Biomolecular Sciences Degree Group

Critical Essay

2015-16

CELLULAR LOGIC AND MEMORY: THE USE OF ɸC31 INTEGRASE AND

RELATED SERINE INTEGRASES IN

GENETIC CIRCUIT DESIGN

Supervised by Dr. Sean Colloms

Dylan MacPhail

Matriculation number: 2022896

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ABSTRACT

The serine integrases are a subfamily of phage recombinases which are capable of integrating,

inverting, or excising a segment of DNA between their recognition sites with a high degree of

efficiency. Integration occurs between the phage and bacterial attachment sites (attP/B), and excision

occurs at the resultant attL/R sites to resolve the original state, requiring a recombination

directionality factor (RDF). Inversion of a segment of DNA is also possible by flanking with inverted att

sites. The directionality of inversion can be tightly controlled by expression of an RDF, and thus serine

integrases allow ‘flipping’ of a segment of DNA between two states. Due to the binary nature of

computational logic this control of directionality makes these proteins particularly attractive as a

method of implementing logic and memory into genetic circuit design. This review details aspects of

the origin, structure, and function of the widely utilised serine integrase from bacteriophage ϕC31

and discusses its application in synthetic genetic circuitry.

ABBREVIATIONS

CTD – C terminal domain

LSR – Large Serine Recombinase

NTD – N terminal domain

RAD – Recombinase addressable data

RDF – Recombination Directionality Factor

INTRODUCTION

The field of biology is vast and diverse, and billions of years of trial and error through evolution

have yielded multifaceted designs integrating a near infinite assortment of complex

functionalities. It is therefore not surprising that humans often look to biodiversity to find

inspiration when engineering new materials. One area where this has traditionally not been the

case however is circuitry and computing; an industry which has grown exponentially since its

conception. Despite the vast complexity now achievable by electronics, biology still has much

inspiration to offer to this field in terms of robustness and redundancy in complex networking

(George et al., 2003).

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The fact that all biology, on the cellular level, is a product of complex interactions with

and information storage in DNA is too often overlooked. At this level all events can be viewed

as the result of the labyrinthine circuitry of promoters, repressors, activators, and genes which

operate in harmony to produce all components of an entire organism from the same basic DNA

programming. This is achieved through developmental and regulatory switches which are not

dissimilar in function to those found in modern electronics (reviewed by Bonnet and Endy,

2013).

In computing information is stored and processed using binary algorithms in which the

basic unit is one bit. One bit of information represents a switch for which there are two possible

states: 0, or 1. Two bits of information therefore represents the number of switches needed to

record one of four possible combinations (00, 01, 10, or 11), while three bits of information can

record double this amount of patterns (000, 001, 010, 011, 100, 101, 110, or 111). The number

of possible states doubles with each bit of ‘memory’ added to the system (n bits = 2n states) such

that 8 bits represents the capacity to store one of 256 possible patterns of 0 and 1; this is known

as one byte, which can instruct the display of one of 256 characters, numbers, or colours; or

performance of one of 256 possible actions (Horowitz and Hill, 2015). DNA, however, is not a

binary system because each position can be occupied by one of four possible bases, thus

allowing each base to represent one possible combination of a 2-bit system. If the information

storage capacity of DNA were to be fully exploited the region of DNA required to hold the

average bacterial gene (1,100bp) (Parakhia, 2010) could store 2,200 bits of information, or 275

bytes.

The capacity for DNA to securely store information in a stable state is the foundation of

a new field in information technology. Goldman and co-workers (2013) have demonstrated the

in vitro storage and recovery of 739 kilobytes (739x103 bytes) of information in DNA. DNA

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cryptography seeks to fully utilise DNA as a stable, low cost storage and encryption tool for

sensitive information which does not require energy input to maintain (Jacob and Murugan,

2013). This information is written into the DNA by chemical synthesis and read by sequencing

however, and as such is not suitable for use in the autonomous regulation of gene networks

within cells through in vivo read and write functions.

With the fields of synthetic biology and genetic engineering expanding rapidly

researchers are searching for methods of increasing control over gene networks such as

metabolism, and as such have begun looking at electronics and computing for the answers they

need. Integration of memory into a circuit (a non-transient switch of state in response to a

certain input) allows programmability as both transient and ongoing inputs can inform the

output of a circuit, while layering of circuits such that the output of one circuit is an input for the

next allows complexity to be achieved (Moon et al., 2012). Circuits which can modulate their

binary output based on the states of two distinct binary inputs demonstrate Boolean logic, for

which a full range of functions can be seen in Table 1.

Such programmable gene networks utilise a variety of methods stimulating promotion

and inhibition of genes and memory within the circuit (reviewed by Brophy and Voigt, 2014)

including protein-protein interactions (Moon et al., 2012; Stricker et al., 2008), protein-DNA

Table 1: Truth table showing the full range of Boolean Logic Functions. Output is determined by the

binary state of two inputs, A and B. The pattern of outputs for each possible input is dependent on

the architecture of the ‘logic gate’ which they inform.

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interactions (Lohmueller et al., 2012), RNA – based methods (Liang et al., 2011), and even use

of the recently discovered CRISPR-Cas system (Bikard et al., 2013; Mimee et al., 2015). These

circuits often encounter problems during development not only due to their design, but also

their context within living cells where interaction with other proteins and their high demand for

cellular resources in order to function can impair both the health of the host organism, and the

stability and predictability of the circuit (reviewed by Brophy and Voigt, 2014; Cardinale and

Arkin, 2012). This has led to a drive for the standardisation of components for synthetic biology

applications akin to a ‘parts list’ for electronic circuitry, as well as development of the

computational tools necessary to design and build predictable genetic programming (Rodrigo

and Jaramillo, 2013).

Recombinases in particular have become particularly useful as tools for genome

engineering and synthetic biology in recent years due to their ability to mediate the conservative

inversion, integration, or excision (resolution) of large segments of DNA, allowing efficient

cloning and DNA modification in vivo (reviewed by Fogg et al., 2014). Examples of recombinases

include invertases, resolvases, and integrases; classifications related to their native biological

function. Serine Integrases, a subfamily of the Large Serine Recombinases (LSRs), could be

enormously valuable in genetic circuit design. These are particularly useful due to their

predictability within a vast range of cell types, as well as the low-maintenance cost in terms of

cellular resources, small circuit size, and heritability associated with building circuitry directly

into DNA (reviewed by Fogg et al., 2014). By manipulating their attachment (att) sites phage

integrases can be made to controllably and reversibly invert DNA such that the coding strand is

non coding, or change the directionality of a gene promoter. The binary nature of this

manipulation makes them particularly amenable to the implementation of logic, memory, and

programming in living cells. This review will discuss both structural and functional aspects of the

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ϕC31 Serine Integrase and its relatives as well as their current applications and future potential

in integrating logic and memory into synthetic gene networks.

INTEGRASES IN CONTEXT

Temperate bacteriophages encode an archetypal genetic switch which allows them to cycle

between lytic growth and dormant lysogeny within prokaryotic cells in response to changing

environmental cues (reviewed by Fogg et al., 2014; Oppenheim et al., 2005). During lysogeny

lytic genes are suppressed, the phage genome is not transcribed and is replicated within a

specific site of the host genome, and lysogens are immune to superinfection (Oppenheim et al.,

2005). In 1962 Campbell was the first to suggest that the λ prophage is integrated into the host

genome at a specific site by ‘recombination’ during the lysogenic switch (Campbell, 1962). λ

integrase is as such the best understood of the phage integrases, a subset of recombinases

characterised by their ability to mediate both the host integration and excision of prophage DNA

from the host genome under separate conditions (Esposito and Scocca, 1997).

λ integrase is a tyrosine recombinase, one of two large families of recombinase (the

other being the serine recombinases), which are named after the conserved residue within their

active site used to form a covalent phospho-linkage with the DNA backbone during

recombination (reviewed by Fogg et al., 2014). Integrases insert phage DNA into host genomes

by binding the phage DNA attachment site (attP) and the attachment site of the bacterial host

DNA (attB), mediating a recombination reaction in which the end product is integrated phage

DNA flanked by the left and right attachment sites (attL & attR) (Campbell, 1992). These each

consist of one half of attP and one half of attB with a small overlap of complimentary nucleotides

in the middle. This process is unidirectional, and so in order to excise the phage DNA and resolve

the prophage (attL and attR recombination) an excisonase or recombination directionality factor

(RDF) is required (Lewis and Hatfull, 2001). Bacteriophage λ encodes the excisonase Xis, which

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it uses to mediate phage resolution, however like all tyrosine integrases a range of host cofactors

are also needed for both integration and excision of the λ prophage (Lewis and Hatfull, 2001).

Naturally att sites are direct repeats when flanking a sequence, however inversion of one att

site such that they are palindromic allows a section of DNA to be ‘flipped’ using a recombinase

(see Figure 1).

The phage encoded serine integrases belong to the LSR family which the retain catalytic

recombinase N terminal domain (NTD), but all exhibit a large, structurally diverse C terminal

extension of around 300-500 amino acids (aa) compared to a typical C terminal domain (CTD) of

40aa in other serine recombinases (Smith and Thorpe, 2002, Smith et al., 2010). While tyrosine

integrases require host co-factors for integration or resolution to occur, serine integrases do

not, and need only their cognate RDF for excision (Smith et al., 2010). In addition to this the

serine integrases also require less genomic space for their att sites, which are both <50bp and

consist of a core TT crossover site flanked by two quasi-symmetrical inverted repeats (Thorpe et

al., 2000). This is compared to >200bp attP, <30bp attB in tyrosine integrases (see Figure 2).

Serine integrases also have no topological restrictions on their att sites, while λ integrase

requires attP to be supercoiled (reviewed by Fogg et al., 2014; Smith et al., 2010).

Properties such as these make Serine integrases like the integrase (Int) from the

Streptomyces phage ϕC31 attractive targets for applications in synthetic biology and genome

Figure 1: att site positioning effects function. Integration causes the attL and attR sites to be arranged

in a directly repeating conformation, allowing excision when Int is expressed with the RDF. If att sites

are arranged to be inverted repeats inversion occurs when Int or Int + RDF are expressed.

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engineering due to the smaller sequence requirement to encode all factors needed for in vivo

activity. ɸC31 Int is among the most studied of the serine integrases; first described in 1991

(Kuhstoss and Rao, 1991), it was extensively utilised to mediate recombination within

Streptomyces strains, and also found use as a reliable unidirectional recombinase within other

prokaryotic and eukaryotic cells (reviewed by Smith et al, 2010). While the first RDF for a serine

integrase was discovered for Bxb1 in 2006 (Gosh et al., 2006), the RDF for ϕC31 Int, gp3,

remained elusive until 2011 (Khaleel et al., 2011); a discovery which finally allows full utilisation

of ϕC31 Int as a reversible integrase in synthetic biology applications.

STRUCTURE AND MECHANISM OF THE ΦC31 INTEGRASE

ɸ C31 Int is a 613 amino acid protein which mediates the unidirectional recombination of attP

and attB sites with high specificity (Kuhstoss and Rao, 1991). ɸC31 Int is a dimer in solution, and

binds each att site as such, while studies in the related serine integrase TP901-1 suggest that

both sites are then brought together to form a synaptic tetramer (Yuan et al., 2008). Unlike the

synapsis mechanism of the tyrosine integrases, which relies upon the formation of a Holliday

Junction – like intermediate (reviewed by Fogg et al., 2011), ϕC31 Int and the Serine integrases

cause a staggered double stranded break in the crossover region of each site which leaves a two

Figure 2: att site structure comparison between tyrosine and serine integrases. While Tyrosine

integrases recognise a smaller attB site, their attP site is many times larger than that of the serine

integrases, and contains binding regions for integrase, RDF, and host cofactors. The overlap region of

the tyrosine integrases is also larger.

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base pair overhang of Thymine or Adenine bases at each half site (reviewed by Fogg et al., 2011;

Smith and Thorpe, 2002; Smith et al., 2010). Following synapsis, it is thought that a right handed

‘gated rotation’ mechanism rotates each half site 180° relative to the other within the synaptic

tetramer and the overhanging bases are re-ligated and released, forming the attL and attR sites

(Olorunniji et al., 2012). Figure 3 shows the methods of synapsis and strand transfer used by

tyrosine and serine integrases.

Figure 3: Synapsis methods of phage integrases.

(A) Tyrosine integrases bind attP and attB sites forming a synaptic tetramer, and make a single

stranded ‘nick’ in each site (at black arrowheads) by attack with their catalytic residue. The

free 5’OH then attacks the 3’ phospho-tyrosine of the opposing att site nick, knocking off the

integrase monomer bound to it and forming a Holliday junction-like intermediate. This is then

repeated by the remaining integrase subunits (at white arrowheads) to fully integrate the

phage.

(B) Serine integrases also form a synaptic tetramer, however they attack all strands

simultaneously (black arrowheads), causing a double stranded break in each genome with a

two base pair overhang. Bound tightly to the integrase monomers by their 5’ phospho-serine

linkage, strands are rotated as each integrase dimer rotates 180° with respect to one another.

The free 3’OH of each strand then attacks the phosphoserine of the neighbouring half-site,

completing integration.

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Mutation of the key active site Serine in the NTD, S12A, abolishes the ability of ϕC31 Int

to induce strand transfer, however synapsis still occurs (Rowley and Smith, 2008), suggesting

regulation is not coupled to catalysis. Sequence alignment has shown the NTD of ϕC31 Int to be

homologous to those of other recombinases (Rowley and Smith, 2008), however only

unpublished data exists for the crystal structure of ϕC31 Int NTD (McMahon et al., 2013). Figure

4 shows the crystal structure of the activated transposon serine recombinase γδ resolvase which

clearly indicates the interface at which gated rotation might occur, and the crystal structure of

Figure 4: Evidence supporting gated rotation of ϕC31 tetramer

(A) Amino acid sequence alignment of ϕC31 Int against γδ resolvase and TP901-1 Int. Conserved

residues are highlighted, showing homology between proteins. Alignment was generated

using CLUSTAL Ω sequence alignment tool.

(B) Adapted from Yuan et al. (2008). Tetramer of activated γδ resolvase NTD bound to DNA.

Interface for rotation can be clearly seen and is indicated by arrowheads.

(C) Adapted from Yuan et al. (2008). Tetramer of TP901-1 Int NTD (unbound) showing structural

similarity to γδ resolvase tetramer. Arrowheads indicate possible rotation interface.

(D) Monomer of TP901-1 Int NTD analogous to subunit II in (C). Structure is coloured in a

spectrum with blue representing the N terminus, and red representing the C terminus.

Monomer was isolated from crystal structure of a tetramer (Yuan et al., 2008). PDB ID: 3BVP.

Image was generated using The PyMOL Molecular Graphics System, Version 1.7.2.2

Schrödinger, LLC.

(E) Monomer of ϕC31 Int NTD showing similarity to TP901-1 Int. Structure is coloured in a

spectrum with blue representing the N terminus, and red representing the C terminus. NTD

was isolated from unpublished crystal structure of N terminal and recombinase domains

(McMahon et al., 2013). PDB ID: 4BQQ. The largest difference between (D) and (E) is the

orientation of the red helix, which may be due to differences in the structures from which the

segment of protein shown was isolated. Image was generated using The PyMOL Molecular

Graphics System, Version 1.7.2.2 Schrödinger, LLC.

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a tetramer of the serine integrase TP901-1 Int which is consistent with this being the mechanism

used by serine integrases (Yuan et al., 2008). While it has been suggested that multiple rounds

of rotation are possible within other Serine integrases (Bai et al., 2011) this was demonstrated

to be unlikely during the canonical action of ϕC31 Int as long as the two core att site base pairs

match (Olorunniji et al., 2012).

The default function of ϕC31 Int is therefore recombination of the attP and attB sites,

and band-shift assays by Thorpe, Wilson, and Smith in 2000 showed that attL and attR

recombination (resolution) could not be mediated by ϕC31 Int alone. The mechanism by which

this directionality is controlled is poorly understood, and while it is now known that the RDF gp3

is needed for attL/attR recombination, the CTDs of serine integrases are also known to play a

large role in this regulation (Rowley et al., 2008; McEwan et al., 2009; reviewed by Smith et al.,

2010; Fogg et al., 2011; Rutherford and Van Duyne, 2014). Indeed, it has been shown that a

single amino acid substitution in the CTD of ϕC31 Int, E449K, produces a hyperactive Int which

can not only catalyse attP/attB recombination, but also attL/attR, attL/attL, and attR/attR, and

could still mediate formation of these synapses in the background of an S12A mutation (Rowley

et al., 2008). Several of such hyperactive mutants, including Int E449K, were identified in a coiled

coil domain on the CTD, a motif which commonly mediates protein-protein interactions.

Furthermore, experiments with the purified histidine tagged CTD show that while the CTDs

alone are monomers in solution they interact co-operatively to bind att sites, and that L460P

and Y475H mutations abolished inter-CTD interaction, DNA binding, and synapsis (McEwan,

Rowley and Smith, 2009). The E449K mutant in the isolated CTD could bind DNA, but could not

catalyse synapse formation, suggesting that formation of a CTD synapse is not essential for ϕC31

Int binding, but is intimately involved in the control of directionality.

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Although the full role of the RDF in this directionality remains poorly investigated, it is

likely that gp3 plays a structural role in the interaction, conferring a conformational change to

the CTD which allows resolution of the recombination sites. It has been shown in the serine

integrase Bxb1 that RDF binds Int attached to the attP/attB sites tightly and inhibits

recombination, while promoting excision at attL/attR sites (Ghosh, Wasil and Hatfull, 2006).

Without crystal structures for the full ϕC31 Int the mechanism of its RDF will likely

remain difficult to elucidate. Another area which remains cryptic is the mechanism of action of

the DNA binding domain(s) within this protein. Site-directed mutagenesis of conserved residues

within the CTD has indicated a cysteine rich motif and a valine rich motif to be important in DNA

binding (Liu et al., 2010), however the mechanism of this interaction has not been defined. The

cysteine rich motif in the CTD is a putative zinc finger domain (McEwan et al., 2011). Rutherford

and Van Duyne (2014) hypothesise that the specific orientation of the Int on each half site

Figure 5: Proposed roll of coiled coil domains in directionality of serine integrases. Adapted from

Rutherford and Van Duyne (2014). Binding of Int dimers to att sites positions coiled coil domains on

either side of the DNA. These domains then form inter-dimer interactions, and following rotation and

ligation all coiled coil domains are on the same side of the recombined att sites. When a dimer is bound

to a recombined att site in the absence of an RDF following tetramer dissociation the coiled coil

domains form intra-dimer interactions which prevent reformation of the tetramer without the RDF.

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conferred by the zinc finger domains allows the CTD coiled coil motifs to interact in an inhibitory

manner upon synapsis such that resolution is not possible without the RDF (see Figure 5).

The mode of DNA interaction is of particular interest in synthetic biology applications as

knowledge of this could allow production of integrases which could specifically bind to

endogenous sites, and also improvement of the existing specificity. It has been shown that ϕC31

Int can be targeted to non-native pseudo-sites which resemble att sequences (Combes et al.,

2002; Malla, 2005; Chalberg et al., 2006), and directed evolution through DNA shuffling has

been reported to have yielded versions of the ϕC31 Int protein which have a high binding

specificity and frequency to a pseudo attP site on human chromosome 8, as well as versions

which integrate more efficiently to pre-inserted sites within the human genome (Sclimenti,

2001; Keravala et al., 2008). High specificity targeting of recombinases has also been

demonstrated using chimeric Zinc finger domains (Akopian and Stark, 2005), however this has

not been demonstrated in ϕC31 Int, and would likely affect the regulation of the integrase.

While integrases can be targeted to ‘landing’ sites introduced into eukaryotic genomes

(reviewed by Fogg et al., 2011), concerns have been raised over the efficiency of ϕC31 Int action

within eukaryotic cells despite its widespread usage for this purpose. Inversion of the DNA

between palindromic att sites introduced using this method was used to re-arrange segments

of the human Y chromosome using ϕC31 Int (Malla, 2005), however it was found that

recombination only occurred in 56% of cells, and in some cases the action of Int had left deleted

regions of DNA, or small insertions attributed to intervention of host double stranded break

repair mechanisms such as non-homologous end joining. This would suggest that the synaptic

complex is around for a much longer time in eukaryotes, and is consistent with findings that

although ϕC31 Int can perform integration with 100% efficiency at pseudo attB sites in E. Coli

and other prokaryotes, integration is only 50% efficient in human cells (Chalberg et al., 2006),

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and efficiency in eukaryotic cells varies between organisms. It is possible that bacteriophage

integrases are not well suited to the nuclear environment of eukaryotic cells. In addition to the

lesions observed due to host repair, the efficiency of ϕC31 Int within human cells could be mildly

impaired by interaction with endogenous cell death associated protein DAXX (Chen et al., 2006).

Interestingly, this interaction occurs within the same region of the ϕC31 Int CTD as the putative

coiled coil domain. Despite this hurdle ϕC31 Int continues to be used in eukaryotic cells due to

the ability to predict insertion sites, and reliably preform integration more efficiently than

tyrosine recombinases and other methods of genomic insertion such as homologous

recombination, resulting in long lasting expression of gene products (reviewed by Fogg et al.,

2011).

The reliability of ϕC31 Int in prokaryotic cells and utility in eukaryotic cells, as well as the

reversibility conferred by use of gp3, allows it to be useful in many emerging biotechnologies.

One such advancement is a mechanism for fast and accurate plasmid assembly pioneered by

Colloms and colleagues (Colloms et al., 2013). The SIRA (Serine Integrase Recombination

Assembly) method for plasmid assembly relies on their findings that the two base pairs of the

core att site crossover region do not require conservation so long as both are the same. This

means that one ϕC31 Int enzyme can assemble multiple regions of DNA in an order pre-

determined by careful arrangement of att sites with different crossover regions flanking the

DNA. Through this method a cassette of up to six gene segments can be assembled in one

reaction using different combinations of core nucleotides, abolishing the traditionally arduous

process of using restriction enzymes and ligases in separate reactions for each inserted section

of DNA. In addition to this, any number of the segments can be removed, or replaced entirely

by a previously assembled cassette, or any expanse of DNA flanked by the appropriate

recombination sites. Furthermore, addition of further serine integrases will allow more

fragments to be assembled simultaneously. This technology vastly expands the scope of genetic

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circuit design as any number of functional units can be easily integrated or changed in few steps.

Rates of transcription can also be predictably controlled by varying the distance between a gene

and its promoter, or the positioning of an inhibitory genetic signal within the assembled array.

SIRA assembly demonstrates the extensive utility of ϕC31 Int in synthetic biology and adds to a

growing list of advantages for Int use over many of the traditional methods used for genome

engineering

ENGINEERING LOGIC AND MEMORY IN GENETIC CIRCUITRY

In terms of genetic circuit or metabolic design assembly of components by means of the

SIRA method allows greater predictability of gene expression. This could aid the mathematical

design of genetic circuits and metabolic pathways by computational methods, as the functions

of components which utilise binary and Boolean logic can be predicted efficiently (reviewed by

Brophy and Voight, 2014). Additionally, the use of differing base pairs in the crossover region of

the recombination sites could allow control of multiple outputs simultaneously by one input

which drives ϕC31 Int expression with or without its RDF. Although genetic circuitry assembled

in such a fashion could be highly predictable it is however unlikely that such ‘cellular machines’

will ever achieve widespread use as supercomputers due to the existing utility of such electronic

constructs, although it is worth noting that biochemical networks are capable of Turing Machine-

like functions and can compute large and complex calculations in as little time as simple ones

(Hjelmfelt, Weinberger and Ross, 1991).

Although the aforementioned SIRA assembly method technique efficiently utilises

serine integrases for circuit construction, the full potential of these proteins in this application

can be realised by using them as the functional units of the circuit. Recombinase based

approaches to biocomputation and gene control mostly utilise the ability of these proteins to

flip a section of DNA between palindromic attP and attB sites, or attL and attR sites. This ability

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therefore changes the sequence of DNA in a non-energy dependant, and importantly heritable

and highly efficient way. This allows the recording of, and response to different stimuli, as well

as binary and Boolean logic functions, to be encoded on a vastly reduced piece of DNA real estate

than use of genetics and biochemical pathways alone permits (reviewed by Brophy and Voight,

2014; Fogg et al, 2011).

One of the most important components which must be implemented into complex

circuitry is memory. Memory allows a sustained or heritable response to transient stimuli within

a circuit, and can permit a response which is informed by multiple sequential inputs (reviewed

by Brophy and Voight, 2014; Horowitz and Hill, 2015). Due to their ability to invert a segment of

DNA between two states, memory is an inherent aspect of the recombinases, and has been

achieved within bacterial cells (Ham et al., 2008; Yang et al., 2014). The ability of serine

integrases such as those from ϕC31 and Bxb1 to controllably invert DNA without the need for

host co-factors or large att sites, however, gives them an edge in this application (Bonnet et al.,

2012; Bonnet et al., 2013; Siuti et al., 2013).

While a single recombinase can be used to achieve digital memory with a 1-bit capacity,

(Bonnet et al., 2013; Siuti et al., 2013), layering of the sites for different recombinases allows

complex memory of order, or number, of inputs in complex ‘state machines’ which fit into a

stretch of DNA smaller than the average gene (Ham et al., 2008; Bonnet et al., 2012; Yang et al.,

2014). Recombination addressable data (RAD) modules were designed by Bonnet and co-

workers (Bonnet et al., 2012) which implemented the serine integrase from Bxb1 as a unit of

reversible memory within cells. Ham and colleagues suggest that 10 recombinases with

overlapping RAD modules could form 1010 possible states of DNA thus recording 1010 different

patterns of input from 10 signals (Ham et al., 2008). Bonnet and co-workers estimated that using

RAD modules construction of an 8bit memory system with 1 byte of memory capable of counting

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256 input pulses would require 16 recombinases (Bonnet et al., 2012). Yang and colleagues,

however, have demonstrated recording of 1.375 bytes of information using 11 different RAD

switches designed using serine integrases discovered through data mining for homology to ϕC31

Int (Yang et al., 2014). This is a gargantuan achievement in context as previous studies had only

demonstrated a maximum memory capacity of 2 bits (Bonnet et al., 2013; Siuti et al, 2013).

Although it can be argued that it does not utilise the full 2 bit / base pair capacity of DNA, the

utility of this method of memory is much greater as it can be used in rewritable storage which is

able to record data in living cells, as opposed to the single write functionality of DNA

cryptography (Ham et al., 2008; Goldman et al., 2013). Durational recording has also been shown

to be possible through genetic manipulation, however recording of this information occurred at

the population level in a form of analogue memory based on the increasing number of

responsive cells (Farzadfard and Lu, 2014). This process would be difficult to replicate using

recombinases due to their high efficiency and digital nature, and moreover a population

response would not be amenable to circuit design.

Readouts from large memory modules such as those described above requires

sequencing, digestion, or PCR; however smaller memory modules can also feasibly be

interpreted using fluorescence. The full potential of such memory is only realised however when

it is implemented into genetic circuitry through incorporation of active genetic elements,

permitting logic within the circuit as opposed to single input response (reviewed by Brophy and

Voight, 2014). The ability for logic in recombinases through use of a 2-bit system was exemplified

in 2008 by the ability of bacteria to survive on antibiotic when the associated resistance gene

was not expressed until the constitutively expressed Hin recombinase it encoded (which does

not require an RDF for reversion) was able to solve the Burnt Pancake Problem (Haynes et al.,

2008). The Burnt Pancake Problem is a logic problem whereby a stack of ‘Pancakes’ (DNA flanked

by hix sites in this case in this case) must be sorted into the correct order and each manipulation

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reverses the order of one or more ‘pancakes’ within the stack (see Figure 6). While the involved

recombinases were constitutively expressed in this experiment, and did not require an RDF, it

demonstrates the possibility that multiple recombinases with expression under the control of

different inputs could mediate output only in a specific combination. This demonstration is also

potentially useful in durational memory as the duration of a specific input driving Int expression

could be estimated based on the minimum number of random inversions in a stretch of DNA

with specific recognition sites needed to reach the observed configuration form the starting

sequence (Haynes et al., 2008). Since this demonstration a full range of all Boolean logic

functions has been achieved in 2 bit systems using the TP901-1 and Bxb1 integrases, and the

Figure 6: Solving the burnt pancake problem with integrases. In this logic problem a stack of

pancakes is presented which all have one good side facing up (solid colour in figure), and one burnt

side facing down (hashed colour in figure). The stack is the wrong way around, and the aim is to sort

all pancakes in the stack so that they are arranged from smallest (on top) to largest, all with burnt

side facing down. If the red ‘pancake’ represents an antibiotic resistance gene, and the purple

‘pancake’ is a promoter (both flanked by att sites) then the minimum amount of flips required for gene

expression represents the quickest solution (3 flips in this case) to the burnt pancake problem. Using

att sites with different overlap regions (arrowheads) one serine integrase (and its RDF) could solve this

logic problem in cells grown on antibiotic. If three separate integrases were used on their cognate att

sites (arrowheads) each flip could be controlled by a separate input.

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ϕC31 and Bxb1 integrases respectively in a

genetic device termed ‘the transcriptor’ by

Bonnet et al. (See Figure 7) (Bonnet et al.,

2013; Siuti et al., 2013).

With the ability of serine integrases

to perform logic and memory functions in

living cells demonstrated as described above

achieving any function needed for a desired

gene network is a matter of combinatorial

design using these components and other

known mechanisms of gene regulation

(reviewed by Brophy and Voight, 2014; Fogg

et al., 2011). For example, an important

function in some electronic circuits can be

oscillation of the output signal and this was

achieved in bacteria through negative and

positive biochemical feedback loops

(Stricker et al., 2008). This could

theoretically be achieved through

expression of a serine integrase which flips a

promoter driving the expression of either

the desired oscillatory gene when flanked by

attP/B sites, or the cognate RDF when

flanked by attL/R sites. Oscillation period

could be extended via targeting of the RDF

Figure 7: Creation of all Boolean logic gates using

two serine integrases. Adapted from Bonnet et al.

(2013). By using a one stranded transcriptional

repressor (red/grey T) and a constitutive promoter

(green arrow) all Boolean logic gates can be

created. These logic gates are operated by two

serine integrases which recognise a distinct set of

att sites (blue and yellow or black and white

arrowheads) and flip the DNA between them,

hence modulating the polymerase flow on each

strand of the DNA.

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mRNA by a complimentary non-coding mRNA, which could be controlled independently (see

Figure 8).

Logic gates operated by serine integrases could be layered in any combination with any

number of downstream effects and feedback loops, integrating any number of other genetically

encoded genomic tools, in order to perform an almost limitless range of specific functions within

cells. These simple genetic components integrate the potentials of computational logic and

memory with synthetic biology, thus allowing programmability for a wide range of highly

predictable functions to an extent which is far beyond the scope of anything achievable in this

field by any other currently known mechanism.

Figure 8: Concept for a synthetic gene oscillator using a serine integrase. Oscillation of a gene of

interest (GOI) is controlled by a promoter within inverted attP/B sites. Expression of the Integrase

(which could be constitutively expressed or under controlled expression) flips the promoter, turning

the GOI expression off. This activates transcription of the RDF, which allows the circuit to be reset.

Oscillatory period, and the state of the circuit, can be modified by controlled expression of a microRNA

which targets the RDF mRNA, or control of integrase expression.

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CONCLUSION

This review has discussed aspects of the origin, structure, and function of the serine integrases

with a specific focus on the integrase encoded by bacteriophage ϕC31, establishing them as

powerful tools for synthetic biology and specifically for engineering complex and programmable

behaviours within living cells. The reliability and specificity of these proteins not only allows

efficient site specific integration to occur within cells with higher proficiency than methods

which do not utilise recombinases such as digestion/ligation, but also permits utility beyond

what is capable of traditional recombinases as they do not rely upon host cofactors and

directionality may be controlled. Though larger than their tyrosine relatives, the serine

integrases also allow more efficient use of DNA by requiring recognition sites which can be one

third of the size of those needed by tyrosine recombinases.

ɸC31 integrase mediates recombination in a vast range of organisms, permitting its

prominence in the field of genetic circuit design. It is worth noting however that this integrase

has been reported to be only half as efficient in eukaryotic cells as it is in prokaryotes (Chalberg

et al., 2006), and thus more research is needed in order to improve its eukaryotic stability. Its

modes of recognition and DNA binding are also poorly understood, and thus further study is

needed due to the extensive efficacy which could result from knowledge of how to reliably

target ϕC31 Int to exogenous binding sites. Both of these areas of research would be enormously

aided by complex knowledge of the structure of this protein, however a crystal structure is

elusive.

The ability to predictably programme genetic expression can benefit all fields of

synthetic biology, and will only grow as research continues into this area. Serine Integrases have

been shown to demonstrate all of the components needed for control of a genetic circuit, and

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future research which integrates the use of these proteins with existing methods of control (or

focuses on mimicking the results of these other methods) could allow the construction of a

genetic circuit to control any biological application. Now that predictability can be built into such

circuits computationally aided design should make the proposal and implementation of such

devices relatively swift and straightforward.

OUTLOOK

Further research into the ϕC31 Integrase and other serine integrases is likely to revolutionise

the field of genomic engineering. The application of SIRA in rapid pathway assembly has already

demonstrated the utility of a ϕC31 Int in this area, however the continued discovery of more of

such proteins theoretically extends the capacity for memory and logic achievable in synthetic

circuits by 1 bit for every serine integrase incorporated. The construction of such large and

complex circuitry could one day lead to the production of a completely reprogrammable,

entirely integrase-controlled organism, however more realistic applications are likely just

around the corner.

Using serine integrases cells could be made to re-organise chromosomes; delete segments

of their genomes (including all synthetic circuitry); change lineage via expression of master

transcription factors; deliver drugs only in diseased cell states; cycle through production phases

in industry; indicate and record pollution levels; optimise crops to their environment; and so

much more in response to transient or lasting stimuli – be it complex or simple. Serine integrases

could become the standard unit of biological programming such that circuits could be designed

by computers with minimal human input, resulting in the production of a linear DNA segment

to mediate any reaction, and needs only be incorporated into target cells (possibly using

integrases). Predictable genetic manipulation may one day dominate the needs of the industrial,

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medicinal, agricultural, and public sectors, and it is likely that ϕC31 Int and other family

members will lead the way into this new era of synthetic biology: predictable genetic circuitry.

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LOG OF INVESTIGATION

This review began to form when I first decided I wanted to write about something within

the scope of synthetic biology, as I have a keen interest in the idea of modulation in

biological manipulation as a technological asset. I enjoy the possibility that in the future

biological research could focus on ‘plug and play’ manipulation of genomes facilitating

design of complex networks for new applications.

After looking through the list of university staff who worked in this area at

http://www.gla.ac.uk/researchinstitutes/biology/research/syntheticbiology/staff/ and

reading through the research interests of each staff member I sent an email to Dr. Sean

Colloms on 06/10/15 detailing my interest in synthetic biology and particularly biological

circuitry and chassis organisms. In this email I asked if Dr. Colloms would be willing to

supervise me in writing my critical review and suggested we should meet to discuss this

further.

I met with Dr. Colloms on 15/10/15 and we discussed the power of serine integrases in

genetic circuit design and some key concepts in this area. This meeting solidified the

topic of review, and I left with a list of papers to read which Dr. Colloms had provided:

(Bonnet et al., 2012; Bonnet et al., 2013; Bonnet and Endy., 2013)

On 18/10/15 I submitted “Genetic Circuitry: The Use of Serine Integrases in

Synthetic Logic and Memory” as the working title of my critical review.

In the months that followed I gathered resources with which to write a review of my

chosen topic. Some of the research papers used herein were identified through reading

the primary literature, while others were discovered through internet searches using

Google Scholar and various databases such as PubMed. The work of Professor Maggie

Smith of the University of York proved exceptionally useful when researching Integrase

structure and function.

ɸC31 integrase continued to crop up during my research as a well-utilised serine

integrase, however much about the specific mechanism of its directionality remained

unknown and its RDF was late to be discovered. This interested me as a better

understanding of this mechanism would enhance its utility in genetic circuit design,

however most current applications utilised the integrase for unidirectional integration

into host genomes.

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The use of this integrase in both the SIRA assembly mechanism (Colloms et al., 2013)

and the implementation of Boolean Logic Gates using serine integrases (Situi et al.,

2013) positioned the protein at the cutting edge of the genetic circuitry and synthetic

biology fields, and cemented the focus of this review.

By early January 2016 I had a solid idea of the shape which this review was going to take

and began planning to write specific sections.

A draft version of the review was sent to Dr. Colloms on 18/02/16 for feedback

I met again with Dr. Colloms on 24/02/16 and we discussed his feedback on my review.

I left with plenty of useful suggestions to improve the review. Notably, Dr. Colloms

suggested areas where figures were necessary, and introduced me to a paper which

suggests the role of the CTD coiled coil motifs in Int directionality (Rutherford and Van

Duyne, 2014).

Having incorporated the feedback of Dr. Colloms into the review and also having proof-

read and edited it in some areas, the review was finally submitted to the school office

on 07/03/16.

While there are many reviews which describe emerging applications for phage

integrases, this review focuses directly on genetic circuits integrating logic and memory,

specifically in the context of a serine integrase which is at the forefront of this

technology. As such, this is a unique piece of work which explores a new field of

biotechnology with specific focus on one small group of proteins which are likely to

revolutionise the possible applications of this research.

ACKNOWLEDGEMENTS

I would like to thank Dr. Colloms for his patience and sound advice when conceiving and

reviewing this document. None of this would have been possible without his input.

I would also like to thank my partner Sarah for her continued support and understanding while

writing this review.

My family has also been incredibly patient and understanding of the time commitment it took

to prepare this piece of work.