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Use of Recombinant Self-Associating Proteins for Altering Cellular Fate and Behavior


Kozlowski, Mark Tybalt (2019) Use of Recombinant Self-Associating Proteins for Altering Cellular Fate and Behavior. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3Z3B-V584.


The behavior of mammalian and bacterial cells is governed by their surroundings, and the interactions of cells with their nearest neighbors. In this work, I will demonstrate how self-associating proteins such as leucine zippers or SpyTag and SpyCatcher can be used either in hydrogels for cell culture, or to drive the aggregation of cells into artificial, engineered communities. I further demonstrate how these self-associating protein-based materials can either alter the fate of cultured cells, or directly change cellular behavior through the activation of a quorum sensing circuit.

In the first chapter, I discuss protein-based methods for making different types of organoids. Organoids are groups of cells derived from stem or progenitor cells that form a multicellular structure consisting of different cell types. These organoids are currently of interest as disease model systems, pharmaceutical test platforms, and replacement tissues. However, most studies of organoids to date have derived them from Matrigel-based cultures. While versatile and inexpensive, Matrigel is undefined, suffers from batch-to-batch variability, and its xenogeneic nature means that organoids derived from Matrigel are unlikely to be approved for clinical use. In Chapter 1, I review current state-of-the-art materials developed as alternatives to Matrigel, such as naturally-derived extracellular matrices, synthetic hydrogels, and recombinant proteins serving as artificial extracellular matrices. I consider the advantages and disadvantages of each method, as well as speculate on possible future directions for the field.

Of these alternatives to Matrigel, recombinant protein-based artificial extracellular matrices have the advantage of being easy to engineer, as genetic encoding of the material allows precise control over molecular weight and functionality. Development of these types of materials has long been a focus of work in our laboratory, and in Chapter 2, I discuss the development of two protein-based hydrogels expressed in Escherichia coli, which are based on a previously-reported PEP hydrogel. These new “PEXEP-type” hydrogels are physically cross-linked by leucine zippers derived from rat cartilage oligomeric matrix protein (COMPcc), incorporate chemical cues from fibronectin and collagen IV, and were used for pancreatic cell culture in defined medium. When comparing this defined, protein-based medium to methylcellulose-Matrigel, we find that the growth of endocrine cells is promoted, as opposed to the ductal cells found in methylcellulose-Matrigel culture. We further find a difference in colony types observed based on whether the fibronectin or collagen IV cue is present. More interestingly, the protein-based culture material promotes the growth of endocrine progenitor cells, which may be useful for further studying the formation of the Islets of Langerhans. Finally, we observe that sorted populations of murine cells cultured in our protein hydrogels have a lower rate of colony formation, and this reduction in the number of colonies is not observed in methylcellulose-Matrigel culture. We believe that this might be evidence for a paracrine effect that promotes cell growth, particularly the growth of putative endocrine colonies, though further experiments are required to confirm this effect.

In Chapter 2, I demonstrate how a self-associating protein can be used to change the fate of a cell culture, and give rise to multicellular colonies. However, for the purpose of constructing bioreactors, microbial fuel cells, or systems for environmental remediation, it may be advantageous to design tissue-like systems de novo, making multifunctional communities of bacteria that function as artificial tissues. In Chapter 3, I will discuss the construction of a synthetic microbial community of E. coli cells, whose quorum sensing response is governed by aggregation of the cells. This aggregation in turn is driven by the expression of surface-displayed self-associating proteins, and I will discuss methods developed to control the size, reversibility, and morphology of these aggregates. As the behavior of these aggregates is dependent on cell-cell communication facilitated by proximity, these consortia represent early examples of synthetically-designed artificial tissues that can be governed by engineered cell-cell signaling.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Surface display; recombinant proteins; bacterial assembly; protein hydrogels; programmable materials; pancreatic cell culture
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Tirrell, David A.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Kornfield, Julia A.
  • Wang, Zhen-Gang
  • Tirrell, David A.
Defense Date:13 May 2019
Record Number:CaltechTHESIS:05292019-133006433
Persistent URL:
Kozlowski, Mark Tybalt0000-0003-4714-8697
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11560
Deposited By: Mark Kozlowski
Deposited On:04 Jun 2019 22:38
Last Modified:08 Nov 2023 00:37

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