Graduate Students Mini Symposium III-2024

01:15 PM Haozhe Chen (AG Diepold)
Deciphering the crosstalk of virulence systems in Pseudomonas aeruginosa

Currently, I am working the research project “Deciphering the crosstalk among virulence systems in Pseudomonas aeruginosa”. As a notorious human pathogen, Pseudomonas aeruginosa still causes countless in-hospital infections each year with huge costs. To understand its role as the pathogen, efforts have been made to discover the innate regulation of virulence systems. In this project, we specifically focused on Type III Secretion System(T3SS), which is mainly used for anti-eukaryotic cell killing, and Type VI Secretion System(T6SS), for inter-bacterial competition. Mediated by the universal second-messenger c-di-GMP, T3SS/T6SS are believed to be incorporated into the lifestyle transition model, which might functionally combines the different lifestyles (motile, biofilm) with its infection mode. By designing novel reporters from different perspectives of the virulence activity, we emphasized the involvement of the key second messenger, c-di-GMP in the interplay of the virulence network and compared the discovered pattern with other model pathogens. Aside from the populational discoveries, for the first time we tried to directly visualize the crosstalk of the virulence systems on a single-cell level using microscopy techniques and thus constructed a vivid picture of the division of labor on the behavior of virulence, possibly leading to a finely-tuned virulence dynamics model during the course of complex infection. Combining classic and newly designed molecular biology methods, the current findings and ongoing research can greatly help understand Pseudomonas aeruginosa as a pathogen and provide insights in blocking Pseudomonas infections.

01:45 PM Aukse Gaizauskaite (ENG Niederholtmeyer)
Microfluidic platform for engineering biomolecular condensates in cell-free systems

Intrinsically disordered regions (IDRs) and high multivalency give some proteins the ability to self-associate into liquid-like droplets, called condensates. While living cells employ condensates as membranelles organelles for spatial organization, it is challenging to recreate such compartmentalization in vitro with a complexity matching living cells. In order to facilitate this challenge, we designed a microfluidic platform, which helps to characterize protein-based condensates in cell-free transcription and translation (TX-TL) systems. Micrometer-size environments in the chip with the diffusive supply of TX-TL reagents ensure long-term continuous protein synthesis with a constant supply of fresh reaction components. Taking advantage of microfluidic techniques, this device allows the screening of multiple condensing proteins to be carried out simultaneously and efficiently with minimal usage of reaction components. Easier characterization of protein variants opens an opportunity to design synthetic condensates as programmable and dynamic assemblies. This not only expands the complexity of artificial life-like systems but also allows the creation of biochemically active compartments that are able to carry or sequestrate particular molecules in vivo and in vitro.

Segregation in binary mixtures under flow

Bacteria are often found in heterogeneous communities organized through physical interaction with their surrounding environment. Although external physical constraints like shear flow are frequent in natural situations and have been shown to influence the swimming behaviour of active particles, little is still known about their effect on the distribution of bacteria inside heterogeneous populations. Using fluorescence microscopy, microfluidics, and image analysis techniques we describe how a model heterogeneous bacterial community of motile and non-motile Escherichia coli organizes in a confined environment when exposed to shear flow. We observe that for low shear rates, the collective reorientation of motile bacteria towards the vorticity direction results in a progressive accumulation of non-motile bacteria near the boundaries in the opposite direction. We describe the evolution of the system through advection-diffusion equations and highlight how the strength of accumulation scales with the concentration of motile particles and the applied shear. After that, we present how interactions with the surface affect the backflow generated by the rheotactic drift and discuss the importance of sedimentation for the dynamics of motion and accumulation of non-motile particles. Finally, we discuss the effects of flow on biofilms of a binary mixture and observe the asymmetric formation of biological aggregate on the left side of the channel.