Improving signal generation
After showing that our toeholds could effectively detect RNA and that they could be triggered by single-stranded DNA and aptamers with single stranded DNA extension, we were interested in improving our output signal in order to make the detection result faster.
What was accomplished ?
In order to chronologically summarize our best accomplishments or discoveries during the elaboration of our project before presenting our results, here is a list:
- Testing trigger addition at different time intervals.
- Testing different buffer conditions.
- Testing toeholds in PURE with FDG.
- Testing toeholds in lysate with FDG.
- Signal amplification using T7 aptamer extension.
ssDNA Trigger Addition At Different Time Intervals
Next, we thought that adding trigger at different time points may yield faster output. Since the trigger we used was ssDNA short, it will not be transcribed in the cell lysate reaction (only double stranded templates can get transcribed in lysate). Instead it will bind to the toehold as soon as they are both present in the solution. However, since the toehold first needs to be transcribed into its RNA form, some time passes before the trigger can bind to it, which could lead to its degradation in the lysate. Adding the trigger at a later stage of the reaction would prevent the trigger from degrading during the transcription of the toehold. To test this hypothesis and whether it could improve our output, we set up 9 different reactions:
- Adding the ssDNA trigger directly at the start of the incubation of the reaction
- Adding the ssDNA trigger after 5 minutes of at the start of the incubation of the reaction
- Adding the ssDNA trigger after 10 minutes of the start of the incubation of the reaction
- Adding the ssDNA trigger after 15 minutes of the start of the incubation of the reaction
- Adding the ssDNA trigger after 20 minutes of the start of the incubation of the reaction
- Adding the ssDNA trigger after 30 minutes of the start of the incubation of the reaction
- Adding the ssDNA trigger after 45 minutes of the start of the incubation of the reaction
- Adding the ssDNA trigger after 60 minutes of the start of the incubation of the reaction
- No trigger control
Upon those results, we did not think it was necessary to add the trigger at a later time in the reaction because there was not significant difference between the first 3 reactions (adding the trigger at the start and adding it after 5 or 10 minutes).
Buffer Z in different lysate reaction conditions
To achieve the best signal possible we set up 5 different reactions:
- Usual lysate reaction (lysate T7M15 + lysate Top10Gams+ Energy solution + BufferA+Substrate)
- Lysate T7M15+ Lysate Top10GamS+ BufferA+ Enegy Solution+ BufferZ+Substrate
- Lysate T7M15+ Lysate Top10GamS+ Enegy Solution+ BufferZ+Substrate
- Lysate T7M15+ Lysate Top10GamS+ BufferA+ Enegy Solution+Substrate eluted in BufferZ
- No trigger control
Table summarizing the different buffer conditions across reactions :
|Reaction Number||Trigger||Buffer A||Buffer Z||Substrate||Substrate + BufferZ||Final Volume [µl]|
These results show that the best signal is obtained when using a substrate eluted in Buffer Z. After acquiring this new result, we repeated the experiment to compare one more time our usual reaction set-up (substrate eluted in water) to using a substrate eluted in Buffer Z.
We conclude that, unequivocally, using chlorophenol red substrate eluted in buffer Z leads to faster and clearer output.
Adding Buffer Z at different time intervals
As 2-Mercaptoethanol is known to protect the enzymatic activity of proteins, we thought that adding the substrate+ Buffer Z combinations at different times (i.e not only at the start of the reaction) may result in higher beta-galactosidase yield. We performed 7 different reactions at different time points:
- 1 : Adding Buffer Z+ Substrate directly at the start of incubation of the reaction
- 2 : Adding Buffer Z+ Substrate after 15 mins of incubation of the reaction
- 3 : Adding Buffer Z+ Substrate after 30 mins of incubation of the reaction
- 4 : Adding Buffer Z+ Substrate after 45 mins of incubation of the reaction
- 5 : Buffer Z+ Substrate after 60 mins of incubation of the reaction
- 6 : Adding Substrate eluted in Buffer Z at the start of the incubation of the reaction
- 7 : No trigger control
Upon these results, we concluded that adding the Substrate + BufferZ after 45 to 60 mins of the start of the reaction was a good way to maintain beta-galactosidase activity but adding just the substrate eluted in Buffer Z remained the best output that we got.
Testing FDG in PURE
FDG (short for Fluorescein di[β-D-galactopyranoside]) is a substrate that emits fluorescence after being cleavage by beta-galactose, our reporter protein. FDG was first tested in a aptamer trigger/LacZ toehold reaction in PURE.
These results show that fluorescence clearly increases with time, which corresponds to toehold being triggered and translated into beta-galactosidase. Those results encouraged us to continue with FDG.
Testing FDG in lysate
After having had first results with FDG in PURE, we went on to test it in lysate. A usual lysate reaction with FDG as a substrate, toehold LacZalpha and aptamer trigger was set up. We had a no trigger control as well as a no toehold control, to check whether FDG is stable in lysate and a reliable substrate.
Both negative controls show no expression compared to the reactions containing both trigger and toehold. This lets us conclude that FDG can be used in lysate and that chip experiments with the fluorescent microscope should be further explored.
For the single-stranded T7 aptamer trigger to be transcribed by an RNA polymerase,
a primer first needs to anneal to the T7 promoter region to make it double stranded.
After letting them anneal, they are incubated with a T7 polymerase and dNTPs at 37°C
for two hours.
After following the protocol for T7 aptamer trigger amplification, we looked at the amplification product on a 2% agarose gel and compared it to several controls. Since the T7 aptamer trigger product will be RNA, we ran an RNA ladder (Lane 1) as well as a DNA ladder (Lane 5). Next to the amplified product (Lane 2) we ran the transcription control (T7 aptamer trigger without the primer, Lane 3). Lane 4 is untreated T7 aptamer trigger at the initial concentration of 3 µM, to compare the strength of the bands.
The strong band in Lane 2 clearly shows that the amplification process was successfull as well as specific. This opens up possibilities for limits of detection that can be a lot lower than our currently detected ~1 µM.