InterLab

Bronze Medal Criterion #4

Standard Tracks: Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.

For teams participating in the InterLab study, all work must be shown on this page.

Introduction

Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.

Results

Cell Competency

Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 10⁸ to 6 ⋅ 10⁸ cfu/µg. The transformation efficiency was 6.6 ⋅ 10⁵ cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.

Parts

The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter. Device Part Number Negative control BBa_R0040 Positive control BBa_I20270 Test Device 1 BBa_J364000 Test Device 2 BBa_J364001 Test Device 3 BBa_J364002 Test Device 4 BBa_J364007 Test Device 5 BBa_J364008 Test Device 6 BBa_J364009

OD ₆₀₀ Reference Point

Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results.

Particle Standard Curve

Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results.

Fluorescence Standard Curve

The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results.

Cell Measurements

Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process.

Absorbance

Fluorescence

[1] http://parts.igem.org/Help:Protocols/Competent_Cells
[2] http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit
[3] http://parts.igem.org/Help:Protocols/Transformation
[4] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf