Experiments

Protocols

Experiments

On this page, you can read about the two main experiments we conducted:

(1) POC assay

(2) GOI abundance test via fusion-PCR

You can go through the main steps and procedures we performed in each experiment. (for more detailed information go to our Protocols or Lab notebook page).

Both of the experiments were conducted on two species of bacteria - E. coli and B. subtilis and the tested GOI was mCherry.

POC assay

We initially calibrated our system conditions, such as the type of minimal medium in which both bacterial species grow up to the stationary phase, bacterial density, experiment duration, and measurements parameters. In parallel, the modeling team designed several optimized and deoptimized gene components to be tested.

Once the system was calibrated and new versions of ORF and promoters arrived, we were ready to go!

First, the plasmid containing the original mCherry gene was purified from the bacteria we got from Avigdor Eldar’s lab. Then, we performed PCR to linearize the plasmid with primers complementary to the gBlocks (software-designed gene components), and that exclude the sequence to be modified (figure 1).

Figure 1: Construction of the tested gene components into our plasmid.

Original GOI or its promoter (marked in blue) was substituted with their modified versions that were obtained by our software (gBlocks), via PCR and Gibson assembly.

Next, a portion of PCR products was examined by DNA electrophoresis, and the rest has treated with DpnI in order to remove the remaining of the original plasmid.

Then, Gibson assembly was performed with the linearized plasmid and its matching gBlock to establish the final plasmid. This plasmid was transformed into competent bacteria.

Colony PCR was conducted to screen transformed colonies for the positive inserts, which was finally confirmed by Sanger sequencing. Following sequence validation, plasmids were prepared from positive colonies and then transformed into our bacterial strains. Transformed colonies were picked and glycerol stocks were prepared. When completed, we proceed with the assay to measure whether our software-designed sequences alter gene expression as predicted. See the experimental design here.

It is important to note that we successfully managed to construct plasmids with optimized ORFs for B. subtilis, which are deoptimized for E. coli. Plasmid construction for the reciprocal modification- ORFs optimization for E. coli and deoptimization for B. subtilis, was failed. When we screened for insert-positive colonies, we found only colonies containing the original vector, except for one ORF version. We suspected that DpnI treatment didn’t work. Due to iGEM’s time frame constraints, we decided to continue with another gene component, and we constructed plasmids with new promoters. In this case, even if DpnI treatment was useless, the original vector was eliminated by extracting the linearized vector directly from the gel, see the notebook.

Figure 2: Main steps of the fluorescence assay experiment

GOI abundance test

To fully verify if our technological approach is capable of restricting plasmid abundance within microbial communities despite HGT, we implemented the fusion-PCR method. To date, we are still calibrating the primers sets necessary for the fusion reaction. For detailed in-silico primers and fused-product design go to protocols.