We purchased a new Promega S30 kit to test our new Arsenic 2.0 plasmid. We compared this against Ars1.1 and the PArsRGFP 1.0 and 2.0. The manufacturer’s protocol was followed, 10ng/uL DNA was used, and each well contained 2uL of master mix. Again, there was no noticeable difference among the wells. Since this was a brand new kit, the reagents should be working. This suggests that our incubation method is flawed or that our DNA doesn’t produce a lot of GFP. One other possibility is that it does make GFP, but that the signal is very low. We may need to think about ways to modify the DNA circuit to improve the GFP signal. This could be through using a mutant GFP that is brighter, or replacing the ribosome binding site with one that recruits more ribosomes to the mRNA.

Building on the last experiment, we repeated the Light Cycler test using the Promega S30 kit. We followed the same protocol and the difference now is that we incubated the reaction using a 96-well plate. We also added an additional DNA control of BBa_J364001, otherwise known as Device 2 in the Interlab Study. We used this plasmid because we know it produces visible GFP, but it was not as intense as the Positive control DNA. Having a positive control that is way beyond our signal would make it harder to interpret our results. This data was acquired every 15 minutes instead of every 30 minutes. The results show a clear difference between the water blank, the master mix only/no DNA control, and the plasmids. The previous results were likely affected by the crushing of the tubes, which is why those water blanks had such high signal. In this experiment, we see that PArsRGFP1.0 duplicates performed the best, PArsRGFP2.0 followed after, and BBa_J364001 was split where one replicate was as bright as PArsRGFP1.0 and the other tracked with PArsRGFP2.0. The No DNA control was lower than all these plasmids, but it was still higher than the water blank. This demonstrates that the master mix does have a lot of autofluorescence.