Team:ULaval/Basic Part
Basic Parts
The parts that we designed for this project are all ToeHold switches used to detect specific target sequences. Initially, we designed them to target an easily accessible E. coli gene, and the ToeHolds were designed by hand. They were cloned in expression plasmids and tested in a cell-free expression system, myTXTL by ArborBiosciences.
Parts that we designed to target viral sequences were designed using our software tool. However, we did not have time to test them using viral nucleic acid extract. All switches were designed using sequence constraint and common sequences from the most efficient switches from the Green et al. 2014 paper from Cell, which introduced synthetic ToeHolds switches.
Figure 1. SBOL map of the ToeHold switches in vector pNZ123. All ToeHold switches designed for this project were designed following this pattern. Only ToeHold sequence varies between switches. pNZ123 was selected because of it high-copy number, small size and versatile replication origin.
Best New Basic Part
Part BBa_K3026001 – ToeHold targeting AmpR gene mRNA
Description:
ToeHold switch targeting mRNA sequence of pBluescript ampicillin resistance cassette. This part is flanked by Gibson homology sites for plasmid pBluescript for insertion in the cloning site. This part, when expressed in a cell or media containing T7 polymerase, will produce a ToeHold switch with a recognition sequence in the mRNA sequence of the ampicillin resistance cassette. When this ToeHold is in presence of this mRNA sequence, the sfGFP will be produced. This system allows for detection of DNA fragments or plasmids expressing this cassette.
Source:
Synthetic construct. Switch sequence comes from pBLuescript SK II. sfGFP sequence comes from part BBa_K1321337. Other ToeHold sequence come from Green et al. 2014 Supp. Data switch #1
Design considerations:
ToeHold were designed with sequence constraints according to the engineered design from Green et al. (2014). sfGFP was chosen according to its expression profile in cell-free media containing T7 polymerase, and according to its excitation and emission spectra.
Usage:
Place in cell-free expression system to detect trigger sequence in media. Can also be used in vivo, in a cell where the T7 polymerase is expressed.
Biology:
Recognition sequence targets an RNA sequence from the AmpR gene. When the ToeHold RNA is in presence of its recognition sequence, GFP is expressed.
Testing and characterization:
This part was tested using the myTXTL cell-free expression media by following the protocol. 5mM of DNA coding for the switch was added to the mix, as well as 20ng of freshly isolated mRNA from E. coli expressing the trigger of interest (RNA for the AmpR resistance gene).
Characterization:
To demonstrate that a part that we designed worked as intended, we chose to concentrate our efforts on part BBa_K3026001, which was a ToeHold switch designed to detect a sequence from the mRNA of an ampicillin resistance gene. We chose this gene as it is easy to confirm that its mRNA sequence will be present in our sample at all times by growing an ampicillin-resistant E. coli in ampicillin-containing media.
As we wanted to prove that our part would work in conditions similar to what will be encountered within the detection chips of the A.D.N device, we decided to do a total mRNA extraction from an overnight culture. This created a complex sample, where our target sequence was in minority. We thought that obtaining a positive signal from this experiment would attest to the sensitivity and efficiency of this part, and therefore of our ToeHold design workflow.
We placed the plasmid harboring this part in myTXTL cell-free expression system from ArborBiosciences, along with a constant amount of the total mRNA sample. As we wanted to replicate the detection apparatus from our tool, we decided to do four biological (BR) and three technical replicates for each biological ones, to a total of 12 replicates. For each biological replicate, we did an mRNA-less reaction to confirm the absence of signal leakage, and also did a control with only myTXTL and mRNA, to confirm that signal was not from autofluorescent proteins produced from the mRNA.
This figure clearly demonstrates that fluorescence was detected in only samples with both the ToeHold switch and the mRNA from ampicillin-resistant E. coli. All negative controls show no fluorescence whatsoever. This data confirms that this part works as intended and that our ToeHold design workflow produces functioning ToeHolds. However, there is a lot of variation between BR and even among technical replicates. For the design of our tool, this confirmed the design choice to incorporate several detection chambers, and treat as much of the initial sample as possible.
Sequencing of the clones used for the BR also showed that the part sequence was as designed for the entire length of the sequence, further confirming the efficiency and specificity of this part.
Green, A.A., P.A. Silver, J.J. Collins, and P. Yin. 2014. Toehold switches: de-novo-designed regulators of gene expression. Cell. 159:925–939.