Team:BGU Israel/InterLab
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InteLab
Our team chose to participate in the Fifth International InterLab Measurement Study in synthetic biology.
The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.
In this study we want to reduce lab-to-lab variability in fluorescence measurements by normalizing it to absolute cell count or colony-forming units (CFUs) instead of OD.
The fluorescence in the bacteria in our experiments occurs due to the presence of a gene that encodes a green fluorescent protein: GFP.
In cell and molecular biology, the GFP gene is frequently used as an expression reporter protein. Reporter genes are often used as an indication that a certain gene has been internalized or expressed in cells or organisms.
Equipment
All of our measurements were taken in a plate reader that can read both absorbance and fluorescence (Neoteck-Tecan-INFINITE M100) without a pathlength correction. The temperature was set to 25̊C, under shaking with a duration of 3 sec and an amplitude of 2 mm. We went through a tutorial and learned how to operate the machine.
In addition, we used the following reagents (Partially supplied by iGEM):
- 1.0 ml LUDOX CL-X
- 150 μL Silica Bead (microsphere suspension)
- Fluorescein (powder, in amber tube)
- iGEM Parts Distribution Kit Plates
- 1 x PBS (phosphate buffered saline, pH 7.4 - 7.6)
- ddH2O
- Competent cells (Escherichia coli DH5α)
- LB (Luria Bertani) media
- Chloramphenicol
- 96 well plates, black with clear flat bottom
Safety procedures
As part of an ongoing work in a biological laboratory, safety gear was used to work with bacteria. The work was done with closed shoes, long pants, gloves and lab coats.
In order to maintain a clean environment, and sterility between the samples the work was performed next to a flame, and all the disposable parts were thrown after a single use into a biological waste bin.
Before and after work, work surfaces were sterilized with 70% alcohol.
ALS conference
As we mentioned previously, the community of ALS research labs in Israel was very limited until recently. Still, as this is a small country, the budding community is still rather small, yet impressive. We were touched by how this community embraced us when we began our project. Any lab we talked to was more than happy to help point us in the right direction, teach us new tools, or provide access to equipment. We were compelled to do something in return, so we organized the annual ALS Research Conference at the Ben-Gurion University of the Negev (our University). The conference was a great success! Not only did we hear from the leading experts in ALS research, we were able to present our project for the first time in a scientific setting. Our presentation received encouraging responses. The community seemed truly excited by our idea and the questions we received were not about the viability of the idea, rather they were insightful thoughts regarding research methods. Many researchers offered their inputs and assistance to aid us in proceeding with our project. One of these researchers, was Dr. Dinorah Friedmann-Morvinski from Tel-Aviv University.
Dino
Around the time of convention, we faced a major lab roadblock. We were having trouble infecting our cells with the plasmids we had designed, due to their size and the sensitivity of the CNS cell lines. Without the ability to insert our plasmids, we had no way of implementing our system. This is when Dr. Friedmann stepped in. Dr. Friedmann heard about our struggles and offered a project-saving solution. Her research group works with plasmids which implement knockdown of genes in the NFkB pathway and delivers these plasmids with viruses. Dr. Friedman was instrumental in teaching us infection techniques and providing us with plasmids from her lab. This collaboration meant, that even if we did not succeed in implementing our designed plasmids before the competition, we would still be able to achieve a proof of concept.
host a group of high-school
Our project would not have succeeded without our Human Practices. Our goals and implementation were all a result of involvement in the communities around us and sharing our ideas through discussions with experts. Science cannot proceed unaided and cannot succeed without public support. As a final gesture, we were delighted to host a group of high-school students in our lab. We had a great time explaining to them about iGEM, our project, and our lab. Although we ourselves as undergraduate students are only at the beginning of our scientific careers, it was wonderful to transfer the knowledge we have gathered to a new generation.
