Team:UGA-Georgia/Interlab
Introduction
Last year, we were able to successfully begin an Archaeal Interlab study and distribute cell extracts of Methanococcus maripaludis containing mCherry mutants to 9 iGEM teams. We asked teams to record fluorescence readings of mCherry protein extracts. What we found was that the fluorescence readings were largely dependent on the type of machinery used to measure fluorescence: plate reader, confocal microscope, Bradford/ BCA and NanoDrop. We decided to make some adjustments this year and start a new project known as the “Formaldehyde Fixed Single Cell Project”.
Instead of lysing our cells and measuring the fluorescence of the mCherry protein extracts from our Methanococcus experiments like before, we decided to fix our harvested Methanococcus cells with 5% formaldehyde before measuring the fluorescence of the mCherry protein. The use of formaldehyde will harden the tissue components of the cells keeping the structure of the proteins intact and ready for measurement of the fluorescence.
Instead of lysing our cells and measuring the fluorescence of the mCherry protein extracts from our Methanococcus experiments like before, we decided to fix our harvested Methanococcus cells with 5% formaldehyde before measuring the fluorescence of the mCherry protein. The use of formaldehyde will harden the tissue components of the cells keeping the structure of the proteins intact and ready for measurement of the fluorescence.
The Experiment
For this experiment, the following cultures: L2C16, L1C18, L1C12 were used as well as Wild Type culture as our negative control. The wild type culture does not contain the mCherry protein and instead just plain plasmid. Since the wild type culture contains no mCherry protein, there was technically no fluorescence; the value recorded was used as the background.
The transformed cells were first grown to a specific absorbance, the cells were centrifuged and the cell pellets were re- suspended in a suspension buffer. The cells were then placed on a shaker overnight for oxygen exposure which allows for the activation of fluorophore and is critical for the expression of the mCherry protein. Afterwards, the cells were then distributed into aliquots and 5% formaldehyde was added to each cell sample for cell fixation. Fixing the cells allowed us to ship the cells at room temperature and made shipping to other iGEM teams go a lot smoother.
Like last year, we used plate reader to measure the fluorescence of our cells. In addition, this year we also decided to use flow cytometry to separate our population of cells based on the fluorescence of our mCherry protein. This method of measurement is a better representation of the fluorescence since it is a more sensitive instrument than a plate reader. With this technology, we are able to measure multiple characteristics of single cells as they pass through a beam of light. The relative size, internal complexity and fluorescence intensity can be measured. To measure the fluorescence intensity, we analyzed a Log mCherry vs. Log SSC plot (side- scatter light) to determine positive expression of mCherry. We used our Wild Type sample for determining the regions for positive vs. negative mCherry regions. We found that using the median of the mCherry values among the positive region of the side- scatter plot would be best to represent the fluorescence intensity of the mCherry for each sample.
The transformed cells were first grown to a specific absorbance, the cells were centrifuged and the cell pellets were re- suspended in a suspension buffer. The cells were then placed on a shaker overnight for oxygen exposure which allows for the activation of fluorophore and is critical for the expression of the mCherry protein. Afterwards, the cells were then distributed into aliquots and 5% formaldehyde was added to each cell sample for cell fixation. Fixing the cells allowed us to ship the cells at room temperature and made shipping to other iGEM teams go a lot smoother.
Like last year, we used plate reader to measure the fluorescence of our cells. In addition, this year we also decided to use flow cytometry to separate our population of cells based on the fluorescence of our mCherry protein. This method of measurement is a better representation of the fluorescence since it is a more sensitive instrument than a plate reader. With this technology, we are able to measure multiple characteristics of single cells as they pass through a beam of light. The relative size, internal complexity and fluorescence intensity can be measured. To measure the fluorescence intensity, we analyzed a Log mCherry vs. Log SSC plot (side- scatter light) to determine positive expression of mCherry. We used our Wild Type sample for determining the regions for positive vs. negative mCherry regions. We found that using the median of the mCherry values among the positive region of the side- scatter plot would be best to represent the fluorescence intensity of the mCherry for each sample.
The Collaboration Study
After finalizing our proposal, contacting the biosafety office, creating a data input form, and creating a universal protocol for measuring mCherry protein fluorescence using plate reader and flow cytometry, we were ready to contact an assortment of iGEM research teams across the United States gauging general interest in our collaboration study. We contacted teams with our proposal and an invitation to join our study asking teams to measure the fluorescence of our formaldehyde fixed cell samples either using flow cytometry or plate reader, which does not require anaerobic chambers or any additional experimentation. We were able to built up a network of 6 participating teams: Columbia University, Georgia State University, University of Tennessee Knoxville, University of Pittsburgh, University of Nebraska and Genspace. We greatly appreciate our participating teams for their support and efforts toward our study.
Figure 1. Geographical Distribution of Archaeal Interlab Teams
Statistical Analysis
Looking at each team’s data inputs, we saw that the fluorescence of the L2C16 Methanococcus mutant stayed consistent among the teams. Therefore, we compared the fluorescence values of mutants L1C12 and L1C18 against the fluorescence value of L2C16 mutant. In doing so, we were able to calculate percentages for the fluorescence of L1C12 and L1C18 mutants and came up with a model displaying the variation among teams that used plate reader and variation among teams that used flow cytometry.
Figure 2. Summary of the percentages of the fluorescence of L1C12 and L1C18 mutants against the fluorescence of L2C16 mutant among teams using flow cytometry.
Figure 3. Summary of the percentages of the fluorescence of L1C12 and L1C18 mutants against the fluorescence of L2C16 mutant among teams using plate reader.
Conclusion
From the collaboration study, we’ve gained information about the reliability and reproducibility of our data. Our collaboration study not only validities our findings with the work we’ve done in the lab, but also gave participating teams awareness of the capabilities of Methanococcus marpaludis and knowledge of the methods of reading mCherry fluorescence. Additionally, the interaction with other teams allowed us to pass on the skills we have developed in lab and obtain feedback on our method of experiments. This season’s interlab allowed our team to implement a standardized protocol for fluorescence measurements for both flow cytometry and plate reader and to look at some of the standard deviation that it may have changed.
The Future
This year was the first time our interlab branched out to iGEM teams in China. Although, the data wasn’t sent back to us in time, it was valuable in creating a protocol for international shipping for our cell extracts. Since it was the first time we had potential teams participating from overseas, this season was a great experience in determining the protocol and process by which genetic materials could be shipped overseas with the additional requirements in customs. Next year, the team plans to extend our interlab to more teams overseas and bring in more participants in order to better measure the effect of our standardized protocol on standard deviation.
