Contribution

Our team has made several contributions to the iGEM and synthetic biology communities. First, we have done extensive work in designing, and to a degree synthesizing, new parts to produce a phagemid that can be used in synthetic biology work with cyanobacteria. See our Part Collection page for more information on the CyanoSpectre Toolkit. The second contribution came about through our participation in the Phototroph Community, described on our Collaborations page. Finally, we contributed to the broader synthetic biology community by participating in the Sixth International InterLaboratory Measurement Study, detailed below.



Jacob keeping records for the Interlab Measurement study.
The complete log of this work can be found on the Notebook page.

Participation in the Sixth International InterLaboratory Measurement Study

iGEM’s 2022 InterLaboratory Study was designed to answer several key questions concerning reproducibility in the field of synthetic biology. The calibration study, which all InterLab participants completed, examined the performance of commonly used calibrants for fluorescence. In addition, our team decided to perform Experiment #2. In this experiment, a series of fluorescent protein constructs were studied to answer the question, “Does the order of transcriptional units influence their expression strength?” The SUNY Oneonta iGEM team decided to participate in this national study for two main reasons. First, the study provided a great opportunity to contribute to the synthetic biology community. Second, we have considered using fluorescence in future work on our project, CyanoSpectre. Thus, any experience gained working with fluorescent calibrants and proteins would contribute to the success of our project. Finally, we selected Experiment #2 because we are interested in the effect of transcriptional unit order on expression due to the importance of virion protein expression stoichiometry for our project. Our work on the InterLab study followed the protocols provided by iGEM, which are summarized below with any alterations and instrument specifications noted.

      
Bill and Jacob working on the fluorimeter.

Calibration Analysis: Establishing Baseline Measurements for Fluorescence Instruments

Part 1a: Fluorescence Calibration The purpose of this procedure was to test fluorescence detection using a series of known fluorescent dyes at varying concentrations. Three dyes were used to correlate to three commonly used fluorescent proteins (Table 1).

Part 1b: Control for Cell Count Calibration

Test absorbance using silica microspheres as an optical density/bacterial cell count calibrant.

Calibration Procedure:

1. The standard dyes, Fluorescein, Sulforhodamine 101 (Texas Red), and Cascade Blue were obtained in powder form. NanoCym 950nm monodisperse silica nanoparticles were obtained already suspended in ultra-pure water.
2. Fluorescein and Sulforhodamine 101 were resuspended in phosphate buffered solution (PBS).
3. Cascade Blue was resuspended in ultra-pure water.
4. A series of ten 2-fold serial dilutions were executed for each standard in a 96-well plate to prepare a range of concentrations for measurement. For Fluorescein and Cascade Blue, the concentrations ranged from 10μM to 10 x 10-3 μM. For Sulforhodamine, the concentrations ranged from 2 μM to 2 E-3 μM. For the silica nanoparticles, the concentrations ranged from 3 x E9 particles/mL of water to 3 E6 particles/mL of water.
5. Spectral measurements were made using the procedure described below.

Experiment #2: Using the three color calibration protocol: Does the order of transcriptional units influence their expression strength?

Eight different cell lines containing different DNA constructs were grown to examine how much fluorescent protein they produce. This measurement was done to compare the relative expression levels of the different DNA constructs.

Experiment #2- Cell Culturing Procedure:

1. DNA constructs were transformed into E. coli DH5α competent cells to make 8 different test cell line strains (TCLS).
2. TCLS were grown overnight in liquid media at 37 °C. Duplicates of each TCLS were prepared.
3. All TCLS cultures were diluted to the same starting concentration. A small sample was removed for the “Time Zero” measurement. Plate setup is depicted in Figure 1.
4. TCLS cultures were incubated at 37 °C for 6 hours. A sample was removed for the for “6 Hour” measurement.
5. Spectral measurements were made using the procedure described below.

NOTE: While preparing cell cultures, the tubes were wrapped in tin foil to maintain a dark environment and kept on ice to halt cell growth. 


Figure 1: Schematic for TCLS fluorescense measurements.
This diagram applies to both the time zero 96-well plate
and the 6 hour 96-well plate. Figure from (2).

Measurement Procedure:

1. For both the calibration and culture measurements, the fluorescence was measured using a Perkin Elmer LS55 Fluorescence Spectrometer. Excitation and emission wavelengths are given in Table 1.
2. Optical absorbance was measured using a Thermo Scientific Multiskan FC Microplate Reader.  The latter measurements were taken at 595 nm due to the filter limitations of the instrument. This is a deviation from the iGEM standard procedure, which specifies 600nm.
3. Before measuring, the plates were gently shaken to ensure suspension of the cells (or nanoparticles for the calibration procedure).

Table 1: Excitation and emission wavelengths used for fluorescence measurements.


Results Our calibration curves are shown in Figures 2-5. Representative results from Experiment #2 are provided in Tables 2 and 3 and Figure 6.


Figure 2: Calibration curve for Fluorescein. The chemical structure
of the dye is shown in the bottom right corner of the graph.




Figure 3: Calibration curve for Cascade blue. The chemical structure
of the dye is shown in the bottom right corner of the graph.




Figure 4: Calibration curve for Sulforhodamine 101. The chemical structure
of the dye is shown in the bottom right corner of the graph.


Figure 5: Calibration curve for NanoCym 950nm monodisperse silica nanoparticles.
Table 2: Experiment #2 Results. The average blank absorbance readings
subtracted from the average absorbance readings of the four aliquots
for each test device, time, and colony. Wavelength of analysis: 595 nm.



Table 3: Sample Time Course Data for BFP (Counts Per Cell). Data obtained
by dividing the fluorescence readings (excitation wavelength of 561 nm,
emission wavelength of 610 nm), by the absorbance readings at 595 nm for BFP.




Figure 6: Difference in counts per cell between the 6h and 0h measurements for BFP (see Table 3).

Conclusions based on our results The calibration study appears to have worked as expected, for the most part. For Cascade Blue and Fluorescein, fluorescence increased with increasing dye concentration (Figures 2 and 3). For Sulforhodamine, little fluorescence was observed at any concentration (Figure #4). Due to the unexpected results for Sulforhodamine, the calibration procedure was repeated for all four calibrants. There was no significant change in the results indicating that the Sulforhodamine calibrant must be defective. Because the rest of the calibrants produced data according to the expected trend, increasing signal with increasing concentration, we concluded that our fluorescence spectrometer and the UV-Vis microplate reader were functional.

Unexpectedly, we did not observe enhancement of fluorescence following incubation in any of the test cell lines (Table 3 and Figure 6). This could be explained in several ways. First, the cell dilution might not have been done appropriately and the cells did not grow sufficiently. Alternatively, the cells did grow but did not produce fluorescent protein. We looked to the absorbance data to determine whether the former was true. We noted an increase in signal from the 0 hour measurements to the 6 hour measurements for all of the different cell line cultures (Table 2). This indicates that the cells did grow. It can be concluded, then, that though the cells multiplied, they did not synthesize a significant amount of fluorescent protein.

References

1. Multicolor Fluorescence Per Particle Calibration Protocol, 2022 iGEM InterLab Study. https://old.igem.org/wiki/images/a/a4/InterLab_2022_-_Calibration_Protocol_v2.pdf
2. Experiment 2 - Using the three color calibration protocol: Does the order of transcriptional units influence their expression strength?, 2022 iGEM InterLab Study. https://old.igem.org/wiki/images/f/ff/InterLab_2022_Exp2.pdf
3. PubChem. (n.d.). Retrieved August 29, 2022, from https://pubchem.ncbi.nlm.nih.gov/