions and Gene Cloning

The selected Anderson promoters were fused to the designated GFP coding sequence using the BioBrick assembly method (step-by-step experimental detail as well as gel electrophoresis images can be found at the InterLab section of our lab notebook).

Briefly, the promoter-containing pSB1C3 plasmids BBa_K823005 (a.k.a. BBa_J23101), BBa_K823008 (a.k.a. BBa_J23106), BBa_K823013 (a.k.a. BBa_J23117), and RBS-GFP-containing pSB1A2 plasmid BBa_I13504 (RBS BBa_B0034 fused to GFP BBa_E0040) were obtained from the 2015 iGEM Distribution Kit and transformed into E. coli DH5α. Upon plasmid extraction (using Omega Biotek E.Z.N.A. Plasmid DNA Mini Kit I, Spin Protocol according to manufacturer specifications), restriction digest was performed on the SpeI and PstI sites of the promoter plasmids to linearize them, and the XbaI and PstI sites of the RBS-GFP plasmid to isolate the RBS-GFP sequence. Subsequent ligations were performed to insert the isolated RBS-GFP sequence into the linearized promoter plasmids, yielding the composite parts BBa_K823005+I13504, BBa_K823008+I13504, and BBa_K823013+I13504, each containing an 8bp assembly scar sequence TACTAGAG between the promoter sequence and the ribosome binding site. The identities of the plasmids were confirmed through sequencing (SourceBioScience, UK). The sequencing data is available for perusal in the Supplementary Information section below.

The three composite parts were then each separately transformed into the three different E. coli expression host strains.

Experimental Controls

BBa_I20270, a constitutively-promoted GFP expression device, and BBa_R0040, a pTetR promoter with no coding sequence linked downstream were cloned into the aforementioned expression host strains to serve as positive and negative controls for the experiments respectively.

Plate Reader: Calibration against Chemical Standard

Sodium fluorescein was the chemical standard of choice for the plate reader fluorescence readings as it fluoresces in the same green colour as GFP at concentrations below 0.05M (above 0.05M, sodium fluorescein has a red tint).

1.66g of fluorescein (free acid) was first dissolved in 5mL of pH 8.0 Tris HCl to obtain 1M fluorescein acid solution. The solution was then neutralized with 5mL 2M NaOH (fluorescein is a dibasic acid) to obtain 10mL of 0.5M sodium fluorescein solution (hereon referred to as NaFluo).

Serial dilution of NaFluo was performed for the construction of calibration curve. 100µL of NaFluo solution is measured at each time in the wells of a 96-well plate.

Plate Reader: Culture Growth Conditions

Stationary cultures for expression measurement were grown overnight for 18 hours at 37°C in 5mL chloramphenicol-supplemented Knight's M9 Supplemented Media contained in glass test tubes oriented to be standing at an angle with 225 rpm orbital shaking.

Plate Reader: Experimental Setup

A BMG LABTECH FLUOstar Omega was used for all plate reader measurements. For fluorescence readings, the excitation filter used was 485-12 nm and the emission filter used was 520 nm. Gain was set to 550 and number of flashes per well was set to 20. For OD600 readings, the excitation filter used was 600 nm and number of flashes per well was set to 22.

The type of 96-well plate used was Corning® 96-well black well, clear bottom, tissue-culture treated sterile plates. 100µL of stationary culture was added into each well, and each culture was measured in triplicate. Each device was also measured in biological triplicate, whereby cultures were set up from three different colonies on the same streaked plate and measured accordingly. Reported fluorescence values are normalized against cell density through the OD600 values read at the time of the fluorescence measurements.

Flow cytometry

Culture Growth Conditions

Stationary cultures for expression measurement were grown overnight for 18 hours at 37°C in 5mL chloramphenicol-supplemented Knight's M9 Supplemented Media contained in glass test tubes oriented to be standing at an angle with 225 rpm orbital shaking. They were then diluted by a 1:20 dilution and grown in conical flasks, shaking at 225rpm till OD600 = 0.6.

Experimental Set Up

  1. Load the preset settings of FSC 560, SSC420, BLH1 200 and flow rate 100µl/min on the flow cytometer.
  2. Start the performance test for the flow cytometer with performance beads and machine solutions.
  3. Vortex the samples and then measure them by running it through the flow cytometer with excitation wavelength at 488nm and emission detected at wavelength 530nm.
  4. Data was analysed using the Attune Nxt software.

Microscopy (using MicrobeTracker analysis)

Culture Growth Conditions

Stationary cultures for expression measurement were grown overnight for 18 hours at 37°C in 5mL chloramphenicol-supplemented Knight's M9 Supplemented Media contained in glass test tubes oriented to be standing at an angle with 225 rpm orbital shaking. They were then diluted by a 1:20 dilution and grown in conical flasks, shaking at 225rpm till OD600 = 0.45.

Experimental Set Up

  1. Prepare 1% agarose gel (1g of agarose in 100mL of milliQ water) as platform for viewing cells on.
  2. Pour agarose gel from previous step onto a glass slide between two square cover slips and push down from above using another square cover slip.
  3. Once agarose gel has set, pipette 2µL of cell culture onto it.
  4. Remove the two cover slips on the sides but keep the one on top of the gel.
  5. Apply a drop of immersion oil (n = 1.5) onto the cover slip and mount the slide on the microscope, cover slip side down.
  6. View images on microscope and adjust focus accordingly.
  7. Once satisfied with images, capture them using the ANDOR camera using GFP excitation at a wavelength of 476nm and an emission filter of 525nm.
  8. Export images into MicrobeTracker.
  9. Use MicrobeTracker to locate cells on the image and quantify their fluorescence in mean pixel intensity/ cell or total intensity/cell.

Results and Discussion

Plate Reader: Calibration against Sodium Fluorescein




Calibration curve for microplate reader settings based on NaFluo concentration. Each data point is the mean of a hexplicate set of wells at a given NaFluo concentration.



The fluorescence of NaFluo at concentrations of 20, 10, 5, 2, 1, 0.2, 0.1, 0.01 µM were measured and the linear relationship between fluorescence and NaFluo concentration was established using the "Add Trendline" linear regression function in Microsoft Excel. The trendline was fitted to pass through the origin and the gradient of the line was found to be 9669.1 arbitrary fluorescence units per µM of NaFluo.

The LINEST array function in Microsoft Excel was used to calculate the standard error in the gradient, which was found to be 177.9 arbitrary fluorescence units per µM of NaFluo, or 1.8% of the value of the gradient.

Plate Reader: Device Measurements



The normalized fluorescence value for each device presented here is the mean value across the three biological replicates and the three measurement replicates within each biological repeat (i.e. averaged across nine values). The error bars constructed for each device take into account the standard error across measurement replicates within each biological repeat as well as the standard error across biological replicates (methodology for computation is detailed in the data sheet attached in the Supplementary Information section below).


In this study, we set out to verify the relative measured strengths between three Anderson Promoters against their published values in the BioBricks registry as well as investigate any potential strain-to-strain variations in terms of across-the-board expression levels as well as relative promoter strengths which may arise. The relative strengths measured in our study is compared against the original Anderson study in the table below:


Study E. coli
strain		 K823005
(J23101)		 K823008
(J23106)		 K823013
(J23117)
Anderson TG1 1 0.67 0.09
Our study DH5α 1 0.91 0.05
Our study MG1655 1 0.75 0.02
Our study RP437 ∆FliC 1 0.26 0.05

It is interesting to note that only ''E. coli'' MG1655 responds to the Anderson Promoters to roughly the same strength ratio as Anderson's original study.

Flow Cytometry Measurements

Raw data is reported here in arbitrary fluorescence units.

Device Strain Biological Sample 1 (averaged across three measurements) Biological Sample 2 (averaged across three measurements) Biological Sample 3 (averaged across three measurements) Mean value across biological triplicate Standard deviation in biological triplicate
J123101 + I13504 DH5α 8127.5 8479.57 10540.24 9049.1 1064.1
J123106 + I13504 DH5α 3069.9 5012.05 3738.44 3940.1 805.6
J123117 + I13504 DH5α 55.91 76.20 90.91 74.34 14.35
Positive control DH5α 1931.12 3092.17 2328.05 2450.45 481.83
Negative Control DH5α 64.59 75.09 47.26 62.31 11.48
J123101 + I13504 RP437 ∆FliC 6996.35 11072.54 10985.48 9684.79 1901.35
J123106 + I13504 RP437 ∆FliC 3552.87 4337.23 4201.13 4030.41 342.21
J123117+ I13504 RP437 ∆FliC 48.26 54.77 50.87 51.3 2.68
Positive control RP437 ∆FliC 1435.36 4000.61 2485.39 2640.45 1052.98
Negative Control RP437 ∆FliC 42.01 41.28 55.98 46.42 6.76
J123101 + I13504 MG1655 11725.07 11809.75 8946.79 10827.20 1330.10
J123106 + I13504 MG1655 7837.80 4887.64 3108.24 5277.89 1950.45
J123117 + I13504 MG1655 101.08 166.74 86.24 118.02 34.98
Positive control MG1655 1363.03 8363.59 3399.49 5543.99 3015.73
Negative Control MG1655 46.81 24.05 51.12 47.10 3.17

Microscopy Results

Fluorescence units are reported in mean pixel intensity per cell.

Device Strain Biological Sample 1 (averaged across three measurements) Biological Sample 2 (averaged across three measurements) Biological Sample 3 (averaged across three measurements) Mean value across biological triplicate Standard deviation in biological triplicate
J123101 + I13504 DH5α 0.0018439
 0.046289 0.0958 0.029686 0.044367
J123106 + I13504 DH5α 0.0018485 0.010835 0.11865 0.025659 0.043008
J123117 + I13504 DH5α 0.0018407 0.0064267 0.0041919 0.0048402 0.0019329
Positive control DH5α 0.019789 0.054828 0.056155 0.043427 0.037385
Negative Control DH5α 0.0027105 0.0029861 0.0029476 0.0028108 0.00025374
J123101 + I13504 RP437 ∆FliC 0.028333 0.11151 0.017413 0.030826 0.053064
J123106 + I13504 RP437 ∆FliC 0.013095 0.035656 0.026299 0.02725 0.035996
J123117+ I13504 RP437 ∆FliC 0.0031156 0.0088561 0.0029594 0.0050305 0.013291
Positive control RP437 ∆FliC 0.058934 0.027682 0.027994 0.037593 0.025714
Negative Control RP437 ∆FliC 0.0027362 0.0024896 0.0025544 0.0026392 0.00019896
J123101 + I13504 MG1655 0.081568 0.12061 0.079278 0.086298 0.070526
J123106 + I13504 MG1655 0.072505 0.092069 0.22325 0.082413 0.096407
J123117 + I13504 MG1655 0.0034456 0.0036499 0.0099427 0.005435 0.005435
Positive control MG1655 0.050019 0.021582 0.21424 0.057183 0.062221
Negative Control MG1655 0.0026454 0.0030729 0.0029368 0.0029536 0.00018618

Comparison of fluorescence between different devices:

Comparison of fluorescence between different E. coli strains:

Discussion

Flow Cytometry

Our results for flow cytometry were in close agreement to both the order and the ratios of the strength of the promoters as in Anderson et al’s initial characterization. The relative strengths measured in different strains in our study is compared against the original Anderson study in the table below.

Promoter Anderson’s
Study		 DH5α
(our study)		 RP437 ∆FliC
(our study)		 MG1655
(our study)
K823005
(J123101)		 1.0 1.0 1.0 1.0
K823008
(J123106)		 0.47 0.35 0.38 0.41
K823013
(J123117)		 0.06 0.0086 0.0046

It also appears that there were minor strain-to-strain variations in expressions levels.

Microscopy

Our microscopy results showed that the strengths of the three promoters investigated are in the same order as Anderson et al's initial characterization across the three E. coli strains which we studied. The relative fluorescence magnitudes we measured however do not agree with the ratios presented in the Anderson study as shown in the table below.

Promoter Anderson’s
Study		 DH5α
(our study)		 RP437 ∆FliC
(our study)		 MG1655
(our study)
K823005
(J123101)		 1.0 1.0 1.0 1.0
K823008
(J123106)		 0.47 0.86 0.88 0.95
K823013
(J123117)		 0.06 0.16 0.16 0.063

While DH5α and RP437 ∆FliC gave similar results for microscopy, MG 1655 gave significantly different results as the other two strains. This could be a potential source of investigation by future iGEM teams.

Another observation we made was that several E. coli cells (from all the different strains) with the constructs J123101 and J123106 had unusually long sizes and large deposits of proteins. This could potentially indicate the burden placed on the cell by the strength of these promoters.

Image showing unusually E. coli with J123101 construct. Note also the protein deposits

Conclusions

Our methods have all given promoter strength in the same order as Anderson’s initial characterization studies although our ratios are not entirely in agreement. Through the use of different strains of E. coli, we have also discovered strain-to-strain variations which were clearly demonstrated in the histograms for microscopy results. This, in addition to the fact that microscopy results could be reported in absolute units without the need for a calibration curve, leads us to suggest that microscopy taken up by other iGEM teams in future years. Furthermore, the aforementioned strain-to-strain variation is another interesting point worth exploring by iGEM teams in future years.