Team:Groningen/InterLab

• ≡ Menu
• Home
• Project
  • Description
  • Model
  • Design
  • Results
  • Notebook
  • Future Research
  • Protocols
  • InterLab
• Human Practices
  • Overview
  • Human Practices (Silver)
  • Integrated (Gold)
  • Public Engagement
  • Applied Design
  • Future Outlook
  • Safety
• Parts
• Collaborations
• Team
  • Team Members
  • Achievements
  • Attributions
  • Fun Activities
• Sponsors

Interlab Groningen 2017 Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The iGEM Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last three years. GFP was chosen as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein. Team Groningen aims to help improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole by participating in the Interlab 2017 study. One of the big challenges in synthetic biology measurement is that fluorescence data usually cannot be compared because it is reported in different units or because different groups process data in different ways. Often we work around this by doing some sort of “relative expression” comparison; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls. The InterLab protocol aims to address these issues by providing researchers with a detailed protocol and data analysis form that yields absolute units for measuring GFP in a plate reader. To start the devices and the control DNA were dissolved from the iGEM 2017 distribution plate. The plate contained 6 test devices and both a positive and negative control (fig 1). These plasmids were transformed into K12 DH5α cells.

These transformations resulted in 8 plates containing either green or opaque colonies (fig. 2 and 3). To be positive that the incorporation of the correct plasmid enabled colony formation, a plasmid isolation was done using the Fast-n-Easy Plasmid Mini-Prep Kit from Jena Bioscience. A portion of the isolated plasmid DNA was then digested using the restriction enzyme EcoRI for 30 minutes at 37°C. Then, the enzymes were denatured by a 20-minute 80°C heat shock. 10 ng of each digestion product was loaded onto a 1% agarose gel (fig. 4). It visualises that every picked colony contained a plasmid with a length of either ±2 or 3 kb. This confirms the incorporation of the correct plasmid.

Figure 2 Interlab transformant plates. Luria-Bertani plates with 25 µg/ml chloramphenicol.

After confirming the genetic makeup of the transformants, both the necessary optical density conversion factor and the fluorescence emission/concentration ratio were determined by measuring LUDOX solution at 600 nm and a fluorescein dilution series at …nm/…nm in the Biotek Synergy™ MX plate reader. Unfortunately, the detection limit for the fluorescein dilution was not high with these settings, so additional dilutions were made. This resulted in a conversion factor of 3.86 and a fluorescence ratio of 7.41∙10-6. With these known conversion factor and ratio, GFP expressing cells were grown for 6 hours at 37°C, starting with identical optical densities of 0.2. Culture samples were taken after 0, 2, 4 and 6 hours of growth (fig. 5). After collecting all the samples, the concentration of GFP per cell was determined by plate reader measurements (fig. 6). These measurements on average shows that the most GFP per cell is expressed after 2 hours. After 4 and 6 hours this ratio is lower, as less GFP per cell is expressed. When comparing individual devices with each other, device 1 and 5 result in the highest GFP per cell expression. Device 1 has a J23101 promotor and a B0034 RBS incorporated before the GFP gene and device 5 a J23106 promotor and a J364100 RBS. So, this would suggest that the combination of these particular promotors and their RBS’ results in the most optimal GFP expression.

Figure 6: Histogram of the measured fluorescence over the optical density of cell samples.