Team:Edinburgh/Project/Results


Team Edinburgh Finding NEMO

Fluoresence of T7 basic construct

This is a proof of concept testing, which illustrated our basic linear construct can generate functional RNA aptamer and emit fluorescence. This construct will later be used as a positive control in other experiments.
Figure 1: This figure shows the fluorescent intensity change with time after the start of in vitro transcription. Pictures with yellow background were taken under the blue light with amber filter using a phone camera. The fluorescence became visible 5 mins after the IVT reaction. .


ArsR downstream construct

50ng DNA template + 7 μM Arsenic (III) + 1.5 U/μl T7 RNA polymerase in MG1655 and CH34 cell extract
Figure 2: This image illustrates normalized fluorescence readings for the construct with ArsR binding site downstream of the T7 promoter in two cell extracts. The Cupriavidus metallidurans CH34 cell extract has better performance than E. coli K12 MG1655 in sensing As (III). No fluorescent difference can be observed for the system with or without the presence of metal in MG1655 extract. .



At 30 mins, the fluorescent difference for the metal present and no metal system in Cupriavidus metallidurans CH34 cell extract has started to become significantly different (P=0.0025).


However, the overall fluorescent difference in the E. coli K12 MG1655 system is lower than that in the CH34 cell extract, also there are no significant differences observed with addition of metals.

Despite changing the concentration for T7 RNA polymerase or DNA template, the overall fluorescent signal is still relatively low in ArsR downstream construct comparing with the positive control. It is possible that the ArsR binds downstream of T7 promoter cannot be released efficiently upon metal binding, which completely blocks the T7 RNA polymerase. Thus, trying the ArsR upstream construct would give us some insights in this aspect.

ArsR upstream construct

100 ng DNA template + 7 μM Arsenic (III) + 1.9 U/μl T7 RNA polymerase in MG1655 and CH34 cell extract
Figure 3: This image illustrates normalized fluorescence readings for the construct with ArsR binding site upstream of the T7 promoter in two cell extracts. In agreement with the previous results, the Cupriavidus metallidurans CH34 cell extract has better performance than E. coli K12 MG1655 in sensing As (III). The overall fluorescent signal has increased to a level that is almost identical to the positive control. .


The significant fluorescent difference (P=0.0042) in the CH34 extract can be observed at 20 mins in this case.

This construct showed higher transcription leakiness indicated from high fluorescent signal when no As (III) was present. Further optimization, such as reducing the amount of DNA template might be required to alleviate the situation.