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Team:Amsterdam/Human Outreach/Collaboration/Construct Verification
From 2011.igem.org
Construct verification
The Wageningen 2011 team has asked us to verify one of their more recent constructs: (F2621-E0422-F2621-K082014). This construct includes a BioBrick which causes the bacterium to express CFP. This expression of CFP can be verified using a microscope. As the Wageningen team did not have such a microscope available for them they asked us if the expression of GFP for them.
The sample was sent to us as an agar stab which we grew to high concentration overnight at 37°C in LB. In order to have a control and to verify the microscope settings we also included one of our own samples with known RFP expression.
Four samples were prepared:
- Our control RFP cells
- The Wageningen CFP cells
- The Wageningen CFP cells with at their border our control RFP cells
- The Wageningen CFP cells mixed with our RFP cells.
At first, we tried to verify CFP expression by using the CFP filter and observing the cells visually. We checked for the distinctive blue light which should come from bacterium expressing CFP. For some bacteria there appeared to be a faint blue light to be present. However, it was not clear to distinguish this faint blue light from “background noise” and as such we checked our RFP cells using the CFP filter as well. We found that this faint blue light was also present in the cells that did not contain a CFP gene and as such was background noise. To verify the microscope was set up correctly, we also checked our RFP cells for RFP expression. For our RFP cells we could easily identify the red fluorescence as most cells appeared bright red. The intensity differences between the faint blue light and the bright red light were extreme. This also indicates that in case the CFP cells were truly expressing CFP the light intensity should have been a lot higher.
However, comparing different samples using a microscope is not easy. Ideally you would like to have both type of cells in one sample for easier verification. As such, we also had prepared samples in which the RFP and CFP cells were both mixed and closely separated.
The CFP and RFP cells were closely separated by immersing both cells in different gel solutions. Upon contact on the sample plate, the solutions were not able to mix. The border between the RFP and CFP cells was located using the microscope and using the CFP and RFP filters the differences between both types of fluorescence was observed. Using the CFP filter, the border could not be located as there was no clear difference between the CFP cells and RFP cells. However, using the RFP filter, the border could easily be located because of the clear difference between the bright red RFP cells and the colorless CFP cells.
As a last check, we also prepared a sample in which the CFP and RFP cells were mixed. We tried to switch between the CFP and RFP filter in order to observe a difference in fluorescence. We only saw such a difference with the RFP filter as half of the cells would turn red. A similar effect with the CFP filter in which they would turn blue was not observe. Our conclusion is that the cells not appear to express any fluorescence.
