Team:CLSB-UK/InterLab

InterLab

The easiest way to get RSI

InterLab Measurement Study

The InterLab aims to standardize fluorescence measurements. We performed a relative expression fluorescence comparison, as fluorescence measurements cannot be compared directly due to different processing and units.

Parts

Interlab test devices used, as set by the iGEM measurement committee:[1]

Device Promoter Relative promoter strength RBS Relative RBS strength
+ve control (BBa_I20270) J23151 B0032 Medium
-ve control (BBa_R0040) R0040 Medium, repressible None None No CDS
TD1 (BBa_J364000) J23101 1791 B0034 Medium
TD2 (BBa_J364001) J23106 1185 B0034 Medium
TD3 (BBa_J364002) J23117 162 B0034 Medium
TD4 (BBa_J364003) J23101 1791 J364100 Strong BCD
TD5 (BBa_J364004) J23106 1185 J364100 Strong BCD
TD6 (BBa_J364005) J23117 162 J364100 Strong BCD


Parts J23100 through to J23119 are a family of constitutive promoter parts of varying strength. The strength of the promoters is given as the relative fluorescence of GFP when the TG1 E. coli strain containing these plasmids are grown in LB media to saturation.[2]

Methods

We used a BMG Fluostar Optima plate reader to measure OD600 and fluorescence values. The instrument settings were:

  • Excitation wavelength: 485nm
  • Emission wavelength: 530nm
  • Software version: 2.20R2
  • Firmware version: 1.26

OD600 measurements were taken in Corning 96 well assay plates (Lot. No. 25210007). The experiments were performed at the University of Westminster, under supervision of their PI (Dr Caroline Smith). We appreciate the use of their plate reader they offered us - see our collaborations page for more details.

We followed iGEM’s InterLab study protocol to collect our results.

Results

We submitted the iGEM’s completed spreadsheet to the measurement committee before the deadline, and received confirmation that the results were accepted on the 28th September.

OD600 calibration

OD calibration LUDOX-HS40 H2O
Replicate 1 12 11
Replicate 2 12 11
Replicate 3 11 11
Replicate 4 12 11
Arith. Mean 11.75 11
Corrected Abs600 0.75
Reference OD600 0.0425
OD600/Abs600 0.0567

Fluorescein standard cuves


Plotted on a base 10 logarithmic scale it becomes a nice straight line:

Fluorescence


For both colonies fluorescence decreased drastically between 0-2 hours, and stayed constant after that except for TD1 which increased slightly from hour 4 to hour 6.

Collaboration

To carry out our measurements, we collaborated with University of Westminster’s iGEM team. We were allowed to use University of Westminster’s laboratory to perform our InterLab study. The amazing Dr Caroline Smith (Ph.D., MA HE, SFHEA) helped us with the experiments. See our collaborations page for more details.

References

  1. (2017). InterLab Study, 2017.igem.org. Retrieved October 30, 2017, from http://2017.igem.org/Competition/InterLab_Study
  2. (2006, August 4). Part:BBa J23101. Registry of Standard Biological Parts. Retrieved October 30, 2017, from http://parts.igem.org/Part:BBa_J23101