Cell-Free System

Linear versus Plasmid DNA

We obtained promising data from adding plasmid DNA to our cell-free system. However, because the Collins paper specified the use of linear DNA in their experiments, we compared the signal from GFP in linear and plasmid form in a cell-free reaction. There is no siginificant difference between the two constructs.

Reaction Volume Reduction

We decreased the cell-free reaction volume from the 25 µL given by the PURExpress protocol. To make their sensor, the Collins group only freeze-dried 1.8 µL of reaction onto paper discs, so our system would have to produce a signal with a much smaller reaction volume. In addition, performing experiments with a smaller reaction volume would make PURExpress last longer. PURExpress was one of the most expensive reagents we used, yet it was also central to our project. We set up reactions with a range of volumes from 1 to 25 µL and measure the fluorescence from GFP. The reaction produces an appreciable signal when the volume is as low as 5 µL.

Dilution

To obtain a good reading from the plate reader, we had to add at least 10 µL of liquid to the plate wells. For endpoint assays, we diluted the completed cell-free reactions by two before reading, which gave us good results. We wanted to try diluting the reaction before incubation to run a time course without increasing the amount of PURExpress used. However, cell-free reactions to not proceed nearly as quickly when diluted.

DNAzyme

We performed our DNAzyme assays in two ways. We used polyacrylamide gel electrophoresis (PAGE); however, the gels rarely provided clear, informative results. Thus, we also put the DNAzymes in cell-free reactions with the toehold switch. If the toehold switch was not triggered, the substrate strand of the DNAzyme remained sequestered by the catalytic strand. If the toehold switch was triggered, the substrate strand must have activated the switch, either because cleavage had occurred or because the substrate strand was not properly sequestered.

Substrate Sequestration

Hairpin versus Duplex

Cleavage

Toehold Switch

Our experiments were performed with the D and G LacZ switches from the paper from the Collins group, "Paper-Based Synthetic Gene Networks".

RNA Trigger

In Week 5, we showed that LacZ is expressed when the plasmids for the switch and trigger constructs are combined in a cell-free system.

DNA Trigger

In Week 6, we showed that toehold switches are activated by single-stranded DNA triggers, but activation levels are lower than with RNA triggers.

Sequestered DNA Trigger

In Week 7, we showed that single-stranded DNA triggers do not activate toehold switches if they are part of a hybridized complex.

Reporter

amilCP

amilCP is a chromoprotein added to the iGEM Registry by Uppsala Sweden, 2012. We were unable to produce amilCP in bacteria or in our cell-free system.

Amplifier