InterLab

Introduction

Last year, we continued our Archaeal Interlab study which entailed distribution of cell extracts of Methanococcus maripaludis containing mCherry mutants to other iGEM teams and asking them to record fluorescence readings of mCherry protein extracts. However, in this extension of the project, we treated the harvested Methanococcus cells with 5% formaldehyde before measuring the fluorescence of the mCherry protein to harden the tissue components of the cells, keeping the structure of the proteins intact and ready for measurement.

This year, we decided to contribute to the replication of fluorescence measurements in equivalent units around the world. Our findings from last year proved that fluorescence readings were largely dependent on the type of machinery used to measure fluorescence. Therefore, we wanted to do our part in improving the effectivity of sharing data between labs and interpreting them in iGEM community and the synthetic biology community. Alongside other iGEM teams all over the world, we followed a strict protocol and sent in green fluorescent protein (GFP) fluorescence measurements from our plate reader to help develop a standard measurement procedure for GFP.

Experiment

For this experiment, we were given six test devices, a positive control, and a negative control in our Kit Plates 6 and 7. All of the devices coded for GFP except for the negative control which was used to calculate the baseline for background noise. Each DNA sample was resuspended and transformed into Escherichia coli DH5α competent cells using heat shock. After a 1 hour incubation at 200 rpm, the cells were spread onto petri plates containing LB agar and Chloramphenicol to be incubated again at 37°C overnight. The next morning, the colonies on the 100μl positive control plate were counted to calculate our competent cell efficiency and verified that our competent cells had an efficiency that met the iGEM standards (between 1.5x10^8 and 6x10^8 cfu/µg). Single colonies were picked from each transformation for PCR to verify the part sizes and make glycerol stock.

Immediately following the overnight incubation of the competent cells with the transformed plasmids, 2 colonies were picked from each plate and inoculated in 5 mL of LB medium and 5µL of Chloramphenicol to grow overnight at 37°C and 220 rpm. The following day, the cultures were all diluted to a target OD600 of 0.02 in 12 mL of LB medium and Chloramphenicol in 50 mL falcon tubes. The cultures were further incubated at the same conditions as before with 500µL samples taken at 0, 2, 4, and 6 hour timepoints. The samples were then measured for OD and FI values in the plate reader. These values were submitted to iGEM using the Google Form links provided on the iGEM website.

NOTE: Prior to utilizing the plate reader, LUDOX-S40 was used to create a single point reference to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD600 measurement. The plate reader was then calibrated using a fluorescein fluorescence standard curve using a dilution series of fluorescein. All calibration steps followed the Interlab 2017 Plate Reader Protocol, officially set by iGEM.

Collaboration Study

In an effort to work together and support another iGEM team, we teamed up with Lambert High School in Georgia. During our data collection period, we invited the members on their team to collect their data as well using our plate reader. By extending the courtesy of the UGA research labs, we facilitated meaningful interaction between Lambert and UGA and subsequently developed a strong, healthy relationship that can allow both teams to succeed in the future. That said, by sharing our previous experiences from iGEM, we helped Lambert develop a more efficient protocol for data collection in the interlab study.

Results

Fluorescein fluorescence standard curve using a dilution series of fluorescein:

Values should form a straight (1:1) line on both linear and log scale. However the values show that at low concentrations, there is a linear pattern, but level out at higher concentrations. This shows an oversaturation of the fluorescein at around 10 µM.

As expected, the negative control colonies show little to no fluorescence. Test device 1 colonies displayed the highest levels of fluorescence over the 6 hour time period while test device 3 and 6 colonies display the lowest levels of fluorescence. For test device 2 and 4 colonies, there was a steady increase in fluorescence over time while test device 3 and 6 colonies showed decreasing fluorescence over time.

Conclusion

From submitting and sharing our results with the other iGEM interlab teams, we will gain information about the reliability of our data. When the results of all the 2017 interlabs are released, we will be able to see how our information compares on a global scale.

Additionally, the interaction with the Lambert team has allowed us to pass on the skills we have developed in lab and obtain feedback on our method of experimentation. This allowed our team to implement a standardized protocol for fluorescence measurements using a plate reader and to pass on this information to future teams.

The Future

Going forward, we hope that our data contribution will help future researchers in the synthetic biology field to interpret and standardize their data. Completion of the protocol set forth by the iGEM committee was important in setting an official order of steps to obtain consistent data across the globe. Using the plate reader was also a great opportunity for our team members to familiarize themselves with calibrating and taking measurements. For future iGEM projects and research, our team members can use plate readers with confidence and help train other students. We will also have an established connection with a lab on campus that has access to a plate reader.