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Team:TU Eindhoven/RCA/Testing
From 2014.igem.org
Testing DBCO-PEG4-DBCO Product
Once the DBCO-primer product is constructed, there is a need to validate that it is functional. To do so, two functions of the DBCO-primer have to both be tested and shown to function, the ability of DBCO to click to azide-groups in COMPx and the DNA to form hydrogen bonds with complementary DNA. Both of these were evaluated in a single step in which the product was reacted with cells expressing COMPx and later labelled with a fluorescent DNA-probe. This was then put in the FACS machine to determine the difference between unlabelled cells, cells with the primer clicked to the surface and cells labelled with DBCO-PEG4-5/6TAMRA. From these results it was concluded that the product still functioned as expected.
right side means higher average fluorescence per sample. This
shows that our clickable DNA oligo is still functioning, however
it also means that the bond is less effective than a covalently
attached fluorophore.
Overview
The previously created DBCO-PEG4-primer has to be tested in order to determine if it’s still functioning, thus making sure it can be used as a starting point for the Rolling Circle Amplification. To test this, cells expressing the COMPx protein where cultured and filtered from 2YT medium by down spinning. The cells were then resuspended in cold PBS so to stop cell division. All samples were then diluted to a concentration of 109 cells/mL. One sample of 200 µL was then kept on ice and not reacted any further, to serve as a negative control. One 200 µL sample was reacted with DBCO-PEG4-5/6TAMRA for 1 hour at 4°C and 500 rpm to check the effectiveness of the primer compared to a positive control. The last 200 µL sample was reacted with the DBCO-PEG4-primer for 1 hour at 4°C and 500 rpm. The last two samples were then isolated from PBS by down spinning once again. The sample labelled with primers was then incubated with a complementary fluorescent primer for 1 hour at 4°C and afterwards the unbound fluorescent probe was removed by spinning the cells down. All three samples were then run through the FACS machine to test for fluorescence. The graph below indicates that while the fluorescent primer didn’t give the same shift in measured fluorescence as the positive control, it did clearly bind to the complementary probe which was clicked to cells showing that the product formed was still fully functional.
For a complete protocol for this step click here
