The Baltimore Bio-Crew’s participation in the iGEM 2018 interlab study was not only an informative experience, but also quite empowering for our team. Contributing to the efficiency of experiments and data production of scientific labs across the world was an amazing experience. This year, our participation in iGEM’s 2018 Interlab encouraged us to gain skills in necessary research procedures, learn to use scientific lab equipment, and pushed us to produce reputable results.
ObjectiveThe objective of the Interlab was to improve the methods in which labs can reproduce experiments and repeat measurements. In synthetic biology, the idea of a “reproducible experiment” is crucial to obtaining valid data and sharing protocols that can be conducted in other labs.
One of the most commonly used markers for certain measurements in synthetic biology is Green Fluorescent Protein (GFP), though there are limitations when using this as a bio-marker. Fluorescence is not a measurement that can be consistently obtained, as it is measured with different protocols, units, and scientific equipment.
The goal of the interlab was to find a more easily regulated way of measuring data. Our team conducted experiments to compare the validity and efficiency of fluorescence measurements against that of absolute cell counts.
Findings from Data Collected:
After completion of the experiments, and a lot of dedication in the lab as a team to complete the requirements of the interlab we reached some many findings about the nature of our data. When measuring the growth of bacterial cell colonies we determined the fluorescence measurement, absorbance measurement, and finally the absolute cell counts. We used the Tecan GENios Plus plate reader for our fluorescence and absorbance measurements. The data from these experiments provided many insights into the reliability of certain measurements over others.
We measured fluorescence with constant parameters: room temperature, reading from the top of the plate, and set for an “optimal reading”. At hour 0, the negative controls had a lower measured fluorescence than the positive controls. We also found that at hour 0, the fluorescent measurements for all of our devices as well as the “blank” measurement were quite similar with about a 3000 fluorescence measurement. At hour 6, though the fluorescence measurements of the controls did increase, the negative control once again had a lower average fluorescence measurement than the positive control. We also saw an increase in fluorescence measurement for all of our devices, though the devices had varied measurement of fluorescence. We measured absorbance with constant parameters: room temperature, ABS filter 550 nm, and excitation of 535. At hour 0, the average of our positive control, negative control, and 6 devices had a quite similar absorbance measurement. With approximately a .400 OD. At hour 6, we saw an increase in OD for our positive control, negative control, and six devices. We did notice that the absorbance measurement for our positive and negative control were very similar, which was not expected considering the negative control was not supposed to grow as much as the positive control did after incubation.
We finally measured the absolute cell counts for each of the colonies grown, which ended up being a very involved process. We found that after incubation, our positive controls grew far more than our negative controls. We also found that as the bacterial cells were diluted, the amount of colonies that grew decreased for each of our six devices. One colony is an indication of one cell growing from the original solution. We found that the dilutions created a quite proportional decrease in cell colonies formed on each of our plates.
Positive and Negative Chart
Measuring Growth of Cell Colonies: Fluorescence and Absorbance
|Measuring optical density provides an immediate ability to collect data||There can be an over saturation where the fluorescence is too bright to produce exact results|
|Although the experiment required numerous steps, we were able to perform each step of the experiment quicker than the colony counting protocol||There will always be variability in these measurements, since different lab equipment and methods might be used to obtain these measurements|
|The experiment required less use of scientific materials, as we only needed to use approximately four clear bottom 96-well plates to complete measurements for fluorescence and absorbance at hour 0 and hour 6||Our data was not consistent with our hypotheses at all times, at some points there was illogical data produced|
|Mistakes can be more prevalent when measuring colony growth through OD, as the parameters of each measurement have to be consistent and if they are not then measurements are not reputable|
|More accurate data is able to be obtained, as there is less error to happen within this experimental protocol. Relatively, the same methods, units, and materials are being used. Better dynamic range( more accurate data over a larger range)||This method of measurement is more involved and requires more manual time. Our team not only had to endure long lab days to wait for the incubation periods to end, but we also spent a lot of time in the lab counting each individual colony.|
|There is a better dynamic range, as the colony counts can be measured for any device or condition. A larger range of data can be obtained.||There is a longer duration that must occur to obtain results, compared to measuring OD through fluorescence and absorbance.|
|There is room for human error when counting each individual colony, as determining each single colony can be quite subjective.|
Final Thoughts and Conclusions
Our experience with the 2018 iGEM Interlab proved to be extremely useful. We integrated some of the skills that we developed during our participation in the Interlab within our own project. The ability to learn how to do a series of dilutions, appropriately measure bacterial colony growth, and measure optical density proved to be very practical. There were only a few issues that we seemed to encounter during our experimental process. The first issue was that our plate reader, the Tecan GENios Plus, did not originally come with an OD 600 filter. The only filter that we had within our lab that was close to an OD 600 filter, was an OD 550 filter. Although we attempted to purchase the filter that we were instructed to use in the protocol, we could not find any local facility with this filter or any online science laboratory supply store with this filter. Instead, we had to develop a conversion factor that would make our absorbance measurements more accurate to that of an OD 600 filter. This could have potentially impacted our data. The second issue that we faced was determining the meaning of an inconsistency within the protocol. Though the antibiotic Chloramphenicol was referenced as “Chlor” throughout most of the steps of the protocol, there was an issue towards the end. We saw that Chloramphenicol had a different abbreviation as “Cam” which left us not completely sure of the antibiotic necessary for the experiment. After doing a bit of research we found that “Cam” is another abbreviation for Chloramphenicol. Our team unifiedly agreed that the protocol would have been easier to follow with optimum consistency.
In all, we enjoyed our experience with the Interlab for iGEM 2018 and we hope that our crew will continue to participate in iGEM’s Interlab study in the years to come. The contributions that we were able to make to the world of synthetic biology were extremely rewarding for us.