Team:NJU-China/InterLab
Introduction
As we all know, repeatability is one of the key elements in biological experiments. It is the first time for NJU-China to participate in the InterLab Measurement Study in order to test some RBS devices this year. GFP (green fluorescent protein), a widely used and mature marker in synthetic biology, was used for the measurement study.
Results
Calibration
In order to take measurements under the consistent conditions, we performed the calibration measurements first. LUDOX-S40 was used to obtain a conversion factor, which enabled us to transform the Abs data into OD values. To perform subsequent experiments, a standard cure of fluorescence for fluorescein concentration was necessary. We prepared a dilution series of fluorescein and measured the fluorescence in a plate reader at the excitation of 485nm and emission of 530nm (Figure 1A and 1B). In the following studies, we used the curve to correct our cell-based measurements to values of corresponding fluorescein concentrations.
Fig 1. Fluorescein Standard Curve. (A) Fluorescein standard curve measured by plate reader with recommended filters (excitation of 485nm and emission of 530nm). (B) The same fluorescein standard curve on log scales.
Cell measurements
Abs600 as well as fluorescence of the eight samples of cultures were measured by plate reader at 0, 2, 4 and 6 hours. Based on the standard curve we obtained, we yielded the values of fluorescence. The time-course analysis of values of OD600- background, fluorescence background and fluorescence /Abs600 of samples of cultures from two colonies demonstrated that all values of OD600– background increased consistently (Figure 2A and 2B). However, those of fluorescence background increased remarkably before 4 hours of culture, after which most of them reached a plateau or began to decline (Figure 2C and 2D). Interestingly, fluorescence backgrounds from test device 1 and 2 were significantly different from others.
To have a better understanding of the results, the changes of fluorescence /Abs600 were also monitored at four time points (Figure 2E and 2F). The data suggested declines between 0 and 2 hours of incubation, followed by slight rises and decreases.
Next, we calculated the summary statistics of both arithmetic means as well as geometric means of the values of fluorescence /Abs600 (Figure 3A and 3B). The further investigation revealed that consistent with previous results of fluorescence /Abs600, the values of test device 1 and 2 were relatively higher than other 6 devices, probably suggesting higher efficiencies of the RBS devices (BCDs).
To have a better understanding of the results, the changes of fluorescence /Abs600 were also monitored at four time points (Figure 2E and 2F). The data suggested declines between 0 and 2 hours of incubation, followed by slight rises and decreases.
Next, we calculated the summary statistics of both arithmetic means as well as geometric means of the values of fluorescence /Abs600 (Figure 3A and 3B). The further investigation revealed that consistent with previous results of fluorescence /Abs600, the values of test device 1 and 2 were relatively higher than other 6 devices, probably suggesting higher efficiencies of the RBS devices (BCDs).
Fig 2. Measurements of OD600– background, fluorescence background and fluorescence /Abs600 at 0, 2, 4 and 6 hours. (A) Values of OD600– background of samples from colony 1 was measured. (B) Values of OD600– background of samples from colony 2 increased continuously in 6 hours. (C) Graph depicts the fluorescence backgrounds of samples of cultures picked from colony 1 with respect to time. (D) The fluorescence backgrounds of samples of cultures picked from colony 2 with respect to time were measured under the same conditions. (E) Data of fluorescence/ Abs600 of samples from colony 1 were qualified after 0, 2, 4 and 6 hours of incubation at 37°C and 220 rpm. (F) Fluorescence/ Abs600 of cultures from colony 2 were qualified after 0, 2, 4 and 6 hours of incubation under the same condition.
Fig 3. Summary statistics of arithmetic means as well as geometric means of fluorescence /Abs600. (A) Arithmetic means of fluorescence /Abs600 of eight samples after 0, 2, 4 and 6 hours of incubation. (B) Geometric means of fluorescence /Abs600 were calculated based on the experimental data.
Discussion
A ribosome-binding site (RBS) plays an important role in gene expression through mediating recruitment and biding of ribosomes. The efficiencies and reliabilities of RBS devices (BCDs) associated with numerous factors, which are largely unclear. In this study, we use GFP as a reporter to demonstrate that test device 1 and 2, representing part BBa_J23101 and BBa_J23106 respectively, have the stronger ability to improve downstream GFP expression. However, our study does not unravel the detailed roles or properties of the RBS devices (BCDs). Therefore, additional studies including collective statistical analysis may be required to address such complications.
Materials and Methods
Transformation
10 μL ddH2O was used to resuspend DNA Distribution Kit wells. Pipet up and down several times and let sit for a few minutes. The solution turned red due to the cresol red dye. 2mL tubes were placed in floating tube racks for better support when working on ice and the heat shock. 2mL tubes and the competent cells were pre-chilled with ice in bucket. 50μL competent cells were pipetted into 2mL tubes, together with 1μL DNA. Tubes were incubated on ice fro 30 min, followed by a heat shock at 42℃ for 1 min. The tubes were incubated on ice for 5 min and added 200μL LB media. The transformations were incubated at 37℃ for 2 hours on a shaker. Each transform was added on two petri plates for a 20μL and 200μL plating. Overnight (14-18 hours )incubation of transformations at 37℃. Single colonies were picked and counted for control transformation.
Calibration
We add 100 µl LUDOX- S40 (provided in kit) and 100 µl of H2O into 96 well plates with flat and transparent bottoms. Absorbance 600 nm of all samples in all standard measurement modes were measured in instrument whose path length correction was turned off (Spectramax m2e). This instrument measured all cell density readings with the same settings and volume.
Fluorescein stock tube was spun down to make sure pellet is at the bottom of tube. 2x fluorescein stock solution (100µM) was prepare by resuspending fluorescein in 1 mL of 1xPBS. The 2x fluorescein stock solution was diluted with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50µM. A serial dilution by consecutively transferring 100 µL from column to column with good mixing was performed. Fluorescence of all samples were measured in all standard measurement modes in instrument (Spectramax m2e).
Fluorescein stock tube was spun down to make sure pellet is at the bottom of tube. 2x fluorescein stock solution (100µM) was prepare by resuspending fluorescein in 1 mL of 1xPBS. The 2x fluorescein stock solution was diluted with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50µM. A serial dilution by consecutively transferring 100 µL from column to column with good mixing was performed. Fluorescence of all samples were measured in all standard measurement modes in instrument (Spectramax m2e).
Cell measurements
Escherichia coli DH5α was transformed with these following plasmids.
Positive control
Negative control
Test Device 1: J23101+I13504
Test Device 2: J23106+I13504
Test Device 3: J23117+I13504
Test Device 4: J23101.BCD2.E0040.B0015
Test Device 5: J23106.BCD2.E0040.B0015
Test Device 6: J23117.BCD2.E0040.B0015
2 colonies from each of plate were picked and inoculated on 5-10 mL LB medium + Chloramphenicol. The cells were grew overnight (16-18 hours) at 37°C and 220 rpm. The instrument was set to read OD600 (as OD calibration setting). OD600 of the overnight cultures was measured and recorded data. The cultures were diluted to a target OD600 of 0.02 in 12 ml LB medium + Chloramphenicol in 15 mL tubes covered with foil to block light. The cultures were incubated at 37°C and 220 rpm. 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation were taken. (At each time point, a sample from each of the 8 devices, two colonies per device, for a total of 16 samples will be taken). Samples were placed on ice. Samples were measured (OD and Fl measurement) at the end of sampling point.
Negative control
Test Device 1: J23101+I13504
Test Device 2: J23106+I13504
Test Device 3: J23117+I13504
Test Device 4: J23101.BCD2.E0040.B0015
Test Device 5: J23106.BCD2.E0040.B0015
Test Device 6: J23117.BCD2.E0040.B0015
Statistical analysis
Error bars indicate ±SEM between biological replications. Technical and biological triplicates of each experiment were performed. Detailed methods used in the study are in the supplemental information.
Supplemental Information
Supplemental information is in a excel table, which can be found and downloaded online at here.
Acknowledgements
This study was supported by M3 Laboratory, School of Life Sciences, Nanjing University. We thank Prof. Xi Chen and Prof. Chen-yu Zhang for their supervision.
Reference
Beal, J., Haddockangelli, T., Gershater, M., Mora, K. D., Lizarazo, M., & Hollenhorst, J., et al. (2016). Correction: reproducibility of fluorescent expression from engineered biological constructs in e. coli. Plos One, 11(3).
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