The human gut microbiota is one of the most densely populated ecosystems of microorganisms on earth. With an estimated 100 trillion microorganisms, the gut is an extraordinarily complex system of microbe-microbe and microbe-host interactions. A growing body of research is beginning to elucidate the diverse impacts the gut microbiota plays in human health and development, from nutrition, to disease, and even cognition. Recently, with the success of fecal matter transplants (FMTs) to treat infectious disease, microbes are emerging as a unique therapeutic. Model systems to both prototype and study complex polymicrobial systems are a necessity for producing robust microbial communities that can be engineered at both the genetic level (subcellular) and population level (multicellular).
Microfluidic & millifluidic, or lab-on-a-chip systems, have been used for several decades as a methodology for miniaturizing and automating biology and chemistry experimentation. Devices can be fabricated a variety of size schools using myriad materials depending upon the functional goals of the device. Active components can be fabricated using polymeric materials (e.g., poly-dimethyl-siloxane (PDMS)) or can be static and more easily fabricated using, for example, 3D printing.
In this homework, we were tasked to design a micro- or milli fluidic device, that could be applied for our final project.
For my final project I want to design and test Arc protein capsids as potential drug delivery systems.
To efficiently analyse Arc-based capsids I wanted to develop an assembly pipeline that would allow to assemble Arc capsids from Arc protein monomers and mRNA (Figure 1). Since the capsid has been only structurally resolved for drosophila Arc so far, I decided to use it as a model for protein engineering of Arc capsids and similar gag-based capsids. I therefore developed my initial experiments around drosophila Arc capsids.
At the start of the pipeline both, mRNAs and Arc protein monomers, are produced in cell cultures. In my constructs, Arc protein monomers are expressed with a cleavable His-Tag, to allow for subsequent purification via metal-ion columns. Specific mRNAs can be isolated and purified through binding to specific oligo-dT and a subsequent washign and elution step (most of the time provided in a specialized mRNA isolation kit).
Using Autodesk Fusion 360, I designed two devices: a first device in which purified Arc protein and up to three different mRNAs are inserted and combined to induce oligomerizationand a second device, in which the Arc nanoparticles are seperated by size, to enrich for (almost) fully formed capsids.
The collected Arc capsids can then be further analysed, for example to identify which mRNA was encapsulated by analysing a barcode, or used for further experiments.
With this pipeline I want to test and answer the following questions among others:
Can Arc capsids form around all types of mRNA? Or does the mRNA have to have properties similar to Arc mRNA, such as size?
Can engineered Arc capsids form in the first place?
Do Arc capsids that were engineered for different cargo specificity actually show this changed specificity?