Results Summary – TUDA iGEM 2021

Results Summary

In this section we want to briefly summarize the results we acquired during the time we had in the wet lab. We aimed to conduct a proof of concept of our adaptive immune-like system for the protection of functionalized biofilms. Due to restrictions at our university caused by the ongoing pandemic, we had very limited lab access. On this page we only present a short summary of our results. If you want to find out more about the experiments planned for our separate research topics, have a look at the corresponding pages. (Bacteriophages, Biofilm, Biosafety and Pathogen Sensing)

We used computational methods to also acquire results for our project and guide the development of our methods and assays. Especially, our genetic circuit model for the sensing of pathogens helped us to fully understand the genetic circuit and adapt the assay design.

Biofilm

To conduct a proof of concept for a multi-functional biofilm containing bacteria carrying different genetic elements we cocultured B. subtilis DK1042 cells with different fluorescence markers. DK1042 is a strain that forms biofilms, thus the resulting biofilms were analyzed by fluorescence microscopy. We were able to create stable cocultures without the need for synthetically introduced dependencies between the different genotypes. These results displays a proof of concept for the integration of multiple bacteriophages inducible by different signaling molecules. Each signaling molecule in this connection could trigger expression of a phage specific to the invading pathogen species.

Figure 1. Picture of the samples of the overnight culture in the 96-well plate, taken with the Operetta High Content Imaging System form Perkin Elmer at the Cardoso lab at TU Darmstadt. The well-column on the far left contains the GFP labelled variant of DK1042; the well-columns on the far right contains the mKATE labelled variant of DK1042; the well-column next to the column with the mKATE labelled variant of DK1042 contains the ComI variant; the well-column next to the column with the ComI variant contains the co-culture with both fluorescently labelled variants of DK1042 and the horizontal well-column on top of the well-plate contain the negative control in form of pure LB 5 media. In the microscope picture GFP, due to its higher fluorescent intensity, is that much enlightened because the contrast the pictures was set the same value to be able to compare them. Otherwise, pattern in co-culture would not have been visible.

Bacteriophages

Bacteriophages can stably integrate into the genome of bacteria as prophages and remain unactivated throughout multiple cell division cycles. The lytic stage is induced commonly by stress reactions such as DNA damage and the connected signaling cascades. This induction of phages from a stable, genomically integrated state towards the active, lytic stage is crucial for the activation of our sleeper cell. We therefore want to use these established, evolutionary developed systems and put them under the control of our pathogen sensing circuit. As a proof of concept for these kinds of circuits we tested the inducible control of RecA730, a constitutively protease-active variant of the lytic state trigger of the lambda phage, and its effect on a cI controlled fluorescence marker. cI is a tight transcriptional repressor keeping the phage in its lysogenic cycle upon cleavage by RecA and thus deactivation.

Figure 2. Ratio of the emitted fluorescence per optical density [FI/OD600] of our sleeper cell before and after the induction with IPTG. The measurements were carried out at 30 min intervals. Between these measuring points the samples were shaken at 225 rpm and 37°C. The induction with 0.5 mM IPTG took place after 195 min. Two biological replicates and five technical replicates per sample per measuring point have been measured.

Pathogen Sensing

We conceptualized a genetic circuit for the sensing of P. aeruginosa by recognition of a quorum sensing molecule. We were able to assemble this construct, but unfortunately didn’t collect any data on the genetic circuit.

For the validation and further improvement of the genetic circuit we set up a model describing the reporter output based on literature and computed parameters.

Figure 3. Genetic circuit for the sensing of AHL expresses egfp in the presence of AHL. The activated transcriptional factor signaling molecule complex initiates transcription of the P_PA1897 promoter.

Biosafety

To secure biosafety and biocontainment of our system, we planned to further optimize the kill-switch from iGEM TU Darmstadt 2020. To do so, we planned experiments for the assessment of the switch and adapt the single components. We were able to assemble the first constructs for these measurements but again were unable to test them in the lab to due very limited access we got. The initial part for assessment was the activation of the kill-switch by the irreversible inversion of a promoter cassette. This part was supposed to be screened by fluorescence measurements of our genetic construct.

Figure 4. Genetic circuit for the assessment of a Cre-mediated promoter cassette flip. After the flip, a constitutive promoter initially controlling sfgfp expression is exchanged with a QS activated promoter sequence. The reporter gene is to be exchanged with a essential gene in the actual construct to couple cell viability to QS reporter concentrations only reached in biofilm cell communities.

Team:TU Darmstadt/results

Biofilm

Bacteriophages

Pathogen Sensing

Biosafety