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Wil V. Srubar III Editor
Engineered
Living
Materials
Engineered Living Materials
Wil V. Srubar III
Editor
Engineered Living Materials

Editor
Wil V. Srubar III
Department of Civil, Environmental, and
Architectural Engineering
Materials Science and Engineering
Program, University of Colorado Boulder
Boulder, CO, USA
ISBN 978-3-030-92948-0 ISBN 978-3-030-92949-7 (eBook)

https://doi.org/10.1007/978-3-030-92949-7
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland
AG 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
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Contents
Network Formation of Engineered Proteins and Their Bioactive

Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Seunghyun Sim
Living Synthetic Polymerizations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Austin J. Graham and Benjamin K. Keitz
Programmable Self-Assembling Protein Nanomaterials:
Current Status and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Kelly Wallin, Ruijie Zhang, and Claudia Schmidt-Dannert
Engineered Living Conductive Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . 95
Lina J. Bird, Fernanda Jiménez Otero, Matthew D. Yates,
Brian J. Eddie, Leonard M. Tender, and Sarah M. Glaven
Photoswitchable Bacterial Adhesions for the Control
of Multicellular Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Fei Chen and Seraphine V. Wegner
Additive Manufacturing of Engineered Living Materials . . . . . . . . . . . . 149
Lynn M. Sidor and Anne S. Meyer
Engineered Living Materials for Construction . . . . . . . . . . . . . . . . . . . . 187
Rollin J. Jones, Elizabeth A. Delesky, Sherri M. Cook,
Jeffrey C. Cameron, Mija H. Hubler, and Wil V. Srubar III
v
Network Formation of Engineered Proteins
and Their Bioactive Properties
Seunghyun Sim
Abstract Proteins are one of the main components of the extracellular matrix in
natural biological materials. They confer a unique advantage in creating engineered
living materials (ELM) because they can be genetically encoded and rationally
designed for constructing bioactive network structures. Advances in the design,
characterization, and engineering of protein networks have been an important
multidisciplinary endeavor and should be considered when designing ELM and
understanding their behavior. This chapter describes the network-forming behavior
of recombinant proteins, as these proteins, in principle, can be genetically
programmed and synthesized directly from living cells residing in ELM. There are
three major classes of protein network-forming mechanisms relevant to this topic:
(1) phase separation and aggregation-induced recombinant protein networks,
(2) self-assembling multi-domain artificial protein networks, and (3) chemically
cross-linked recombinant protein networks. We will begin by introducing protein
hydrogels and discuss their mechanism of network formation, which is a critical
element in designing functionalities and mechanical properties of ELM. After
introducing the network-forming mechanisms in protein hydrogels, we will discuss
examples of bioactive protein networks equipped with various functionalities before
concluding with future directions and remaining challenges in this field.
Keywords Protein engineering · Protein network · Protein hydrogel · Bioactive

protein network · Artificial extracellular matrix
1 Introduction
Natural biological materials have been a key inspiration in the broad scientific field
due to their unique ability to self-regulate, self-organize, self-heal, and respond to
complex environmental cues. We see this in bone, wood, skin, and biofilm, where
S. Sim (*)
Department of Chemistry, University of California, Irvine, Irvine, CA, USA
e-mail: s.sim@uci.edu
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

W. V. Srubar III (ed.), Engineered Living Materials,
https://doi.org/10.1007/978-3-030-92949-7_1
2 S. Sim
living cells produce, regulate, and repair their own surrounding matrix. These
emergent properties result from the living cells functioning as an active component
in these materials and have yet to be demonstrated in man-made synthetic materials.
Inspired and motivated by this striking functionality and complexity of natural
biological materials, many efforts have been made in recent years to construct
engineered living materials (ELM) where living cells are the producers and modu-
lators of their surroundings. In nature, the main components of these networks made
by living cells are self-assembling proteins and exopolysaccharides. Although
exopolysaccharides produced by living cells are often the most abundant structural
component, engineering and production of designed networks are challenging as
they involve a variety of biosynthetic machinery that is not necessarily shared across
different species. Proteins, on the other hand, can be genetically encoded, rationally
designed for specific purposes, and ported between species. In addition, proteins are
an attractive building block for networks interfacing living cells from both functional
and structural aspects. They fold into a defined three-dimensional structure, can bind
specific partners even in a complex cellular environment, and form protein-protein
interactions. As a result, they can assemble into a higher-order network responsible
for the mechanical property of the ELM. Moreover, many functional protein motifs
are amenable to use in conjunction with structural units that undergo network
formation.
Self-assembled protein networks in the context of extracellular materials can be
classified as hydrogels, as they are extensively hydrated networks housing constit-
uent living cells. Therefore, throughout this chapter, we will use the terms “network”
and “hydrogel” interchangeably. Hydrogels are physically or chemically cross-
linked polymer networks that swell in water. Their ability to hold large amounts of
water stems from a delicate balance of good water solubility and interchain cross-
linking. Self-assembling proteins and aggregation-prone proteins constitute physi-
cally cross-linked hydrogel as they rely on the physical association of particular
structural motifs for the network construction. Proteins that form a covalent bond
between two separate domains upon association can serve as a building block for
chemically cross-linked hydrogels. Protein hydrogels have a significant advantage
over synthetic or bio-derived hydrogels in that one can rationally design protein
networks with specific functions and properties in mind, as protein structures can be
precisely engineered via genetic modification of DNA. For example, a protein
domain known to confer an appealing functionality can be rationally fused to
other protein domains that form a network. In addition, protein-based hydrogels
tend to be more biocompatible and biodegradable than synthetic polymeric
hydrogels. For these reasons, protein hydrogels have been extensively studied for
their utility in injectable delivery vehicles, implantable scaffolds for soft-tissue
engineering, and matrices for in vitro cell culture. Conformational changes of pro-
teins in response to temperature, pH, light, ligands, and mechanical force further
prompted the development of stimuli-responsive protein hydrogels with sensing
capability.
The main focus of this chapter is understanding the network-forming behavior of
proteins as a structural component in engineered living materials, as these proteins,
Network Formation of Engineered Proteins and Their Bioactive Properties 3
in principle, can be genetically programmed to be produced from living cells

residing in ELM. We identify three major classes of protein network-forming
mechanisms relevant to this topic: (1) phase separation and aggregation-induced
recombinant protein networks, (2) self-assembling multi-domain artificial protein
networks, and (3) chemically cross-linked recombinant protein networks. We will
begin by introducing protein hydrogels and discuss their mechanism of network
formation, which is a critical element in designing functionalities and mechanical
properties of ELM. After introducing the network-forming mechanisms in protein
hydrogels, we will discuss examples of bioactive protein networks equipped with
various functionalities before concluding with future directions and remaining
challenges in this field.
2 Protein Structure and Self-Assembly
Proteins are sequence- and length-controlled linear chains of amino acids joined by
peptide bonds. This well-defined linear chain is the most basic structure of proteins,
also known as the primary structure. Proteins are synthesized by a sequential
biological process. First, the genetic information stored in DNA is transcribed into
a messenger RNA (mRNA). The second step is the translation of mRNA by
ribosomal catalysis into a linear polypeptide chain, transferring genetic information
written in a nucleic acid sequence into a protein sequence. It is possible to produce
recombinant proteins with a varying production yield by modifying the DNA of host
organisms and harnessing their transcription and translation machinery. Innovation
in synthetic and chemical biology gave birth to many tools to manipulate this
process, such as engineered ribosomal binding sites, split RNA polymerase, and
mutant tRNA synthetase. Notably, advances in co-translational incorporation of
noncanonical amino acids have allowed the expansion of native functionalities of
proteins, for example, introducing bio-orthogonal reactive handles and photo-
reactive moieties into recombinant proteins (Link et al. 2003).
2.1 Protein Structures and Their Assembly
Proteins organize themselves into specific three-dimensional structures. The local

structural element of proteins with a regular arrangement of peptide backbone is
called secondary structures. Their regularity is maintained by the local conformation
of the peptide backbone arising from their rotational degree of freedom, hydrogen
bonding between amide moieties, and other supramolecular interaction between side
chains. The most well-known secondary structures are the α-helix and β-strand.
A typical α-helix structure is shown in Fig. 1a: The polypeptide backbone forms a
right-handed helix by regular intramolecular hydrogen bonding. Each backbone
carbonyl oxygen in an α-helix is hydrogen-bonded to the backbone amide hydrogen
4 S. Sim
Fig. 1 (a) A schematic illustration of an α-helix. It is a right-handed helix with 3.6 amino acid
residues per helical turn. Each backbone carbonyl oxygen in an α-helix is hydrogen-bonded to the
backbone amide hydrogen four residues down from it toward C-terminus. The side chains of the
amino acids (designated as R) extend outward from the helical backbone. (b) Schematic illustration
of a coiled-coil dimer and its helical wheel representation. Dimer formation is driven by hydro-
phobic interaction of residues at a and d positions. Residues at e and f positions can stabilize coiled-
coil dimers by electrostatic attractions. (c, d) Hydrogen-bonding networks between two β-strands to
form (c) parallel and (d) antiparallel β-sheet
four residues down from it toward the C-terminus, resulting in 3.6 amino acids per
one complete turn of the helix. While the amide backbone is hydrogen-bonded to
itself almost parallel to the helical axis, the side chains of each amino acid extend
outward from the backbone. The unique ability of α-helix presenting side chains of
its constituent amino acids in a regular fashion has attracted significant interest as a
model system for protein folding studies and a design template for creating protein
assembly. In particular, coiled-coil structures are formed when two or more α-helices
self-assemble (Fig. 1b). Depending on the relative orientation of helical axes, coiled-
coil forms either parallel or antiparallel arrangement. The primary structures of a
coiled-coil domain comprise heptad repeats—repetition of seven amino acids, often
represented as (abcdefg)n. In each helix in coiled-coil, hydrophobic amino acids,
including leucine and isoleucine, occupy every three or four residues (a and d ),
resulting in hydrophobic “face” of the helix. The interaction (burial) of these

hydrophobic faces is the major driving force for the self-assembly of coiled-coil
domains. The supramolecular interactions of amino acid side chains on each helix
affect a helical orientation, oligomerization state, and binding partner specificity of
coiled-coil domains. For example, ionic residues at e and g position can stabilize
coiled-coil by electrostatic attractions (Fig. 1b). Leucine zippers, a sub-type of
coiled-coil and also a common motif in DNA binding proteins, contain a series of
leucines spaced seven residues apart. Leucine zippers form coiled-coil structures via
paired contacts between hydrophobic faces on the constituent helices. It has been
shown that the oligomerization state of coiled-coil configurations in leucine zippers
can be affected by the packing characteristics of the hydrophobic residues at
positions a and d (Harbury et al. 1993). Examples of leucine zipper coiled-coils
with high association numbers have been studied, including a pentamer (n ¼ 5)
based on cartilage oligomeric matrix protein (Malashkevich et al. 1996) and a
heptamer (n ¼ 7) by engineering GCN4 leucine zipper (Liu et al. 2006). Other
minor forms of helix include the 310-helix and polyproline type II (PPII) helix. 310-
helix is similar to α-helix, but with three residues per turn. PPII helix occurs in
proteins with repeating proline residues. It is an extended left-handed helix distinct
from other helical secondary structures as it has no internal hydrogen bonding. It
adopts a more extended form, with a helical pitch of 9.3 Å per turn, compared to
5.5 Å in the α-helix, and has three residues per turn. The collagen triple helix, as it
consists of proline, hydroxyproline, and glycine, adopts a similar conformation to
the PPII helix. Examples of self-assembling structures of various helices in naturally
occurring protein materials include honey bee silk, collagen, and keratin.
The other common secondary structure is the β-strand. β-strands are domains
with a fully extended polypeptide chain, and multiple β-strands can laterally self-
assemble into β-sheets. Typically, alternating sequences of hydrophobic amino acid
and polar amino acid constitute hydrophobic and hydrophilic faces, which drive
lateral assembly of β-strands into β-sheets stabilized by hydrogen bonding between
carbonyl oxygens and amide hydrogens of adjacent β-strands. Similar to coiled-coil
domains, β-sheets are either parallel (same N- to C-terminal direction, Fig. 1c) or
antiparallel (opposite N- to C-terminal direction, Fig. 1d) depending on the relative
orientation of the constituent β-strands. Sequences containing many amino acids
with branched side chains, such as valine, threonine, and isoleucine, are known for
their propensity to form β-strands. β-strands can also form β-hairpins, in which two
β-strands are connected by a short loop and adopt an antiparallel arrangement, β-
spirals, or β-turns. Self-assembly and physical association of β-strands can be found
in natural structural proteins, including silk, resilin, and elastin.
The overall three-dimensional configuration of multiple secondary structures in a
single polypeptide chain is called tertiary structure. In other words, it refers to the
fully folded state of a protein, with three-dimensional arrangements of peptide
backbone and side chains. Therefore, it is closely related to a specific protein
function. The quaternary structure of proteins refers to a precise association of
more than one polypeptide chain. Non-covalent interactions via hydrogen bonding,
hydrophobic interactions, and ionic interactions are commonly found at the interface
6 S. Sim
of subunits. One of such examples is GroEL, a barrel-shaped molecular chaperone. It

consists of identical 14 subunits arranged into two heptameric rings that are stacked
onto each other. Each subunit is held together by elaborate salt bridges between polar
amino acids. Alternatively, in some cases, covalent bonding through disulfide bond
formation or enzymatic catalysis can be formed between protein subunits. Hemo-
globin is a notable example, comprising two pairs of α and β chains, where each
subunit is covalently linked to a heme molecule.
Recent advances in the de novo computational design of proteins have expanded
the toolbox for protein-based materials (Huang et al. 2016). Based on the hypothesis
that proteins fold into the lowest energy conformation of the defined sequences,
the computational approach using a set of physical principles has now advanced to
the point where we can accurately predict the folding of a prescribed sequence. The
major driving force for protein folding is the burial of hydrophobic residues away
from the solvent, typically water molecules. As a result, we are witnessing striking
examples of artificial proteins with sequences unrelated to naturally occurring ones.
One notable example is computationally designed multimeric, water-soluble, and
channel-forming coiled-coil α-helical barrel (Thomson et al. 2014). Another exam-
ple is Keating and colleagues’ work in developing a computational framework for
designing protein-interaction specificity (Grigoryan et al. 2009). They also reported
pairs of synthetic coiled-coils undergoing heterodimeric association called SYNZIPs
(Thompson et al. 2012). As discussed in the following section, well-defined inter-
actions between secondary structures have been extensively studied and employed
as cross-linking motifs for artificial protein hydrogels. An advanced modeling
algorithm, AlphaFold, based on artificial intelligence technology, has been shown
to accurately predict protein structures even when no similar structure is known
(Jumper et al. 2021).
3 Network Formation of Recombinant Proteins
Motivated by the structural role they accomplish in nature, natural proteins have
been examined as building blocks for biomaterials in the form of hydrogels, films,
and others. In particular, those derived from extracellular matrices, such as collagen
and gelatin, were extensively studied in the context of in vitro tissue culture. As our
understanding of protein structure, recognition, and self-assembly deepens with the
aid of computational frameworks, engineering recombinant proteins offers the
unique opportunity to control network structure and functionality. Protein network
formation is driven either by non-covalent interactions between domains or by
covalent linkages between specific residues. The non-covalent interaction of proteins
in a network can be further classified into molecular recognition (self-assembly) and
aggregation. Although hydrogels made with short synthetic peptides also constitute
an important class of biomaterials, this chapter will only describe recombinant
protein networks and the resulting biomaterials. Considering the relevance of protein
network in constructing ELM, three important categories of recombinant proteins
hydrogels, focusing on their network-forming mechanism and some of the physical

properties, will be discussed here: (1) phase separation and aggregation-induced
recombinant protein hydrogels, (2) self-assembling multi-domain artificial protein
hydrogels, and (3) chemically cross-linked recombinant protein hydrogels.
3.1 Phase Separation and Aggregation-Induced

Recombinant Protein Hydrogels
Elastin is one of the essential structural components in the extracellular matrix,

forming fibers responsible for tensile strength and elasticity of tissues. It is abundant
in organs that require constant elastic expansion and contraction as a part of their
function, including skin, lungs, and blood vessels. Structurally, elastin combines
with other proteins to form elastic fibers, for example, ropelike structures in liga-
ments. Exported tropoelastins, the elastin precursor protein, assemble with microfi-
brils comprising fibrillin 1 and are then further cross-linked to each other. Inspired by
the elastic mechanical property of organs conferred by elastin, there have been
attempts to isolate elastin protein from natural sources. However, the purification
of natural elastin poses a multitude of challenges due to its low solubility and
tendency both to calcify and aggregate with other structural components (Daamen
et al. 2007). Alternatively, the desired mechanical properties of elastin can be
recapitulated in recombinant proteins containing multiple elastin-like polypeptides
(ELP). ELP contains [VPGXG]n repeats where the X residue can be any amino acid
except proline. Notably, ELP proteins exhibit inverse temperature transition behav-
ior: At a lower temperature, ELPs are soluble and adopt random-coil conformation.
Upon increasing temperature, ELP proteins become less soluble and eventually
aggregate in coacervate phases above a critical temperature. The temperature
where this transition from soluble to an aggregated state occurs is called the lower
critical solution temperature (LCST), a well-known phenomenon often observed in
hydrophilic polymers, including poly(N-isopropylacrylamide). The LCST phenom-
enon is thermodynamically driven by entropy gained by losing the bound water
molecules to the bulk solution. It has been proposed that ELPs adopt a β-spiral
structure in higher temperatures above LCST (Urry et al. 1981). The LCST of an
ELP is a function of the protein length, concentration, hydrophobicity, and mole
fraction of the guest residues (Urry 1997; Meyer and Chilkoti 2004). Therefore,
varying the length and the guest residue composition of an ELP alters the protein’s
LCST behavior. Due to its LCST behavior, recombinant ELP proteins can be
purified via several rounds of temperature cycling and selective centrifugation
(McPherson et al. 1996). Temperature-sensitive phase transition leads to aggregation
of the more hydrophobic blocks above the LCST and drives the physical self-
assembly of ELP networks. Conticello and co-workers have described the physical
cross-linking of triblock ELP proteins comprising more hydrophilic mid-block
ELP, A, and more hydrophobic end-block ELP, B (Wright and Conticello 2002;
8 S. Sim
Wright et al. 2002). These sequences were selected based on their phase transition
temperature, with the LCST of B being lower than that of A. At temperatures above
its LCST, end-block B undergoes aggregation, while the hydrophilic mid-block A
remains solvated, yielding physical and thermoreversible protein hydrogels. The
critical temperature and other variables affecting sol-gel transition can be engineered
through sequence variation of the triblock proteins. In a study by Chilkoti and
co-workers (Betre et al. 2002), recombinant ELP without explicit “more hydropho-
bic” aggregation domain forms a gel-like coacervate at 37 C, above its LCST
(35 C). The ELP coacervate showed three orders of magnitude increase in the
complex shear modulus and dynamic viscosity and exhibited similar mechanical
properties of the gels that were formed with cartilage extracellular matrix compo-
nents, suggesting their potential utility for cartilage tissue engineering. As described
in Sect. 3.3, ELP hydrogels have also been formed by chemical cross-linking
methods after varying the ELP guest residue to incorporate reactive moieties.
Silk is another natural protein that features impressive mechanical strength and
extensibility. For example, spider dragline silk forms robust and elastic fibers and is
three times tougher than synthetic bulletproof material Kevlar (Rising and Johansson
2015). Silk protein contains a repetitive sequence rich in glycine and alanine, which
forms β-sheet crystalline domains responsible for high mechanical strength and
hydrophilic amorphous regions. Physically cross-linked β-sheet-rich protein
hydrogels can be produced from naturally derived silk, such as silkworm fibroin.
This process involves chemical and mechanical perturbations, including low pH
(Fini et al. 2005), high temperature (Kim et al. 2004), and sonication (Wang et al.
2008). However, harvesting native silk from natural sources faces multitudes of
challenges, such as batch-to-batch variation, impurities, and difficulties in farming
particular silk proteins, especially spider silk. Successful recombinant productions of
silk proteins have been reported using bacterial (Xia et al. 2010), plant (Scheller et al.
2001), and mammalian (Lazaris et al. 2002) hosts. Although a few studies have
demonstrated hydrogel formation of recombinant silk proteins (Rammensee et al.
2006; Schacht and Scheibel 2011), most of the efforts to create biomaterials with
recombinant silk have focused on processing them into fibers, films, and foams. An
alternative approach involves a chimeric recombinant protein containing tandem
repeats of the silklike sequence GAGAGS as well as ELP domain (Megeed et al.
2002; Nagarsekar et al. 2003). The addition of ELP reduces the degree of crystal-
lization of the silklike domain and introduces flexibility and solubility. These pro-
teins spontaneously form physically cross-linked hydrogels due to crystalline
domains comprising the silklike region and show ELP-like properties, such as
temperature responsiveness.
3.2 Self-Assembling, Multi-domain Recombinant Protein

Hydrogels
Coiled-coil associations have been extensively studied as network-forming motifs

for generating recombinant protein networks. In work by Tirrell and colleagues, a
telechelic triblock protein debuted as the first example of a rationally designed
recombinant protein that forms a hydrogel (Petka et al. 1998). This protein, namely,
ACnA, contains two leucine zipper end-blocks (A) separated by a protein spacer
mid-block comprising n-repeats of C unit (Cn) that adopts mostly random coil
geometry and is highly water-soluble. The leucine zipper forming sequence of A
comprises six heptad repeats. It is engineered based on the a/d residue pattern of the
Jun oncogene product and a database constructed with naturally occurring coiled-
coil proteins for determining residues at b/c/f position. Nine Glu and three Lys
residues occupy 12 e/g positions of the A sequence in order to solubilize the
coiled-coil structure and control their assembly with pH. The mid-block was based
on an alanine and glycine-rich sequence [(AG)3PEG]10. This telechelic multi-
domain protein, ACnA, forms a hydrogel by the physical association of A blocks,
while the mid-block linker [(AG)3PEG]10 remains fully solvated. Temperature and
pH affect the association of leucine zipper domains, and as a result, drive the sol-gel
transition of this protein. At low pH, the acidic residues at the e and g positions are
protonated, and the stability of coiled-coil aggregates increases. With increasing pH,
deprotonation of these residues and increased electrostatic repulsion between helices
destabilize the association of coiled-coil domains. At high temperatures, ACnA
behaves as viscous liquids due to the thermal unfolding of leucine zipper domains.
Although ACnA forms a physical protein network by the coiled-coil association of A
end-blocks, their low aggregation number (n ¼ 4) and the transient nature of
association resulted in a soft hydrogel that erodes rapidly in open solutions near
physiological pH. In addition, triblock telechelic proteins tend to form intramolec-
ular loops that do not contribute to network elasticity (Fig. 2a). Intramolecular loops
usually form an antiparallel association of the two end-blocks joined by a mid-block.
To overcome this limitation, the Tirrell lab showcased several different engineering
strategies. Carefully placing cysteine residues on the hydrophobic face of the helix in
order to preferentially stabilize intermolecular associations resulted in coiled-coil
aggregates that were stabilized via disulfide bond formation (Shen et al. 2005).
Extension of a mid-block, Cn, suppresses loop formation, and as a result, ACnA
with more extended linker regions were mechanically stiffer (Shen et al. 2007).
Similarly, loop formation is reduced in pH or ionic strength conditions that favor the
mid-block extension. Kopeček and co-workers designed a series of telechelic
triblock proteins, namely, ABA, CBA, ABC, and CBC, with a mid-block spacer B
as [(AG)3PEG]10 (Xu et al. 2005; Xu and Kopeček 2008). A and C block sequences
are (VSSLESK)6 and (VSSLESK)2-VSKLESK-KSKLESK-VSKLESK-VSSLESK,
respectively. Changing one valine and three serine residues from the A block to
lysine (underlined) resulted in the C block sequence. These added lysine residues in
10 S. Sim
Fig. 2 (a) Schematic illustration of coiled-coil domains. Intermolecular loop formation occurs and
competes with network formation in ACnA proteins that form antiparallel coiled-coil aggregates.
On the other hand, loop formation is suppressed in PCnP proteins with P domains that aggregates in
parallel orientation, as it requires chain stretching of the mid-block. (b) Shear-thinning and elastic
recovery of PCnP hydrogels. The shear storage modulus of PCnP hydrogels decreases upon
oscillatory strain and recovers to its original modulus within seconds (left). PCnP forms a shear-
thinning, yet self-supporting, gel (right). Reproduced with permission (Olsen et al. 2010). Copy-
right 2010, the American Chemical Society. (c) Schematic illustration of the mixing-induced,
two-component hydrogel (MITCH) where two domains in component 1 and 2 assemble via
molecular recognition. Reproduced with permission (Foo et al. 2009). Copyright 2009, the National
Academy of Sciences
the C block changed the thermal stability, pH responsiveness, and self-association

behavior of the protein hydrogel.
Controlling network structure by protein engineering effectively modulates net-
work elasticity and erosion rates of the artificial protein network formed by the
physical association of the coiled-coil domain. Another type of telechelic triblock
protein, PCnP, contains a P zipper domain sequence derived from a rat cartilage
oligomeric matrix protein as end-blocks (Shen et al. 2006). PCnP hydrogels are
stiffer, exhibit a lower erosion rate than ACnA, and prefer to form homo-oligomeric
associations when mixed with A. As A forms interactions with an antiparallel
orientation in the aggregate, intramolecular loop formation always competes with
network formation. In contrast to A, the P block shows a higher aggregation number
(n ¼ 5) and prefers parallel alignment of individual helices. The fact that P exclu-
sively forms parallel orientation for the pentameric coiled-coil aggregate results in
PCnP forming a stiffer network (Fig. 2b). The formation of loops in PCnP network is
energetically unfavorable because the mid-block needs to stretch. As a result, the P
block prefers intermolecular associations that contribute to the network elasticity. In
contrast to the trend observed in ACnA, the stiffness decreases with increasing length
(n) of the mid-block of the PCnP proteins (Olsen et al. 2010). In the case of ACnP
triblock proteins containing both A and P domains, intramolecular loop formation is
further suppressed as both A and P helices form homo-oligomeric associations. As a
result, ACnP showed the lowest erosion rate and the highest normalized plateau
moduli among the three telechelic proteins—ACnA, PCnP, and ACnP—suggesting
that ACnP indeed has the fewest number of intramolecular loops (Shen et al. 2006).
Another desirable feature of these telechelic triblock PCnP hydrogels is a strong
shear-thinning behavior and rapid recovery of elastic strength upon discontinuation
of shear stress. In addition to the biocompatibility of protein hydrogel materials, the
shear-thinning property is advantageous in the context of injectable biomaterials.
Protein hydrogels extruded from a narrow-gauge needle recover to 98% of their
initial elastic strength nearly instantaneously and form self-supporting structures
(Fig. 2c). A different mid-block, En, comprising ELP repeats, yielded PEnP hydrogel
with similar viscoelastic behaviors as PCnP proteins (Dooling et al. 2016; Rapp et al.
2018).
Collagen comprises three left-handed helices intertwined to form a right-handed
triple helix and further assembles into higher-order fibrous structures. As one of the
most abundant proteins found in skin, tendons, and ligaments, collagens are respon-
sible for critical mechanical functions in these tissues. Similar to the case of ELPs,
synthetic or recombinant collagen-mimetic peptides containing glycine, proline, and
4-hydroxyproline have been studied to recapitulate collagen’s self-association
behavior in protein materials. This motif has also been utilized to construct telechelic
triblock protein hydrogels (Skrzeszewska et al. 2009, 2010; Werten et al. 2009). The
two end-blocks contain nine Pro-Gly-Pro repeats that form polyproline helices
assembling into a collagen-like triple-helical “knot” at the junction points in the
protein network and flank a hydrophilic mid-block sequence containing glycine,
asparagine, and glutamine. This protein also forms shear-thinning, thermoreversible,
and self-healing hydrogels that are capable of releasing a protein cargo via erosion.
12 S. Sim
Physical gel formation often requires exposure to high or low pH, temperature
change, or high ion concentration, which are not ideal from the cell or
biomacromolecule encapsulation perspective as they might lead to loss of function
or cell death. To circumvent this issue, Heilshorn and colleagues developed a peptide
association pair to trigger gelation upon mixing the two protein components
containing separate domains (Fig. 2d) and designated this system as mixing-induced,
two-component hydrogel (MITCH) (Foo et al. 2009). Domains suitable for this
purpose need to fulfill the three criteria that (1) the association domain sequence
must be short enough to be repeated in a single recombinant protein, (2) the domains
must not interfere with extracellular signaling machinery, and (3) the domain
association should be selective and tunable, and the WW and proline-rich domains
were chosen accordingly. The WW domain, named after the conserved tryptophan
residue, folds into antiparallel β-sheet structures and associates with the proline-rich
domain. A set of two artificial proteins for MITCH comprises several repeats of
either one of the domains separated by linker regions that form mostly random coils.
Two WW domains, CC43 and Nedd 4.3, were selected because they differ by an
order of magnitude in their association constants with a proline-rich domain (PPxY).
The sol-gel transition and the viscoelastic properties of the MITCH system can be
engineered by varying the stoichiometry and binding strength of the two components
(Mulyasasmita et al. 2011). Like other physical protein gels, such as PEnP, MITCH
hydrogels are shear-thinning, injectable, and self-healing. Li and co-workers have
reported a different two-component protein hydrogel with a pair of complementary
leucine zippers, CCE and CCK, that form heterodimeric coiled-coils at neutral pH
(Lv et al. 2012). Engineered bio-recognition by splitting a native protein has also
shown to be effective in creating physically cross-linked protein hydrogels, as these
split protein fragments spontaneously reconstitute the folded conformation of the
native protein. The loop elongation variant of GB1 protein, GL5, can be split into
two fragments, GN and GC, capable of spontaneous reconstitution. Protein solutions
containing repeats of the two fragments produced physical hydrogels (Kong and Li
2015).
3.3 Chemically Cross-Linked Recombinant Protein

Hydrogels
Chemical cross-linking—creating covalent connections between constituent protein

units—provides a more stable and less dynamic network than the physical protein
hydrogels described in the previous section. Although significant progress has been
made in the last two decades in polymer-protein conjugates and cross-linking, here
we limit our scope to the covalent cross-linking of proteins. Covalent cross-linking
of lysine residues is a commonly employed strategy to produce protein hydrogels.
An organophosphorus cross-linker, β-[tris(hydroxymethyl) phosphino]-propionic
acid (THPP), undergoes a Mannich-type condensation with primary or secondary
amine residues on proteins. The reaction of THPP with amine is fast and
cytocompatible and occurs in physiological conditions. ELP proteins were cross-
linked with THPP to yield hydrogels encapsulating various cell types (Lim et al.
2008; Chung et al. 2012a). Resilin is a highly elastomeric protein found in arthro-
pods showing superior resilience and excellent high-frequency responsiveness (Qin
et al. 2012). Kiick and colleagues used the resilin-like recombinant protein, RLP-12,
which contains 12 repeats of the resilin sequence from the D. melanogaster
CG15920 (Li et al. 2011). THPP was employed to cross-link and modulate the
mechanical properties of RLP12 hydrogels, exhibiting from 500 Pa to 10 kPa storage
moduli. Despite its effectiveness in producing chemically cross-linked protein
hydrogel, THPP is no longer commercially available due to its complicated synthetic
procedure. Heilshorn and colleagues reported tetrakis(hydroxymethyl)phosphonium
chloride (THPC) as an inexpensive and widely applicable cross-linker alternative
(Chung et al. 2012b). THPC selectively reacts with amine residues, can modulate the
mechanical properties of ELP hydrogels, and is cytocompatible. Glutaraldehyde is
another type of cross-linker that reacts to amine residues, and it was employed to
cross-link a telechelic protein containing a mid-block with 12 lysine residues
sandwiched by end-blocks forming triple helix. This process produced shape-
memory protein hydrogels dependent on the thermoreversible association of the
end-block triple helices (Skrzeszewska et al. 2011). Thiol groups on cysteine
residues can form a disulfide bridge under oxidizing conditions and be used for
protein hydrogel formation. Chilkoti and colleagues showed that an ELP with
periodic cysteine residues undergoes hydrogel formation under mildly oxidative
conditions (Asai et al. 2012). The reversibility of the covalent linkage between
two sulfur atoms upon exposure to reducing agents such as glutathione confers a
significant advantage for producing biodegradable protein hydrogels.
Inspired by the biological processes that produce tough networks via enzymatic
activities, such as wound healing, extracellular matrix reinforcement, and cell wall
synthesis, it has been shown that carefully selected enzymes can construct protein
networks. Chilkoti and co-workers chose tissue transglutaminase (tTG), which
catalyzes the calcium-dependent acyl transfer reaction between glutamate and var-
ious primary amine residues. ELP recombinant proteins containing a 1:6 ratio of
lysine (ELP[KV6-112]) or glutamine (ELP[QV6-112]) to valine were incubated
with tTG to encapsulate chondrocytes in situ (McHale et al. 2005). On the other
hand, the microbial transglutaminase (mTG) does not require proenzymes or cal-
cium ions and achieves a high level of activity over a wide temperature and pH
ranges. Using mTG, gelatin hydrogels were cross-linked and studied for their utility
in thermal stability, encapsulation of HEK 293 cells, and therapeutic protein trans-
port (Yung et al. 2007, 2010). In addition to transglutaminases, the horseradish
peroxidase (HPO) system was harnessed to create di-tyrosine covalent bridges. HPO
catalyzes conjugation reactions of phenol and aniline derivatives in the presence of
hydrogen peroxide (Teixeira et al. 2012). Cross-linking resilin proteins from Dro-
sophila melanogaster with HPO resulted in both highly elastic and adhesive bio-
materials (Qin et al. 2011).
14 S. Sim
Photochemical cross-linking is another effective strategy for the generation of

protein hydrogels. Visible light irradiation of a protein solution containing a photo-
sensitizer, Ru(bpy)3Cl2, in the presence of electron acceptor, ammonium persulfate,
resulted in efficient cross-linking of proteins via tyrosine residues (Fancy and
Kodadek 1999). Dixon and co-workers used this technique to cross-link recombi-
nant resilin protein and produce a rubber-like biomaterial (Elvin et al. 2005).
Alternatively, reacting ELP recombinant proteins with a heterobifunctional
succinimidyl 4,40 -azipentanoate (NHS-diazirine) and subsequent cross-linking
through ultraviolet light exposure resulted in the formation of protein films (Raphel
et al. 2012).
Howarth and colleagues engineered a genetically encodable, highly reactive
SpyTag/SpyCatcher covalent bioconjugation system (Zakeri et al. 2012; Veggiani
et al. 2014). Fibronectin-binding protein FbaB of S. pyogenes contains a domain
called CnaB2 with an intramolecular isopeptide bond between Lys and Asp. By
splitting this domain into two and subsequent rational modification, a specific
bioconjugation pair, a short peptide (SpyTag) and a protein fragment (SpyCatcher),
was created. Upon mixing, SpyTag and SpyCatcher undergo isopeptide bond for-
mation between Asp117 on SpyTag and Lys31 on SpyCatcher (Fig. 3a). The
reaction between SpyTag and SpyCatcher is highly specific and works well in
diverse pH, temperature, and ionic environments. As the reactive units can be
genetically encoded and incorporated into protein building blocks, this technology
allows for posttranslational modification of the protein topology (Zhang et al. 2013)
and network formation in situ. Tirrell and co-workers employed this strategy to
engineer a covalently cross-linked hydrogel comprising trifunctional SpyTag chain,
denoted as AAA, and bifunctional SpyCatcher protein, BB (Fig. 3b) (Sun et al.
2014). Instead of using unstructured ELP, Li and colleagues used tandem modular
proteins containing folded globular domains such as GB1 and FnIII. Tandem
modular proteins that contain either multiple SpyCatcher or SpyTag sequences
form cross-linked hydrogels upon mixing with their binding partner (Gao et al.
2016). Stress-relaxation behaviors and viscoelasticity of chemically cross-linked
SpyTag/SpyCatcher protein networks can be programmed by incorporating a
mechanically interlocked p53dim domain (Yang et al. 2018). Similarly, Zhang and
colleagues combined SpyTag/SpyCatcher chemistry with split-GFP and reported a
4-arm recombinant protein that presents four reactive SpyCatcher domains (Yang
et al. 2020). Split green fluorescent proteins (GFPs) comprise two fragments of
native GFP: a small, 15-amino acid fragment GFP11 and the rest of GFP, GFP1–10.
They are highly soluble and reconstitute spontaneously when brought together. Two
recombinant proteins, SpyCatcher–ELP–GFP1–10–ELP–SpyCatcher, denoted as
B10B, and SpyCatcher–ELP–GFP11–ELP–SpyCatcher, denoted as B11B, were
encoded on the same plasmid. Recombinant co-expression of B10B and B11B
yielded a 4-arm SpyCatcher with reconstituted GFP (Fig. 3c). After purification,
this protein readily forms hydrogels with bi- or trifunctional SpyTag recombinant
proteins. The utility of the SpyTag/SpyCatcher system will be discussed again in the
next section below.
Network Formation of Engineered Proteins and Their Bioactive Properties
Fig. 3 (a) Splitting of CnaB2 into the protein SpyCatcher and its peptide reaction partner SpyTag (blue). Reproduced with permission (Veggiani et al. 2014).
Copyright 2014, Elsevier. (b) Schematic illustration of covalently cross-linked SpyTag-SpyTag hydrogel formed by mixing the trifunctional protein (AAA)
15
containing SpyTag and the bifunctional protein containing SpyCatcher. Reproduced with permission (Sun et al. 2014). Copyright 2014, the National Academy
of Sciences. (c) Schematic illustration describing cellular synthesis of the 4-arm starlike protein, (SpyCatcher)4GFP. Co-expression of the two fusion proteins,
16 S. Sim
4 Functional Recombinant Protein Hydrogels
Protein engineering offers the unique capability to design bioactive hydrogels, as

functional protein modules can be genetically encoded and attached to structural
protein domains that serve as junction points. In this regard, the advances in
engineering multifunctional protein hydrogels provide a foundation for designing
functional ELM. Because recombinant protein materials have been actively investi-
gated in the context of tissue engineering and drug delivery, the most widely
investigated functionality to date is cell adhesion and retention. The RGD peptide
ligand, in particular, has been utilized as a “minimal cell-binding motif” and encoded
into ELP (Lampe et al. 2013; Chung et al. 2012b; Sun et al. 2014; Liu and Tirrell
2008), RLP (Balu et al. 2014; Li et al. 2013, 2016), silk-like proteins (Kambe et al.
2010; Bini et al. 2006; Yanagisawa et al. 2007), and a flexible spacer in MITCH
hydrogels (Parisi-Amon et al. 2013) to promote interactions of the protein scaffolds
with a variety of cell types. Alternatively, the CS5 cell-binding domain can be
conjugated into a structural protein, including ELP, for this purpose (Heilshorn
et al. 2005; Panitch et al. 1999). A protein domain derived from extracellular matrix
protein, Tenascin-C, binds to integrins. A chimeric protein of this domain and RLP
formed ECM-mimetic hydrogels that promote the spreading of living fibroblasts
(Lv et al. 2013). In addition to cell-adhesion domains, amino acid sequences that can
be cleaved at specific sites by cell-secreted proteases have been installed in protein
hydrogels, mimicking the matrix remodeling phenomena in natural biological mate-
rials. One commonly employed motif is a substrate sequence of the matrix
metalloproteinases (MMPs). Incorporating an MMP substrate sequence into chime-
ric proteins of RLP (Charati et al. 2009; Li et al. 2013), silk/elastin-like proteins
(Price et al. 2015), and silk-like proteins (Gustafson et al. 2013) has been demon-
strated. Similarly, in the context of tissue engineering, peptide sequences sensitive to
degradation by the proteases, such as tissue plasminogen activator and urokinase
plasminogen activator, have been encoded into ELP-based protein hydrogels
(Straley and Heilshorn 2009a, b; Madl et al. 2018).
Banta and co-workers have engineered functional protein hydrogels based on the
coiled-coil association of ACnA proteins. As incorporating fluorescent proteins in
the mid-block region of ACnA architecture does not interfere with the coiled-coil
association and subsequent gelation, they engineered multicolored protein hydrogels
with two different fluorescent proteins and studied network structure by Förster
resonance energy transfer (Wheeldon et al. 2007). Based on a similar telechelic
architecture, two bifunctional co-assembling recombinant proteins were designed to
form a catalytic protein hydrogel (Wheeldon et al. 2008). One protein has a
Fig. 3 (continued) B10B and B11B, and subsequent split GFP reconstitution produced intracellular
(SpyCatcher)4GFP. Upon mixing with AA containing two SpyTag peptides, (SpyCatcher)4GFP
assembles into a Spy-G network. Reproduced with permission (Yang et al. 2020). Copyright 2020,
Elsevier
telechelic architecture in which a mid-block is sandwiched by self-assembling

α-helical leucine zippers and bears a few histidine residues to which exogenously
added osmium ion could be attached. The second protein building block is the same
α-helix fused to the polyphenol oxidase small laccase (SLAC) that performs catal-
ysis upon dimerization. The combination of the oxidase enzyme and
metallopolypeptide yielded an electron-conducting protein hydrogel that reduces
molecular oxygen to water. The association of leucine zipper domains in these two
protein building blocks allows the incorporation of SLAC enzymes into the protein
network, and dimerization of SLAC contributes to additional cross-linking and
catalysis. Potential applications of this hydrogel as a cathode in biofuel cells or
biosensors have been suggested. Other examples of enzymes incorporated into
ACnA design include aldo-keto reductase (Wheeldon et al. 2009) and organophos-
phate hydrolase (Lu et al. 2010).
Chimeric proteins containing a leucine zipper domain fused with other stimuli-
responsive self-associating protein domains have been shown to form stimuli-
responsive protein hydrogels. One such example utilizes calmodulin, which
undergoes a conformational change and binds to calmodulin-binding domains
within other proteins in the presence of Ca2+ ions (Topp et al. 2006). When a
chimeric calmodulin protein fused to a leucine zipper domain is mixed with a
telechelic triblock protein bearing calmodulin-binding domain as end-blocks and
Ca2+, a protein network is formed (Fig. 4a). This network undergoes a reversible
change in viscosity upon addition and removal of Ca2+. Banta and colleagues
engineered various Ca2+-responsive protein building blocks based on the block V
repeats-in-toxin (RTX) domain of B. pertussis adenylate cyclase (Dooley et al. 2012,
2014). This domain is intrinsically disordered in the absence of Ca2+ and folds into a
β-roll in response to high Ca2+ concentration. Upon addition of Ca2+, the engineered
RTX β-roll folds, and the hydrophobic faces which drive oligomerization are
exposed. Banta and co-workers engineered chimeric proteins with an engineered
β-roll domain that self-assembles upon Ca2+ addition and mixed them with a β-roll
mutant that conditionally binds to a specific target, hen egg white lysozyme. This
process yielded a protein hydrogel in which gelation and target protein retention is
controlled in a Ca2+-dependent manner (Bulutoglu et al. 2017).
Conjugating SpyTag and SpyCatcher domains to other functional modules was
demonstrated as an effective strategy to produce functional protein hydrogels. Sun
and co-workers used the CarH protein, a transcriptional regulator that senses visible
light through its C-terminal adenosylcobalamin binding domain (CarHC), for this
purpose (Fig. 4b) (Yang et al. 2020; Jiang et al. 2020; Wang et al. 2017). The light-
sensing CarHC domains form tetramers when binding to adenosylcobalamin
(AdoB12) in the dark and disassemble into monomers upon exposure to a green
(522 nm) light. Recombinant proteins SpyTag–ELP–CarHC–ELP–SpyTag, denoted
as ACA, and SpyCatcher–ELP–CarHC–ELP–SpyCatcher, denoted as BCB, poly-
merize upon mixing, and in the presence of AdoB12, form hydrogels that undergo a
rapid gel-sol transition with light exposure. Encapsulation and release of fibroblasts
and human mesenchymal stem cells, as well as controlled release of proteins, have
been demonstrated with this platform, harnessing in situ gel formation upon mixing
18
Fig. 4 (a) Ca2+-dependent network formation of the triblock artificial proteins. Calmodulin undergoes a conformational change and binds to proteins containing
calmodulin-binding domains, including petunia glutamate decarboxylase (PGD) and human endothelial NO synthase (eNOS), in response to Ca2+ ions.
Reproduced with permission (Topp et al. 2006). Copyright 2006, the American Chemical Society. (b) Synthesis of photoresponsive CarHC hydrogels. CarHC
tetramer undergoes disassembly upon light exposure and releases 40 ,50 -anhydroadenosine. The two telechelic proteins, ACA containing SpyTag and BCB
containing SpyCatcher, assemble into polymers and further form a protein network via AdoB12-induced CarHC tetramerization. This network can reversibly be
S. Sim
disassembled upon light exposure. Reproduced with permission (Wang et al. 2017). Copyright 2017, the National Academy of Sciences. (c) Reproduced with
permission (Huang et al. 2019). Copyright 2019, Springer Nature
and photoresponsive gel-sol transition. The similar architecture of recombinant

proteins bearing metal-binding domains showed the potential of sequestering ura-
nium and chromate from water (Kou et al. 2017). Li and co-workers used
SpyCatcher/SpyTag cross-linking to engineer optically responsive protein hydrogel
networks, where multimerization of a mutant fluorescent protein, Dronpa145N,
serves as an optically controllable physical cross-linker (Lyu et al. 2017). The
fluorescence of the Dronpa145N switches off under a strong 500-nm illumination
and switches on by 400-nm illumination, which is accompanied by the
tetramerization of Dronpa145N. As a result, protein hydrogels produced by
SpyCatcher/SpyTag conjugation and physical association of Dronpa145N undergo
gel-sol transition upon 500-nm light illumination.
Recent pioneering efforts inspired by bacterial biofilms have opened a new
chapter of protein network design in the context of ELM. Many bacteria build
their own physical habitat called biofilms. As bacterial biofilms are extensively
hydrated networks where living cells build and maintain the proteinaceous scaffolds,
they are inherently hydrogels. Bacterial fibers are a key building block of bacterial
biofilms and provide a significant advantage in constructing protein networks in
ELM, as they are genetically encodable, can be secreted from bacterial cells, and in
some cases, form cross-β strand amyloids with high tensile strength and mechanical
rigidity. Isolated bacterial amyloid fibers have been shown to be effective in enhanc-
ing the stiffness of alginate hydrogels (Axpe et al. 2018). Efforts in engineering
bacterial fibers, including curli proteins in E. coli and TasA in B. subtilis, have
yielded bioinspired ELM with tunable properties. Lu and colleagues demonstrated
that conjugation of a short histidine peptide to a major curli substituent, CsgA, yields
an extracellular protein fiber displaying reactive handles to load functional modules
(Chen et al. 2014). Controlling the production of an engineered CsgA by quorum
sensing enabled the autonomous patterning of curli fibers over time. Joshi and
co-workers developed Biofilm-Integrated Nanofiber Display (BIND) by appending
peptide motifs to the CsgA (Nguyen et al. 2014). Their initial work includes
screening the optimal fusion sites on CsgA and a panel of peptide tags that can
bind to various exogenous chimeric species. Combination of the covalent binding
of a target protein using SpyTag/SpyCatcher chemistry and non-covalent attachment
of another target protein via FLAG tag and anti-FLAG antibody allows decoration of
the E. coli biofilms with more than one functionality. In addition to the SpyTag/
SpyCatcher conjugation pair, other covalent and non-covalent protein conjugation
pairs were examined for this purpose: InaD/EFCA, Tip1/WRESAI, calmodulin/
M13, and SZ21/SZ16 (Nussbaumer et al. 2017). InaD specifically binds to a small
peptide EFCA and forms a disulfide linkage, and similarly, Tip1 non-covalently
binds to a small hexapeptide WRESAI with a dissociation constant in the nanomolar
range (Lu et al. 2014). Similar to the examples discussed in the previous paragraph,
calmodulin binds to M13 peptides in a Ca2+-dependent manner (Blumenthal et al.
1985). SZ21 and SZ16 are helical motifs that heterodimerize to form superhelical
bundles with each other (Thompson et al. 2012). This technique has expanded our
ability to incorporate other functionalities to biofilms, including enzymes
(Botyanszki et al. 2015; Nussbaumer et al. 2017), gold nanoparticles (Seker et al.
20 S. Sim
2017), and adhesive proteins (Zhong et al. 2014). Bacillus subtilis, a gram-positive
microorganism, produces biofilms containing bacterial filaments of the TasA pro-
tein. Engineering TasA by fusing it with various other protein motifs enabled
programmable ELM based on B. subtilis biofilm with functional properties, includ-
ing intrinsic fluorescence, enzymatic activity, and the templated assembly of inor-
ganic nanoparticles (Fig. 4c) (Huang et al. 2019).
5 Future Directions and Challenges
This chapter explored achievements in the field of recombinant protein hydrogels in

the last few decades. As discussed in previous sections, a rationally designed,
programmable, and hydrated protein network offers a unique platform for addressing
challenges in biology, medicine, environment, energy, and many other areas.
Advances in protein engineering have enabled important discoveries in this field,
ranging from fundamental studies on protein network structures to developing
functional protein hydrogels. At the same time, the fast-moving frontier of the
computational design of artificial proteins will continue to expand the breadth of
this field and exert a significant impact. With the frontier of ELM moving toward
implementing multifunctional properties, the design capability of recombinant pro-
tein networks could drive a multitude of innovations. In this regard, the remaining
key challenges include (1) expanding the library of genetically encodable and
orthogonal protein-protein interaction/reaction pairs, (2) engineering effective pro-
duction and secretion systems for recombinant proteins from the constituent cells of
ELM, and (3) understanding and designing synergistic effects of protein scaffolds
and living cells.
The development of SpyTag/SpyCatcher and many other protein-protein recog-
nition motifs has enabled the engineering of protein networks with complex topol-
ogy and functionalities. They act as a genetically encodable, proteinaceous “glue”
that creates junction points in a network or serves as anchor points for attaching
functional modules. Expanding the library of employable orthogonal protein-protein
interaction/conjugation pairs for multifunctional decoration of protein networks will
propel future innovations in the field of recombinant protein hydrogels and ELM.
Engineering more efficient or stimuli-responsive bio-recognition, either by rational
or de novo computational design, will create protein networks with desired chemical,
physical, and functional properties.
As seen in artificial telechelic proteins and other protein hydrogels, the concen-
tration of the monomeric protein is a critical factor for the mechanical properties of
the resulting materials. Except in several cases, the yields of secreted recombinant
proteins from host cells are often low. For instance, the production yield of curli
fibers is in the order of milligrams or lower per liter scale (Dorval Courchesne et al.
2017). Although the use of mutant E. coli strains with modified outer membranes has
been shown to achieve promising production and secretion yield of some recombi-
nant proteins, the results vary case-by-case and often rely on trial and error (Kleiner-
Grote et al. 2018). B. subtilis is widely used for the production of industrial enzymes
due to well-defined secretion pathways and the absence of an outer membrane.
However, yields are often milligram or sub-milligram quantities per liter of culture
when it comes to the production and secretion of heterologous proteins (Harwood
and Cranenburgh 2008). Engineering bacterial strains and pathways optimized for
producing a wide range of network-forming recombinant proteins in large amounts
would open possibilities to build ELM with desired mechanical properties.
The synergistic effect of living cells and protein networks in ELM beyond their
production is often overlooked. Not only do the living cells produce the protein
network, but they are also capable of manipulating them. Extensive experimental
characterizations complemented by multi-scale modeling will allow us to understand
how protein networks in the ELM change over time and respond to environmental
perturbations. The ability to understand and engineer the cellular contribution to the
mechanical properties over time will allow predictive designs of ELM.
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Living Synthetic Polymerizations
Austin J. Graham and Benjamin K. Keitz
Abstract Natural materials, such as biofilms and tissues, sense and respond to
environmental signals using genetic, metabolic, and proteomic machinery. This
machinery allows natural materials to actuate changes with unmatched spatiotem-
poral precision. However, natural materials are relatively limited in morphology and
functionality compared to synthetic materials. In an effort to enhance synthetic
materials with the capabilities of living systems, we describe recent efforts to control
synthetic polymerizations using live cells as actuators. Both microbes and eukaryotic
cells have been employed in radical and oxidative polymerizations, significantly
expanding the synthetic scope available to living systems. In addition, these mech-
anisms have enabled construction of polymer networks and hydrogels that resemble
natural materials like tissues. Future efforts in synthetic biology, combined with new
methods for reprogramming metabolism to control abiotic chemistry, will enable
more platforms that synergistically enhance synthetic materials with living
functions.
Keywords Living materials · Synthetic biology · Extracellular electron transfer ·

Shewanella oneidensis · ATRP · Synthetic chemistry · Organometallic catalysis ·
Hydrogels
1 Introduction
Living organisms detect and respond to a variety of inputs within complex environ-
ments. Specifically, living systems exist in a state of dynamic reciprocity, wherein
bidirectional flow of chemical, mechanical, and electrical information between cells
and their environment dictates micro- and macroscopic properties. This relationship
guides the development of cellular “living materials,” including biofilm growth and
A. J. Graham · B. K. Keitz (*)

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA
Center for Dynamics and Control of Materials, University of Texas at Austin, Austin, TX, USA
e-mail: keitz@utexas.edu
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 27

W. V. Srubar III (ed.), Engineered Living Materials,
https://doi.org/10.1007/978-3-030-92949-7_2
28 A. J. Graham and B. K. Keitz
tissue formation. Inputs such as food, water, light, and oxygen are processed by
cellular building blocks, instructing living material properties through growth,
morphology changes, and differentiation. At the single cell level, these environmen-
tal signals are processed and amplified using genetic, metabolic, and proteomic
networks (e.g., biological computation).
Biological computation is a central capability of living systems that enables
autonomy, self-regulation, and non-equilibrium processing. In a simple example, a
physical input such as sunlight serves as a transcriptional activator in plants, kick-
starting metabolism through the expression of photosynthesis genes. A more complex
example would be tissue homeostasis, where multiple extra- and intracellular signals
are simultaneously processed with spatial and temporal precision to maintain proper
tissue function across many length scales. This information processing is actuated and
amplified using specific biomolecules, including proteins, nucleic acids, and small
molecules. Synthetic biology, systems biology, metabolic engineering, and related
fields seek to understand and engineer control over biological computation,
harnessing the responsiveness of living systems toward outputs ranging from phar-
maceutical production to bioremediation (Meng and Ellis 2020; Tang et al. 2020;
Voigt 2020). In platforms that capitalize on the self-regulation and sustained growth
of living cells, programming both native and new functions through synthetic biology
confers added autonomy and intelligence to the engineered application. These cells
can then be deployed in dynamic and intervention-less environments.
With respect to materials, controlling biological computation toward desired
material properties has predominately focused on natural materials already produced
by the host organism. Some leading examples in the field are biofilm- and hydrogel-
based materials formed from the extracellular amyloid protein CsgA in E. coli
(Nguyen et al. 2014). A key advantage of protein-based living materials is the ability
to tailor function using amino acid sequence, such that the same general platform can
be used to design adhesives, conductors, and bioplastics. These gels also benefit
from the flexibility to harbor constituent cells as a method of continued gelation and
propagation, or to easily filter out cells for abiotic application. Another common
microbially produced material is cellulose, which can be engineered into tissue
mimetics or enzymatically functionalized surfaces (Shaffner et al. 2017; Gilbert
et al. 2021). Designer control over polysaccharide properties presents an interesting
challenge, as genetic sequence does not directly encode properties in the same way
as proteins; nonetheless, it has proven an invaluable platform for developing diverse
living materials. Organisms have also evolved relationships with inorganic mate-
rials, including synthesizing magnetic nanoparticles or forming structural elements
like bone and teeth (Furubayashi et al. 2021). In some cases, these relationships can
be metabolically tuned to control inorganic material morphology, which informs
their function. Coupling enzyme function to inorganic materials presents a novel
opportunity to apply bioengineering techniques, such as directed evolution, toward
probing and optimizing the functional landscape of these materials. It is possible that
features of this space are inaccessible to typical chemical syntheses. Overall, the
materials outlined above are notable for their tight evolutionary relationship with the
host organism, potentially facilitating genetic control over their synthesis and
Living Synthetic Polymerizations 29
designed function. However, endogenously produced natural materials suffer from a

potential lack of design flexibility that primarily stems from their basis in biological
building blocks, such as amino acids, carbohydrates, and a few inorganic molecules.
In contrast, synthetic materials offer a wider array of chemical, physical, and
transport properties that are generally unavailable to natural systems.
Given their respective strengths, it would be advantageous to improve the func-
tional versatility of synthetic materials with the dynamic capabilities of biological
computation. These “living synthetic materials” could process diverse signals, self-
regulate, and coexist in bidirectional communication with their environments by
using cells as actuators within the material. However, as nature did not evolve
genotype-phenotype relationships between cells and synthetic materials in the
same way as with natural materials, generating such relationships de novo is not
trivial. This requires reprogramming of metabolic function toward controlling tradi-
tionally abiotic processes, such as catalytic nanoparticle assembly or synthetic
polymerizations. With respect to synthetic polymerizations, herein we outline
some examples that are actuated by living cells (Fig. 1). Specifically, we highlight
Fig. 1 Schematic illustrating an example of a living synthetic polymerization. Biological compu-

tation (i.e., sensing environmental signals and computing those signals using genetic, metabolic,
and proteomic machinery) can be rewired to control synthetic polymerizations through redox-
mediated processes. These mediators can include flavins, peroxides, reduced metals, and glutathi-
one, among others. In addition, extracellular electron transfer pathways, such as the Mtr pathway in
Shewanella oneidensis, can also power these reactions. Figure created with BioRender.com
recent work toward controlling redox-driven polymerization mechanisms using the

diverse reducing power of cells, including generation of electron equivalents, secre-
tion of specific enzymes, and direct electron transfer to polymerization catalysts.
Synthetic polymerizations are some of the most common platforms for generating
scalable, versatile, functional soft materials. Although much of the described work is
still in an early stage, rewiring cellular metabolism for controlling these polymeri-
zations is an important step toward sustainable, self-regenerable, and adaptable
living materials with the functional diversity of synthetic polymers.
2 Extracellular Electron Transfer Bridges

the Biotic/Abiotic Interface
In order to program synthetic polymers using living cells, catalytic activity must be
controlled by natural biological functions. One evolutionary relationship that cou-
ples the biotic and abiotic (i.e., natural and synthetic) worlds is a form of microbial
anaerobic respiration known as extracellular electron transfer (EET). In oxygen-
limited or redox stratified environments, such as the deep soil and ocean, some
microbes have evolved efficient machinery for moving electrons in/out of the cell
and across long (cm) distances. This capability allows life to persist in challenging
and competitive resource-limited environments. For example, the purple photosyn-
thetic bacteria Rhodopseudomonas palustris TIE-1 is a photoautotrophic microbe
that couples photosynthesis and the oxidation of ferrous iron, Fe(II), to carbon
dioxide reduction (Guzman et al. 2019). In this case, extracellular metals or poised
electrodes serve as electron donors to drive carbon fixation. Alternatively, microbes
such as Geobacter spp. and Shewanella oneidensis MR-1 direct metabolic electron
flux from carbon oxidation onto inorganic extracellular substrates. In nature, these
electron acceptors include metals and metal oxides, such as iron and manganese
oxides. However, synthetic materials, including various types of electrodes,
nanocrystals, metal-organic frameworks, and soluble metals, can also act as electron
acceptors (Shi et al. 2016; Dundas et al. 2018; Springthorpe et al. 2019; Graham
et al. 2021). Overall, EET provides an electronic interface between cellular metab-
olism and redox-driven processes occurring outside of the cell.
Geobacter sulfurreducens and S. oneidensis MR-1 are two of the most well-
studied organisms for understanding and applying EET. Despite both species being
electroactive, there are several key differences between these bacteria. First,
G. sulfurreducens is an obligate anaerobe, which cannot survive under highly
oxygenated conditions (Bond and Lovley 2003). In contrast, S. oneidensis is a
facultative anaerobe that is famed for its ability to utilize a variety of electron
acceptors including oxygen, DMSO, fumarate, nitrate, and various metals. Second,
the mechanisms responsible for EET are different between the two bacteria.
G. sulfurreducens and related Geobacter species exclusively use protein-based
nanowires to connect cellular metabolism to extracellular electron acceptors. The
exact structure and mechanism of these wires is somewhat controversial, but they are
required for Geobacter EET. On the other hand, S. oneidensis MR-1 uses a combi-
nation of electron transfer cytochromes bound to the outer membrane and soluble
redox shuttles, such as flavins, to facilitate EET (Coursolle et al. 2009; Coursolle and
Gralnick 2010; Brutinel and Gralnick 2012; Xu et al. 2016). In one specific example,
S. oneidensis uses the Mtr pathway (Fig. 1) to move electrons across the outer
membrane and onto extracellular electron acceptors. A final difference between the
two bacteria concerns genetic tractability. Genetic transformations in Geobacter
species, including the creation of knockouts and the insertion of exogenous DNA,
are possible, but time consuming and laborious. Although S. oneidensis is not a
model chassis, an increasing number of sophisticated genetic engineering techniques
have been demonstrated in this species (Hu et al. 2015; Corts et al. 2019; Dundas
et al. 2020; Li et al. 2020). Overall, the oxygen tolerance, relatively high growth rate,
well-understood EET machinery, and genetic tractability of S. oneidensis MR-1
make it an ideal organism for applying and manipulating EET. However, it is
important to note that Geobacter or other EET active microbes could replace
S. oneidensis in many of the EET-driven applications described below.
Our understanding of the specific EET pathways in S. oneidensis has facilitated
numerous applications using a diversity of metal electron acceptors. The most
apparent and well-studied is current generation on electrodes. S. oneidensis colo-
nizes different metal oxide surfaces to respire, which can be used to design a
microbial fuel cell (Shi et al. 2016). The diverse and facultative respiratory capabil-
ities of S. oneidensis enable long-distance electron transfer to the oxide surface even
in multicellular biofilms, where redox potential is stratified moving away from the
electrode. At appropriate potentials, electron transfer can also be redirected into the
cells through specific proteins, enabling unfavorable intracellular transformations
(termed bioelectrosynthesis). Underpinning these applications is our strong under-
standing of the Mtr and related pathways and their programmability using synthetic
biology. The wide substrate scope of S. oneidensis’ EET machinery enables contin-
ued development of new cell-substrate relationships. For instance, EET can be
directed toward oxidized soluble metals, such as Pd2+, to generate nanoparticles
(Dundas et al. 2018). Manipulating EET through both genetics and metabolism
allows control over nanoparticle synthesis rate and localization (e.g., outer
membrane-bound or intracellular). In addition, this evolutionary relationship to
metals allows cells to tune novel material properties. For example, EET can be
used to regulate the optical response of inorganic nanocrystals (Graham et al. 2021).
Overall, the tight metabolic relationship between electron transfer via the Mtr
pathway and central carbon metabolism in S. oneidensis allows for living control
over traditionally abiotic processes like producing current or programming
materials.
Given even just these few examples, it is apparent that redox-driven chemical
transformations are ubiquitous in both living and synthetic systems. However, in
practice, synthetic reactions driven by electron flow are usually controlled with
chemical reductants or in an electrochemical cell using a potentiostat. Based on
early reports that S. oneidensis could affect dehalogenation reactions in the presence
of soluble metals (Workman et al. 1997), we reasoned that these bacteria may be able
to power a wide variety of material-relevant transformations via EET to synthetic
catalysts. In this chapter, we describe the efforts of several research groups to
validate this and related hypotheses. Specifically, we highlight the ability of
S. oneidensis and other electroactive organisms to control polymerization catalysts
for the synthesis of polymers and polymer networks. Although EET is an efficient
mechanism through which electrogenic bacteria can influence their redox environ-
ment, we also provide examples of non-electroactive organisms that are able to
influence their extracellular redox environment and redox-driven catalytic reactions
through molecule secretion, metabolic activity, and other means. With further
development, materials synthesized via the coupling of cellular metabolism to
synthetic catalysts will capitalize on the versatility, chemical functionality, and
robustness of synthetic materials while still maintaining the autonomy, sensing,
and computational abilities of living cells.
3 Synthetic Polymerizations Mediated by Microbes
Radical polymerizations are central to synthetic chemistry and yield a diverse array
of soft, plastic, and glassy materials. Two well-known classes of “living” radical
polymerizations are atom-transfer radical polymerization (ATRP) and reversible
addition-fragmentation chain transfer (RAFT) (Kamigaito et al. 2001;
Matyjaszewski 2012). In the context of these polymerizations, “living” refers to
the presence of an active/dormant polymer chain end that does not spontaneously
decompose or participate in undesirable radical reactions. Both ATRP and RAFT
utilize polymer chain ends that continually switch between an active (can add
additional vinyl monomers) and a dormant (unreactive) state. The relatively low
concentration of radical species allows these polymerizations to achieve high con-
versions and produce polymers with controlled molecular weights and low polydis-
persity. The presence of a living chain end also allows for the synthesis of block
copolymers, bottlebrush polymers, and other polymer microstructures. There are
several different variants of ATRP and RAFT, but critically, there are examples for
both where an appropriately poised redox environment promotes reduction of a
chemical species, leading to radical initiation. As electrogenic microbes can be
potent reducers, we and others reasoned that endogenous cell metabolism could be
coopted to kick-start these radical polymerizations.
In RAFT, a radical initiator and a chain-transfer agent control the growth of living
polymer chains. Many common radical initiators are appropriately poised within a
reduction window achieved by microbial culture. Capitalizing on this reducing
capability of bacteria, Nothling et al. (2021) employed cultures of E. coli and
S. enterica serovar Typhimurium toward activation of a common redox-active aryl
diazonium salt. This allowed synthesis of p(oligoethylene glycol methacrylate) via a
microbially mediated RAFT mechanism (Fig. 2). The metal-free catalysis employed
nontoxic substrates, such that active cell metabolism was maintained within the
Fig. 2 Controlled radical polymerization (BacRAFT) facilitated by the terminal electron flux of E. coli (strain MC4100) and S. Typhimurium (strain TAS2010).
The reducing potential generated by growing bacteria activates the redox-active diazonium salt (4-bromobenzenediazonium tetrafluoroborate (4-BT)) to furnish
a carbon-centered aryl radical, which subsequently initiates a controlled radical polymerization of methacrylate monomers (OEGMA) in the presence of a chain-
transfer agent (TTC-1) via RAFT (Reproduced with permission from Nothling et al. 2021)
33
living polymerization. The reaction was well-controlled, producing low dispersity

polymers with first-order kinetics and achieving substantial monomer conversion
upward of ~70%. Engineered knockout variants of S. enterica demonstrated the
importance of redox mediators in the polymerization—glutathione biosynthesis
deficiency and accumulation of reduced copper each inhibited and enhanced con-
version, respectively. Although redox-controlled RAFT polymerizations are rela-
tively rare (Wang et al. 2017; Lorandi et al. 2019), this example highlights how the
careful selection of radical initiators and chain-transfer agents can facilitate biolog-
ical control over a synthetic polymerization.
In ATRP, a radical initiator is activated and deactivated by a transition metal
catalyst, and this redox equilibrium controls the polymerization rate of vinyl mono-
mers. By keeping the overall radical concentration low, ATRP yields low dispersity
polymers with tight control over molecular weight and minimal chain termination.
Unlike most RAFT polymerizations, ATRP is controlled using transition metal
catalysts and can be easily actuated using electrochemical control over the catalyst
redox equilibrium, which determines the polymerization rate and polymer charac-
teristics. Thus, microbial secretion of reducing equivalents can initiate ATRP in the
presence of bacteria. In one example, Magennis et al. (2014) “templated” vinyl
monomers on bacterial surfaces for labeling and selection (Fig. 3). The bacteria
instructed reduction of a copper catalyst, yielding polymers bound to the cell
membrane that were specific to those cells and could be used to identify specific
cell populations in co-cultures. Cells could also be fluorescently surface-labeled with
tandem copper-catalyzed polymerization and azide-alkyne cycloaddition reactions.
In a similar reaction scheme, glycopolymers that were cell-adherent and strain-
specific could be polymerized with glycosylated monomers (Luo et al. 2019). The
incorporation of biomimetic architectures like sugar molecules provides an interest-
ing handle with which to tune synthetic polymer interactions within biological
systems. ATRP initiated by a general reducing environment was also demonstrated
using various microbes in the presence of iron, another common organometallic
catalyst that is less cytotoxic than copper (Bennett et al. 2020). Overall, the redox
environment created by living cells has an appropriate reduction potential for
controlling a variety of synthetic polymerizations, and further exploration of reaction
conditions, combined with rewiring new metabolic capabilities to catalyst activation,
will significantly expand the reaction space available to living organisms.
4 Atom-Transfer Radical Polymerization Powered by

Extracellular Electron Transfer
While the general reducing capability of microbes is powerful for controlling the
above reactions, we reasoned that a more specific mechanism for metal reduction
could directly couple synthetic reactions to active cell metabolism. As S. oneidensis
is known to reduce a variety of catalytic transition metals, we hypothesized that EET
Fig. 3 A schematic of the bacteria-instructed synthesis process. (a–c) Bacteria induce polymerization in monomer-catalyst suspensions (a) to generate a
synthetic extracellular matrix of polymers (b). Recovery of polymers from the suspensions leads to two fractions (c), with polymer obtained from the aqueous
35
phase suspension around the bacteria denoted conceptually as non-templated and a second fraction obtained from a wash of the cell surfaces denoted as
could serve as a general electron transfer mechanism for organometallic reactions

(Fan et al. 2018). As a proof of concept, we chose ATRP for the strong conceptual
parallel between EET and a poised electrode (Fig. 4a, b). We first demonstrated that
S. oneidensis was capable of polymerizing a vinyl monomer, oligo (ethylene glycol)
methyl ether methacrylate, in a microbial broth, Shewanella basal medium (SBM),
that was supplemented with mixed metals serving as catalysts (Fig. 4c). Isolating
copper within the metal mix improved the reaction kinetics, and E. coli in the same
conditions did not polymerize monomer, indicating that specifically EET was
required for this process (Fig. 4d). Consistent with previous work, reducing equiv-
alents including glutathione and cell lysate could actuate a small amount of poly-
merization. Indeed, conversion achieved from cell lysate approached ~40%, which is
similar to that achieved by concentrated bacteria cultures that were not performing
EET (Magennis et al. 2014), supporting the hypothesis that the reducing environ-
ment of microbial culture can nonspecifically drive these reactions. However, living
S. oneidensis cells capable of continuous catalyst reduction were required for
significant copper reduction and high monomer conversion upward of 90%
(Fig. 4e). Overall, p(OEOMA500) polymers formed via EET in the presence of live
cells were monodisperse, exhibited first-order kinetics, and achieved high monomer
conversion, all indicative of a well-controlled reaction.
A primary advantage to using EET as opposed to nonspecific reducing equiva-
lents is the development of a genotype-phenotype linkage to material synthesis. To
demonstrate this link, we employed knockouts of specific EET genes in
S. oneidensis in ATRP reactions. Although a variety of cytochromes and molecular
redox shuttles, such as flavins, are responsible for EET activity in S. oneidensis, we
found that the extracellular cytochromes MtrC and OmcA, which anchor on the
outer membrane, were the strongest regulators of electron flux to copper, and
therefore polymerization (Fig. 5a, b). This was evidenced by a greater decrease in
polymerization rate in a ΔmtrCΔomcA knockout compared to a flavin exporter
knockout (Δbfe) and a hydrogenase knockout (ΔhydAΔhyaB). Toward further
engineering of this genotype-phenotype relationship, plasmid-based complementa-
tion of mtrC in the ΔmtrCΔomcA strain almost fully rescued polymerization activity
(Fig. 5c). Future efforts in maximizing the role of the Mtr pathway in synthetic
polymerizations may investigate downregulation or knockouts of other reduction
pathways that may also interact with soluble copper. For example, the flavin exporter
Fig. 3 (continued) templated. (d, e) Incubation of polymers with bacteria results in low binding of
cells for which the polymer is non-templated (d), or where a polymer templated with one cell type
(shown in red) is incubated with a cell (shown in green) of another type (e). (f) Addition of a
polymer, templated by one cell type, with its own “matched” cell population results in the formation
of large polymer cell clusters, as the templated polymers sequester the bacteria that “instructed”
their formation with high affinity. (g) The same reducing environment at bacterial surfaces that aids
the polymer synthesis can also be used to label the cells in situ via pro-fluorescent markers, which
react with cell-surface bound polymers containing “clickable” residues (Reproduced with permis-
sion from Magennis et al. 2014)
Fig. 4 S. oneidensis-enabled ATRP and initial polymerization kinetics. (a) Electron equivalents generated from S. oneidensis MR-1 reduce a metal catalyst
from an inactive state (MOX) to an active state (MRED). The active catalyst reacts with a halogenated initiator or polymer chain to produce a radical (gray arrow)
that can polymerize olefins. The radical can also react with the now-deactivated catalyst (MOX) to form a dormant chain (black arrow, right). (b) ATRP initiator
(2-hydroxyethyl 2-bromoisobutyrate, HEBIB) and macromonomer (OEOMA500) used in this study. (c) Monomer conversion after 24 h under various
conditions with (white) and without (purple) trace metal mix added to bacterial media. (d) Kinetics of monomer conversion in MR-1 or E. coli culture using
37
Cu(II)-EDTA as catalyst (e) Extracellular Cu(II) reduction monitored with the Cu(I)-specific fluorescent dye CF4. Data show mean SD of three independent
experiments. **P < 0.01 (Reproduced with permission from Fan et al. 2018)
Fig. 5 Electron transfer proteins impact polymerization kinetics. (a) Key proteins involved in
extracellular electron transport in MR-1. (b) Effect of gene knockouts on polymerization activity
using Cu(II)-TPMA. (c) Rescue of normal polymerization activity via complementation with a
plasmid encoding mtrC, using Cu(II)-TPMA as a catalyst. Data show mean SD of three
independent experiments (Reproduced with permission from Fan et al. 2018)
knockout could be augmented with a glutathione knockdown strategy to minimize

reducing capacity beyond the specific pathway of interest. Another strategy would
be to physically or chemically tether copper to extracellular cytochromes using
noncanonical amino acids or metal-binding peptides. This work served as an impor-
tant foundation toward controlling living synthetic polymerizations using synthetic
biology.
An outstanding challenge of ATRP, and indeed many organometallic reactions, is
oxygen quenching in aerobic environments. As a result, most of these reactions must
take place under strictly anaerobic conditions to facilitate control over catalyst redox
cycles. For this purpose, it is convenient that EET is an anaerobic respiratory
mechanism. However, inspired by enzymatic oxygen consumption in aqueous
solutions (Szczepaniak et al. 2021), we further hypothesized that the facultative
nature of S. oneidensis would enable polymerization in ambient environments by

effectively “scrubbing” dissolved oxygen through aerobic respiration, followed by
activation of EET pathways (Fan et al. 2020). We first confirmed that dissolved
oxygen was consumed in our reaction mixtures after inoculation with bacteria and
polymerization reagents (Fig. 6a). The creation of this in situ anaerobic environment
enabled synthesis of well-defined polymers in ambient conditions, using only the
bacteria to overcome oxygen inhibition (Fig. 6b). Aerobic polymerization also
suppressed nonspecific radical production from cell lysate, more tightly coupling
catalyst reduction to continuous EET from S. oneidensis (Fig. 6c). Indeed, increasing
the concentration of bacteria in the reaction correspondingly increased polymeriza-
tion rate (Fig. 6d). In contrast to aerobic ATRP powered by enzymatic oxygen
depletion, microbial aerobic ATRP is a living reaction that tethers facultative cell
metabolism to catalyst reduction. This was further exemplified by the ability to
recycle cells after a reaction to complete multiple polymerization cycles. On the
other hand, cells could also be lyophilized and treated as a chemical reagent added to
an aerobic reaction mixture to initiate a polymerization, highlighting the robust
flexibility of this platform.
This work also developed the large synthetic material landscape available to
S. oneidensis through EET-driven ATRP, as a variety of vinyl monomers were
successfully polymerized (Table 1). This included traditionally challenging hydro-
phobic species such as styrene. In addition, a variety of transition metals, including
iron, ruthenium, cobalt, and nickel could all be reduced by S. oneidensis as catalysts
a b c d
100 100 + Cu(II)-TPMA – Cu(II)-TPMA 100 MR-1 E. coli 2.5
OD = 0.2
Polymerization Rate (h-1)
OD = 0.2, Polymerization
Monomer Converion
Monomer Converion
2.0
75 75 75
(% Saturation)
Dissolved O2
after 2 h (%)
after 2 h (%)
1.5
50 50 50
1.0
25 25 25
0.5
0 0 0 0.0
0 1 2 3 Monomer + + + + HK Lysed HK Lysed
05
1
2
5
0.
0.
0.
Initiator + + +
0.
Time (h) Bacteria MR-1 MR-1 supernatant Aerobic Anaerobic

Inoculating OD600
Fig. 6 S. oneidensis rapidly consumes dissolved oxygen and activates radical polymerization in
cultures for which no additional steps were taken to remove oxygen. (a) Dissolved oxygen in
S. oneidensis MR-1 cultures growing in SBM and under typical polymerization conditions. Under
both conditions, oxygen consumption outcompetes oxygen diffusion. (b) Effect of different bio-
logical and polymerization components on monomer (OEOMA500) conversion under aerobic
conditions. Monomer, initiator, catalyst, and S. oneidensis MR-1 are all required to achieve
significant monomer conversion. (c) Monomer (OEOMA500) conversion using heat-killed
(HK) and lysed E. coli MG1655 or S. oneidensis MR-1 cells under anaerobic and aerobic
conditions. Lysed cells release intracellular reductants that reduce Cu(II) to Cu(I), which activates
polymerization. Under aerobic conditions, adventitious reductants and radicals are quenched by
oxygen. Heat-killed cells do not result in polymerization under either condition as they neither
consume oxygen nor generate EET flux to reduce the metal catalyst. (d) Polymerization rate
constants of OEOMA500 using S. oneidensis MR-1 and Cu(II)-TPMA at varying inoculating
OD600 under aerobic conditions. Data show the mean SD of n ¼ 3 replicates (Reproduced
with permission from Fan et al. 2020)
40
Table 1 Monomer scope under anaerobic and aerobic polymerizations that involve S. oneidensis MR-1. Yields are listed as percentages, followed by
experimental Mn and Ð. The target Mn values for the monomer/initiator of 500:1 were polyOEOMA300 (150 kDa), polyOEOMA500 (250 kDa), polyHEMA
(65.1 kDa), polyNIPAM (56.6 kDa), polyDMAEMA (78.6 kDa), polyMMA (50.1 kDa), and PS (52.1 kDa). Average and SD values were obtained from
n ¼ 3 replicates (Reproduced with permission from Fan et al. 2020)
A. J. Graham and B. K. Keitz
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Engineered Living Materials Wil Srubar

[Wil V. Srubar Iii (Editor)]

Advances in Engineered Cementitious Composite:

Handbook of Nanocomposite Supercapacitor Materials III

Data-centric Living: Algorithms, Digitization and

Obtaining and Characterization of New Materials Volume

Handbook on Synthesis Strategies for Advanced Materials

The Living Word of God Rethinking the Theology of the

House of a Million Rooms 1st Edition Wil Mara

Microbial Bioinformatics in the Oil and Gas Industry:

Engineered Living Materials

ISBN 978-3-030-92948-0 ISBN 978-3-030-92949-7 (eBook)

Network Formation of Engineered Proteins and Their Bioactive

Keywords Protein engineering · Protein network · Protein hydrogel · Bioactive

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

in principle, can be genetically programmed to be produced from living cells

2 Protein Structure and Self-Assembly

2.1 Protein Structures and Their Assembly

Proteins organize themselves into speciﬁc three-dimensional structures. The local

resulting in hydrophobic “face” of the helix. The interaction (burial) of these

of subunits. One of such examples is GroEL, a barrel-shaped molecular chaperone. It

3 Network Formation of Recombinant Proteins

hydrogels, focusing on their network-forming mechanism and some of the physical

3.1 Phase Separation and Aggregation-Induced

Elastin is one of the essential structural components in the extracellular matrix,

3.2 Self-Assembling, Multi-domain Recombinant Protein

Coiled-coil associations have been extensively studied as network-forming motifs

the C block changed the thermal stability, pH responsiveness, and self-association

3.3 Chemically Cross-Linked Recombinant Protein

Chemical cross-linking—creating covalent connections between constituent protein

Photochemical cross-linking is another effective strategy for the generation of

4 Functional Recombinant Protein Hydrogels

Protein engineering offers the unique capability to design bioactive hydrogels, as

telechelic architecture in which a mid-block is sandwiched by self-assembling

and photoresponsive gel-sol transition. The similar architecture of recombinant

5 Future Directions and Challenges

This chapter explored achievements in the ﬁeld of recombinant protein hydrogels in

Austin J. Graham and Benjamin K. Keitz

Keywords Living materials · Synthetic biology · Extracellular electron transfer ·

A. J. Graham · B. K. Keitz (*)

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 27

designed function. However, endogenously produced natural materials suffer from a

Fig. 1 Schematic illustrating an example of a living synthetic polymerization. Biological compu-

recent work toward controlling redox-driven polymerization mechanisms using the

2 Extracellular Electron Transfer Bridges

3 Synthetic Polymerizations Mediated by Microbes

living polymerization. The reaction was well-controlled, producing low dispersity

4 Atom-Transfer Radical Polymerization Powered by

could serve as a general electron transfer mechanism for organometallic reactions

knockout could be augmented with a glutathione knockdown strategy to minimize

nature of S. oneidensis would enable polymerization in ambient environments by

Time (h) Bacteria MR-1 MR-1 supernatant Aerobic Anaerobic

Section 2. Information about the Mission of

Volunteers and financial support to provide volunteers with the

Section 3. Information about the Project

The Foundation’s business office is located at 809 North 1500 West,

Section 4. Information about Donations to

The Foundation is committed to complying with the laws regulating