iGEM 2024 — Team evry-paris-saclay


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{% block title %}Engineering Success{% endblock %}
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# ENGINEERING

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The PHAGEVO solution is an improved directed evolution technology that combines the strength of phage-assisted
continuous evolution (PACE) and Evolution.T7, a continuous selection system with auto-amplification of the best fitted
variants for the former **[Liu *et al.*, 2010]** and a targeted *in vivo* mutagenesis system
focusing evolution on a desired gene for the latter **[[https://2021.igem.org/Team:Evry\_Paris-Saclay](https://2021.igem.org/Team%3AEvry_Paris-Saclay%5D)]**.

Based on the latest improvements of the PACE technology, notably the non-continuous version of it (PANCE) and the use
of gene VI as a selection marker, we first focused on building the selection phages strain incorporating the
Evolution.T7 cassette, a synthetic *Escherichia coli* chassis adapted for PHAGEVO, the accessory and mutagenesis
plasmids required for a versatile PHAGEVO platform that could be used for *in vivo* directed evolution of a protein of
interest.

For the proof of concept of our system, we all agreed on searching for a protein that could have a positive impact on
sustainable development. The successful candidate was XylS, a transcription factor that was recently engineered toward
detection of the plastic degradation products phthalic acid (PA) and terephthalic acid (TPA) **[Li *et
al.*, 2022]**. Our goal was to evolve two XylS mutants already reported to have the ability to detect
PA and TPA (K38R-L224Q and W88C-L224Q, respectively) in order to get better variants with
optimized PA or TPA biosensing properties.

This page describes our strategic choices, our designs and our experimental results. It is organized in several
chapters that explain:

1. The background of the project, summarizing our knowledge on the PACE technology and on the M13 bacteriophage it is
   based on and on the Evolution.T7, both with their advantages and disadvantages
2. The design of the PHAGEVO system and its experimental implementation

---

## I. PHAGEVO PROJECT BACKGROUND

### I.1. M13 Bacteriophage

*E. coli*'s M13 bacteriophage is the most studied member of the *Inovirus* genus, part of the
*Inoviridae* family, whose name is derived from the Greek word "ina" meaning fiber or filament **[Knezevic *et al.*, 2021]**. Inoviruses infect Gram-negative bacteria like *E. coli* and have a characteristic elongated
filamentous capsid that packages the genome, a circular single-stranded DNA (ssDNA) of positive sense, ranging from
5.5 to 10.6 kilobases (kb) in length (**Figure 1**).

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-m13-virion-elements.png)
![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-m13-virion-full.png)

**Figure 1**. Schematic representation of the M13 bacteriophage. pIII, pVI, pVII, pVIII and pIX are coat
proteins that form the virion and encapsulate the single stranded circular DNA (ssDNA). The virion consists of
non-enveloped, flexible filaments, measuring between 0.6 and 2.5 µm in length and 6-7 nm in diameter. Its length
varies depending on the size of the packaged DNA, which is an adjustable variable. The major coat protein, pVIII
(CoaB), forms the outer layer of the filament, along with proteins pVII and pIX, while the inner coat is composed of
pVI. The pIII protein (CoaA) plays a crucial role in adhesion, recognition, and the infection process. The 3D
structure of the M13 virion is a helical complex, where pVIII forms an alpha-helix that makes up the viral coat. The
DNA inside the virion is supercoiled and partially folded into a double-helix structure. It adopts both A-form and
B-form conformations of double-stranded DNA (dsDNA), with the top segment in an unfolded, unwound state, the middle
segment in A-form DNA, and the bottom segment in the more common B-form, which is typical of many protein-DNA
complexes. (Figure adapted from **Knezevic *et al.*, 2021**.)

The M13 genome (6.407 kb, GenBank V00604, **Figure 2**) encodes 11 different proteins, numbered from p1
to p11, with a size between 3.3 kDa and 50 kDa.

* pIII, pVI, pVII, pVIII and pIX are coat proteins that form the virion protein structure complex responsible for
  the assembly and stability of the viral capsid.
* pII, pV and pX are proteins that control DNA synthesis (pII and pX) and translation rate of any viral protein (pII
  and pV). This last one, in high concentration, polymerizes around viral DNA and forms the pre-assembly complex
  during virion formation.
* pI, pIV, pV and pXI are proteins involved in the extrusion of virions from the host cell.

Their interplay during the bacteriophage life cycle hijacks cellular processes to produce new virus particles, as
schematised in **Figure 3**.

M13 infection begins with the recognition and adhesion of the phage to the bacterial surface via the phage pIII
protein and the bacterial F pilus (a transmembrane protein) and the TolQRA co-receptor located in the inner membrane
of the Gram-negative bacterial host. This limits the M13 infection only to *E. coli* cells carrying a wild-type F
plasmid or a modified F’ one, a feature we took into account in the choice of the *E. coli* host strain in our PHAGEVO
project (see details below). Following this interaction, the M13 ssDNA is translocated into the cytoplasm where it
remains in a circular, extra-chromosomal form, similar to a plasmid, without integrating into the host genome. Phage
DNA is then transcribed into mRNA by RNA polymerase III, and replicated following a rolling-circle mechanism. The
newly synthesized ssDNA is stabilized through interaction with protein pV, which is later replaced by pVIII, the major
coat protein, during virion assembly and extrusion. In this process, the mature virions are released into the
environment starting with the pVII and pIX end, followed by multiple copies of pVIII, while pIII and pVI are added at
the final stage.

M13 establishes a chronic productive infection, where virions are continuously released from infected cells via
extrusion, while the cells remain viable and intact, although typically exhibiting a slower growth rate.

In the absence of pIII protein, the phage loses its ability to recognize and infect host bacteria, while in the
absence of pVI phage particles remain associated with the host membrane. Our PHAGEVO technology leverages this feature
by making pIII and/or pVI selection markers for phage production depending on the activity of XylS, our protein of
interest.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-m13-genome-3.png)

**Figure 2**. M13 bacteriophage genome map (GenBank: V00604) with:

* pII (46.0 kDa): Replication initiation protein with endonuclease activity, essential for initiating the
  replication process of the viral genome.
* pX (12.0 kDa): A truncated version of pII produced from an internal start codon, it is necessary for the
  accumulation of the genomic (+) single-stranded DNA (ssDNA).
* pV (9.8 kDa): A helix destabilizing protein that binds to the (+) ssDNA as a homodimer, preventing its conversion
  into the double-stranded replicative form (RF).
* pVII (3.5 kDa): A minor coat protein located at the tip of the phage virion, composed of five copies, and is the
  first part to exit the host cell during phage release.
* pIX (3.3 kDa): Another minor coat protein, also present in five copies at the tip of the virion that exits the
  host cell during the phage release process.
* pVIII (5.4 kDa): The major coat protein CoaB that combines with the ssDNA genome to form the elongated filamentous
  structure of the phage particle.
* pIII (43.0 kDa): The minor coat protein CoaA located on the opposite side of the filament from pVII and piX; its
  C-terminal domain helps terminate the assembly process, while its N-terminal domain attaches to host pili to
  initiate infection.
* pVI (12.0 kDa): Another minor coat protein found at the end of the virion along with pIII; it plays a role in the
  termination of virion assembly. In its absence, phage particles remain attached to the host membrane.
* pI (35.0 kDa): Morphogenesis protein (Zot, zonula occludens toxin) with ATPase activity, an inner membrane protein
  that interacts with the outer membrane protein pXI, involved in phage extrusion.
* pXI (8.0 kDa): A C-terminal fragment of pI, translated from an internal start codon. It works with pI in the phage
  extrusion process.
* pIV (50.0 kDa): Forms a multimeric (14-mer) channel in the host membrane to facilitate phage secretion, working
  alongside p1 and p11 in the assembly of the secretion machinery.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-m13-lifecycle.png)

**Figure 3**. Schematic representation of the infection and replication cycle of the M13 bacteriophage and its steps: (1) adhesion of M13 phage via pilus interaction, (2) pilus retraction and phage approach, (3) phage genome entry via TolQRA co-receptor, (4) conversion of (+) ssDNA to dsDNA replicative form (RF), (5) RNA transcription from RF and translation of phage proteins, (6) translation of overlapping reading frames, (7) DNA replication initiation by pII endonuclease, (8) rolling circle DNA synthesis, (9) synthesis of additional (+) ssDNA and RFs, (10) formation of DNA filaments by pV, (11) DNA filament collapsing and packaging signal exposure, (12) structural proteins integration into the cytoplasmic membrane, (13) assembly of pore for M13 virion extrusion, (14) extrusion of virions with pV replacement, and (15) release of new virions. Figure adapted from **Knezevic *et al.*, 2021**.

---

### I.2 PACE & PANCE & PRANCE EVOLUTION SYSTEMS

#### I.2.1 PACE

**Phage-Assisted Continuous Evolution**

PACE is a continuous directed evolution system based on the M13 bacteriophage **[Esvelt *et al.*, 2011;
Miller *et al.*, 2020a]**.

Infectivity of M13 phages is also dependent on the protein VI encoded by gene VI (pVI / gVI) which was developed as
an alternative selection marker **[Brödel *et al.*, 2016; Miller *et al.*, 2020b]**
especially because low levels of pIII expression make the host cell resistant to infection **[Boeke *et
al.*, 1982]**.

PACE operates in a continuous manner through repeated cycles of bacterial infection and POI’s activity-based
bacteriophage production. For this, fresh host bacterial cells are continuously introduced into a reaction vessel
called a lagoon, where phages infect the bacteria, replicate, and then go on to infect new bacterial cells. This
continuous system is set up in a custom-built PACE apparatus, where the inflow of fresh cells into the lagoon and the
outflow mechanism for removing bacteria are regulated at rates carefully adjusted to allow phage replication within
the lagoon without washing away all the phages.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-pance-system-part-1.png)

**Figure 4**. The principle of PACE and PANCE and PRANCE systems for continuous, non-continuous and
robotics-assisted near-continuous evolution with SP for selection phage, AP for accessory plasmid, MP for mutagenesis
plasmid and POI for protein of interest. The red dots correspond to mutations introduced into the genome and plasmids.
Phages are periodically isolated and introduced in a medium with new host cells (adapted from **Badran *et al.*,
2016; Brödel *et al.*, 2018; Esvelt *et al.*, 2011; Miller *et al.*, 2020a**).

#### I.2.2 PANCE

**Phage-Assisted Non-Continuous Evolution**

PANCE is a non-continuous version of the PACE system, operating on the same principles but requiring human
intervention between each evolution cycle: instead of a continuous supply of host cells as in PACE, in PANCE a
subculture is initiated in fresh media with fresh host cells with phages isolated from the previous culture
**[Miller *et al.*, 2020a]**. This discontinuous process makes PANCE slower than PACE in terms of
culture growth and mutant appearance. However, the manual selection of variants allows for better control over each
evolution cycle, including the selection of phages and variants, despite a higher potential for bias. PANCE is easier
to implement and more flexible for various lab environments, as it doesn’t require custom-made continuous flow
bioreactors systems. It is more adaptable and enables multiple evolution experiments to be conducted in parallel in
96-well plates for instance.

#### I.2.3 PRANCE

**Phage and Robotics-Assisted Near-Continuous Evolution**

PRANCE is a high-throughput system developed to automate the PACE process, traditionally performed in a bioreactor,
by utilizing 96-well plates and robotic liquid-handling equipment **[DeBenedictis *et al.*,
2022]**. This setup allows for hundreds of independent evolution experiments to be run simultaneously, and,
by integrating real-time monitoring of biomolecular activity, automated feedback control mechanisms, high-throughput
sequencing, PRANCE allows precise control over environmental factors such as media composition, OD600, timing, and
chemical conditions, enabling experiments under varied conditions, including different temperatures, host strains, and
chemical environments.

#### I.2.4 ADVANTAGES and DISADVANTAGES of PACE / PANCE / PRANCE EVOLUTION SYSTEMS

The elegance of the PACE system and its declinations is that it makes an auto-screening of the best variants that
have an improved activity of the protein to evolve (POI). Indeed, as the expression levels of the essential gIII (or
gVI) are regulated by the POI, the infective phage production levels are also tightly regulated by the POI. This
results in an auto-amplification of the best variants, that will quickly outcompete the ineffective variants in the
lagoon where evolution occurs. With time, the POI will continuously evolve toward the optimal sequence for the desired
properties, and at the end of the experiment only a limited number of variants with increased fitness will be found in
the lagoon.

As for all technologies, PACE has limitations. The major challenge when designing a PACE experiment is to be able to
link the activity of the POI to the expression levels of gIII (or gVI). This is essential for PACE as it provides the
selection pressure toward the best adapted POI. PACE, PANCE, and PRANCE have all been successfully employed to
engineer a wide range of proteins. The easiest targets for evolution using PACE, and the first to be evolved with this
technology, are proteins directly involved in gene regulation, such as T7 RNA polymerase **[Liu *et al.*,
2011]**, transcription factors **[Brödel *et al.*, 2016]** or DNA-binding proteins like
TALEN **[Hubbard *et al.*, 2015]**. More recently, other types of proteins have been engineered
with PACE. This includes biosensors for small molecules **[Jones *et al.*, 2021]**, **[Li
*et al.*, 2022]**, enzymes like proteases **[Dickinson *et al.*, 2014]** or
involved in metabolic pathways like the methanol dehydrogenase Bm Mdh2 **[Roth *et al.*, 2019]**,
the therapeutic target PD1 involved in cancer immune escape **[Ye *et al.*, 2020]** or
protein-protein interactions **[Wang *et al.*, 2018]**.

The latest improvements of the PACE technology includes PRANCE **[DeBenedictis *et al.*, 2022]**,
which enables the high-throughput study of factors influencing evolution, and makes it possible to study how the
conditions of evolution influence evolutionary trajectories of the gene of interest. PRANCE is particularly useful for
protein or RNA engineering and enables the study of evolutionary dynamics in a controlled environment which
facilitates the analysis of complex evolutionary processes.

In PACE, PANCE and PRANCE, mutagenesis is triggered by a mutagenesis plasmid expressing error-prone DNA polymerase,
inhibit DNA repair pathways, increases DNA methylation or inhibit elimination of mutagenic nucleobase **[Miller
*et al.*, 2020a]**. The most potent mutagenesis plasmid increases mutation rates 300000 fold, reaching
approximately 6.10⁻⁶ mutations per base pair per generation. However, these mutations are not restricted to the gene
of interest and occur on the whole genome. Therefore, mutations can accumulate on the phage genome and lead to
defectuous phages and loss of POI mutants. In PANCE, where bacteria are not continuously renewed, mutations on the
genome can also lead to mortality or defects in the plasmids required for PANCE.

To further increase mutation rates and fasten PACE, PANCE or PRANCE experiments, we decided in PHAGEVO to target
evolution on the gene of interest only. Doing this may not only further increase mutation rates, but also reduce
failure rate of the experiment and phage washout, which is a common issue arising during PACE, by preventing
deleterious mutations on the phage or bacteria genome.

---

### I.3. The EVOLUTION.T7 System

Evolution.T7 is a tool developed by the iGEM Evry Paris-Saclay 2021 team **[[https://2021.igem.org/Team:Evry\_Paris-Saclay](https://2021.igem.org/Team%3AEvry_Paris-Saclay%5D)]**.
It is
based on the orthogonal T7 RNA polymerase (T7RNAP) linked to a base deaminase (BD) either a cytosine or an adenosine
deaminase (respectively CD and AD), which allows for the rapid generation of genetic diversity in GOI *in vivo* in *E. coli*. When BD-T7RNAP fusion protein is expressed, the sequence flanked by the T7 promoter and the T7
terminator(s) gets mutated as the CD or AD randomly deaminates the nucleotides mainly on the non template
strand of the T7RNAP. Upon DNA replication, these deaminated bases lead to C→T or A→G transition mutations, depending
on whether CD or AD was used (**Figures 5 and 6**).

MutaT7 was the first BD-T7RNAP-based tool reported in the literature **[Moore *et al.*, 2018]**.
It was using only the rApo1 / rAPOBEC1 cytosine deaminase and thus had a limited mutational spectrum, but, compared to
existing direct mutagenesis tools, this method showed improved on-target mutagenesis. This advantage was the key
reason for its subsequent development and the emergence of more advanced versions like T7-DIVA **[Álvarez *et
al.*, 2020]**, eMutaT7 **[Park & Kim 2021]**, dT7-Muta **[Ting & Ng 2023]**,
T7-DualMuta **[Wei *et al.*, 2023]**, Optimized Muta-T7 **[Mengiste *et al.*,
2023]**, eMuta-T7transition **[Seo *et al.*, 2023]**. The primary host organism for these
studies has been *E. coli*, but the system proved its functionality also in eukaryotic cells like mammalian
**[Chen *et al.*, 2020]**, yeast **[Cravens *et al.*, 2021, Huang *et al.*,
2023]** or plant cells **[Butt *et al.*, 2022]**. This versatility is on account of the
orthogonality of the T7RNAP which is highly specific to its promoter sequence (TAATACGACTCACTATA) and is able to
operate in other organisms than *E. coli* cells **[Imburgio *et al.*, 2000]**.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-mutat7.png)

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-cd-ad-fwd-rev.png)

**Figure 6**. Mutation mechanisms through deamination. (A-C) In DNA, deamination of a cytosine by a
cytosine deaminase converts it to deoxyuridine which pairs with adenosine and leads to a C→T mutation, and, when the
deamination occurs on the reverse strand, a G→A mutation occurs. (D-F) Deamination of an adenine by an adenine
deaminase converts it to deoxyinosine which pairs with C and causes an A→G mutation, and, when the deamination
occurs on the reverse strand, a T→C mutation occurs.

Compared to other T7RNAP-based tools mentioned above, to be able to introduce also T→C and G→A mutations,
Evolution.T7 uses also a mutated T7RNAPCGG-R12-KIRV specific to an altered PT7CGG promoter sequence which was placed in
the reverse orientation downstream of the target region in order to compensate for the above mentioned bias of
deaminations occurring mainly on the non template strand (**Figure 7**).

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-evolutiont7.png)

**Figure 7**. Schematic of the general organization of the Evolution.T7 system. The GOI is flanked
upstream and downstream by the PT7 (sense) and PT7CGG (antisense) promoters, respectively, and by four T7 terminators
(B0015, Sba\_000587, T7wt, Sba\_000451).

---

## II. PHAGEVO = PA(N)CE x EVOLUTION.T7

PA(N)CE is a powerful directed evolution tool notably due to its auto selection process of the best variants of the gene
of interest (GOI) and their subsequent amplification. However, it finds its limit through the fact that mutations can
occur everywhere in the plasmids, bacterial chromosome and on the phage genome and lead to defects in essential
genes, leading to the loss of GOI's mutants.

Our team took advantage of the best aspects of both technologies to design the PHAGEVO system. As summarized in
**Figure 8**, several modifications were implemented in PHAGEVO compared to the PA(N)CE system. On the
selection phage (SP), the GOI was flanked by the two T7 promoters (PT7 and PT7CGG) and the terminators from
Evolution.T7. The mutagenesis plasmid was completely remodeled to accommodate the specificity of the Evolution.T7
targeted mutagenesis and express the T7 RNA polymerase linked to base deaminase. Only the accessory plasmid, which
expresses pIII or pVI phage proteins under the control of the gene to evolve, remains unchanged in PHAGEVO compared to
PA(N)CE.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/es-pance-vs-phagevo.png)

**Figure 8**. PHAGEVO evolution system (adapted from **Badran *et al.*, 2016; Brödel *et
al.*, 2018; Esvelt *et al.*, 2011; Miller *et al.*, 2020a**)

In the development of our project we followed the Design, Build, Test, Learn (DBTL) cycle on each aspect. In the
following parts of this page, we present how we gradually implemented it at each step:

* The construction of the PHAGEVO host *E. coli* strain
* The production of Selection Phages (SP)
* The setup of the XylS evolution experiments
  and
* The selection of evolved XylS libraries

---

### II.1. Construction of *E. coli* S2060 ∆*ung* ∆*nfi*, a strain adapted for the PHAGEVO system

A question that arose early on in the development of our project was the choice of the *E. coli* host strain. Which one
would be better for PHAGEVO between the S2060 developed for PACE and MG1655\* ∆*flu* ∆*pyrF* ∆*ung* ∆*nfi* used in Evolution.T7
?

#### II.1.1. S2060 versus MG1655\* ∆*flu* ∆*pyrF* ∆*ung* ∆*nfi*

S2060 is a strain developed by **[Hubbard *et al.*, 2015]** as an improvement of earlier *E. coli* strains specifically
engineered for PACE, S1030 **[Carlson *et al.*, 2014]** and S109 **[Esvelt *et al.*,
2011]**, all of which were derived from the widely used and well-known DH10B **[Durfee *et al.*,
2008]**. The modifications gradually introduced in DH10B are highlighted in **Table 1**. They are located both at
chromosome level, as well as on the F’ plasmid. The main reasons for these modifications are reducing the biofilm
formation which represents an important issue in continuous cultures, enabling a robust induction of the mutagenesis
or the evolution of specific genes:

* ∆*lacIZYA*: *lacIZYA* is the lac operon. It was deleted in order to allow the mutagenesis experiments on LacI
  described by **Esvelt *et al.*, 2011**.
* *proBA::pir116*: ProB and ProA encode 2 enzymes from the proline biosynthetic pathways (glutamate 5-kinase and
  glutamate-5-semialdehyde dehydrogenase, respectively). They were replaced by *pir116* encoding a allele of the Π
  protein required for the replication of the R6K plasmids and their maintenance at an increased copy number
  **[Metcalf *et al.*, 1994]**. This modification is not required for PACE, but it provides
  flexibility in the type of plasmids that can be used in this strain. Deletion of *proBA* is turning this strain
  proline-auxotrophic, which is used as a selection marker for F’ plasmid maintenance (see below).
* *araE201*: AraE is a low affinity arabinose transporter. According to **Hogg & Englesberg, 1969** and to **Brown &
  Hogg, 1972**, *araE201* is a mutation that makes the strain transport-deficient. **Carlson *et al.*, 2014** use this
  annotation to designate a modification of the *araE* promoter into a constitutive one, in order to ensure a constant
  level of AraE expression in a cell and thus a constant level of arabinose import. Arabinose is used in PACE to
  induce the expression from the MP plasmids.
* ∆*rpoZ*: RpoZ is the omega subunit of *E. coli* RNA polymerase. Its deletion confers a slow growth phenotype
  **[Gentry & Burgess, 1989]**. This mutation was introduced in the PACE strain in order to perform
  one-hybrid, two-hybrid experiments to detect protein–DNA interactions or protein–protein interactions in *E. coli*
  **[Dove & Hochschild, 1998]**.
* ∆*flu*: the *flu* gene encodes the antigen 43 (Ag43) which is an autotransporter protein localized in the outer
  membrane of *E. coli* and is involved in biofilm formation **[van der Woude & Henderson, 2008]**. Its
  deletion reduces biofilm formation, which is a problem in PACE experiments performed in a continuous manner.
* ∆*csgABCDEFG*: *csgBAC* and *csgDEFG* are two divergently transcribed operons of the curli biosynthetic pathway
  **[Barnhart & Chapman, 2006]**. Curli are thin, coiled surface structures that play a role in
  colonizing inert surfaces, forming biofilms, and facilitating bacterial attachment to various extracellular matrix
  components and serum proteins. Deletion of this cluster reduces biofilm formation, which is a problem in PACE
  experiments performed in a continuous manner.
* ∆*pgaC*: *pgaC* encodes the poly-beta-1,6-N-acetyl-D-glucosamine synthase that participates along with PgaD in the
  synthesis of the extracellular matrix polysaccharide poly-N-acetylglucosamine required for biofilm formation
  **[Morrison *et al.*, 2022]**. Its deletion reduces biofilm formation, which is a problem in
  PACE experiments performed in a continuous manner.

In addition, DH10B was modified to become an F' strain, an essential condition for M13 phage propagation as
described above. The original F’ plasmid was acquired by conjugation from the *E. coli* ER2738 strain and had the
genotype F´ *proA+B+ lacIq* ∆*(lacZ)M15 zzf Tn10(TetR)*. It was further modified to keep or acquire new features:

* *proA+B+*: ProB and ProA encode 2 enzymes from the proline biosynthetic pathways (glutamate 5-kinase and
  glutamate-5-semialdehyde dehydrogenase, respectively). Their deletion is turning the strain proline-auxotrophic,
  while their presence on the F’ plasmids acts as a selection marker.
* ∆*(lacIZY)*: the *lac* operon genes present on the original F’ plasmid (*lacIq* ∆*(lacZ)M15*) were removed and later
  replaced by *lacIQ1* and *lacZ* with different regulations of their expression
* *zzf::Tn10(TetR)*: insertion of the Tn10 transposable element carrying the tetracycline resistance genes
* *lacIQ1*: LacI is the transcriptional regulator of the *lac* operon. *LacIQ1* is a detention of the -35 box of its
  promoter that is responsible for increased transcription **[Glascock & Weickert, 1998]**.
* *PN25-tetR*: TetR is the transcriptional regulator of the *tet* operon. Its expression from the F’ plasmid facilitates
  the use of genetic circuits carrying promoters regulated by TetR inducible by anhydrotetracycline.
* *luxCDE*: is an operon encoding the fatty acid reductase complex catalyzing the synthesis the long-chain aliphatic
  aldehyde decanal that is the substrate in the luminescence reaction catalyzed by the luciferase enzyme encoded by
  the *luxAB* operon **[Meighen, 1991]**.
* *Ppsp(AR2) lacZ*: expression cassette of the *lacZ* gene under the control of the promoter of the *pspA* gene inducible
  by stress factors and is regulated by a network of positive and negative mechanisms **[Weiner *et al.*,
  1991; Elderkin *et al.*, 2002]**. This allows the use of LacZ-mediated blue/white visualization in
  response to phage infection.
* *luxR*: LuxR is the transcriptional regulator of the Plux promoter of the *lux* operon inducible by
  N-(3-oxohexanoyl)-l-homoserine lactone. On the F’ plasmid it controls the expression of the *groESL* operon.
* Plux *groESL*: *groESL* is an *E. coli* operon encoding the heat shock proteins GroEL and GroES that act as chaperones
  and play an important role in protein folding **[Koubek *et al.*, 2021]**. Their expression from
  the F’ plasmid improved the folding of LuxAB luciferase and increased the luminescence response.

**Table 1**. The genotype of *E. coli* S2060 strain and of its ancestors. Modifications highlighted in blue are those
introduced early on in the DH10B strain to construct the S109 strain, those in purple were introduced in the S109 to
construct S1030, while those in red were added in S2060.

| STRAIN | GENOTYPE | REFERENCES |
| --- | --- | --- |
| DH10B | F- *endA1 recA1 galE15 galK16 nupG rpsL* ∆*lacX74 ϕ80dlacZ∆M15* araD139 ∆*(ara,leu)7697 mcrA* ∆*(mrr-hsdRMS-mcrBC) λ–* | **Durfee *et al.*, 2008** |
| S109 | F’ *proA+B+* ∆*(lacIZY)* zzf::Tn10(TetR) / *endA1 recA1 galE15 galK16 nupG rpsL* ∆*lacIZYA* *araD139* ∆*(ara,leu)7697 mcrA* ∆*(mrr-hsdRMS-mcrBC) proBA::pir116 λ–* | **Esvelt *et al.*, 2011** |
| S1030 | F’ *proA+B+* ∆*(lacIZY)* zzf::Tn10(TetR) lacIQ1 PN25-tetR luxCDE / *endA1 recA1 galE15 galK16 nupG rpsL* ∆*lacIZYA* *araD139* ∆*(ara,leu)7697 mcrA* ∆*(mrr-hsdRMS-mcrBC) proBA::pir116* |

*araE201* ∆*rpoZ* ∆*flu* ∆*csgABCDEFG* ∆*pgaC*

λ– **Carlson *et al.*, 2014** || S2060 | F’ *proA+B+* ∆*(lacIZY)* zzf::Tn10(TetR) lacIQ1 PN25-tetR luxCDE  Ppsp(AR2) lacZ luxR Plux groESL / *endA1 recA1 galE15 galK16 nupG rpsL* ∆*lacIZYA* *araD139* ∆*(ara,leu)7697 mcrA* ∆*(mrr-hsdRMS-mcrBC) proBA::pir116*  *araE201* ∆*rpoZ* ∆*flu* ∆*csgABCDEFG* ∆*pgaC* λ– | **Hubbard *et al.*, 2015** |

*E. coli* MG1655\* ∆*flu* ∆*pyrF* ∆*ung* ∆*nfi* is a strain developed by **Álvarez *et al.*, 2020** to increase the mutation
rate in BD-T7RNAP-based mutagenesis tools. This strain was derived from the wild-type *E. coli* MG1655 in which several
modifications have been introduced:

* The \* refers to the insertion of a G nucleotide in the *rph* gene after position 671, which corrects a frameshift
  present in the MG1655 genome when compared to other *E. coli* strains. The frameshift results in a truncated *rph* gene
  product that lacks RNase PH activity. This premature stop in translation negatively affects the expression of the
  downstream *pyrE* gene, which is part of the same polycistronic RNA **[Jensen, 1993]**.
  *PyrE* encodes orotate phosphoribosyltransferase, an enzyme involved in uracil metabolism. In MG1655, low *pyrE*
  expression causes pyridine starvation, while its higher expression in MG1655\* increases the toxicity of 5’-fluorotic
  acid.
* ∆*flu*: the *flu* gene encodes the antigen 43 (Ag43) which is an autotransporter protein localized in the outer
  membrane of *E. coli* and is involved in biofilm formation **[van der Woude & Henderson, 2008]**. Its
  deletion reduces biofilm formation. This locus was used for genome integration of synthetic constructs by **Álvarez
  *et al.*, 2020**.
* ∆*pyrF*: the pyrF gene encodes the orotidine 5'-phosphate decarboxylase involved in the uracil metabolism
  **[Lee & Houk, 1997]**. Its deletion makes *E. coli* auxotrophic for uracil, but resistant to
  5’-fluorotic acid.
* ∆*ung*: the *ung* gene encodes the uracil DNA N-glycosylase which is involved in the elimination of uracil from the
  DNA **[Pearl, 2000]**.
* ∆*nfi*: the *nfi* gene encodes the exonuclease V of *E. coli* which is involved in the elimination of inosines from the
  DNA **[Guo *et al.*, 1997; Vik *et al.*, 2013]**.

Heaving all these considerations in mind, we decided to take the best from the two strains and construct the S2060
∆*ung* ∆*nfi*. Indeed, the numerous modifications implemented in the S2060 strain make the monitoring of phage infection
and thus of the evolution experiment easier to follow. In addition, its genetic background (DH10B) is that of a
‘cloning’ *E. coli* strain commonly used in laboratories as a competent cell for its high large plasmid DNA
transformation efficiency and the construction of large DNA libraries. DH10 was constructed from MC1061 by introducing
the recombinase-deficient mutant *recA1*, which reduces recombination with host DNA, and the endonuclease-deficient
mutant *endA1* mutation, which improves plasmid DNA quality. The strain also carries mutations in *mcrA*, *mcrB*, blocking
the restriction of methylated cytosine DNA and *deoR*, a mutant allowing for the efficient propagation of large plasmids
thanks the constitutive expression of genes for deoxyribose synthesis enabling deoxyribose synthesis **[Durfee
*et al.*, 2008]**.

In Evolution.T7 and related BD-based tools, ∆*ung* and ∆*nfi* are important modifications for enhancing the mutation
rate. Without these genes, the DNA repair mechanisms are impaired both in case of cytosine and adenine deamination,
and thus, the mutations generated are maintained.

#### II.1.2 Construction of *E. coli* S2060 ∆*ung* ∆*nfi* through CRISPR/Cas9 and λ Red mediated recombineering

#### II.1.2.1 Design

CRISPR/Cas9 is a powerful genome engineering technology, which makes it our first choice for constructing the S2060
∆*ung* ∆*nfi*.

Using CHOPCHOP (version 3) web tool **[Montague
*et al.*, 2014; Labun *et al.*, 2016, 2019]** we designed CRISPR/Cas9 target sites for both *ung* and *nfi* genes. For *ung*, we
selected as target sequence AATCAGTGGCTGGAACAACGTGG, as the best ranked among 94 proposed for knocking-out this gene
in *E. coli* str. K-12/MG1655. Similarly, for nfi, the highest-ranked target sequence was GATGTCGGGTTTGAGCAGGGCGG,
chosen from 95 proposed sequences for this gene knockout in the same strain. Next, we used the first 20 nt of these
sequences to design the gRNAs specific for *ung* and nfi, [BBa\_K5061015](https://parts.igem.org/Part%3ABBa_K5061015) and [BBa\_K5061016](https://parts.igem.org/Part%3ABBa_K5061016), respectively.

In *E. coli*, the DNA repair system for double-strand breaks is highly inefficient, rendering CRISPR/Cas9-induced
breaks lethal to the cell **[Widney *et al.*, 2024]**. Additionally, the native homologous
recombination system is not effective too, therefore targeted repairs cannot be easily introduced without the use of
phage recombination systems. To overcome this, the λ Red recombination system, derived from lambda (λ) phage, is
commonly used as one of the most efficient tool for facilitating targeted genetic modifications **[Datsenko &
Wanner, 2000]**.

#### II.1.2.2. Build

Following the design of the two gRNA sequences specific for *ung* and *nfi* genes, we constructed two corresponding
transcription units under the control of the J23119(SpeI) promoter ([BBa\_K5061115](https://parts.igem.org/Part%3ABBa_K5061115) and [BBa\_K5061116](https://parts.igem.org/Part%3ABBa_K5061116),
respectively) that we assembled by CPEC (Circular Polymerase Extension Cloning) **[Quan and Tian, 2011]**
in a backbone composed of the ampicillin resistance gene (AmpR) and the thermosensitive version of the oriR101 origin
of replication.
In parallel, we amplified by PCR the *ung* and *nfi* repair templates ([BBa\_K5061215](https://parts.igem.org/Part%3ABBa_K5061215) and [BBa\_K5061216](https://parts.igem.org/Part%3ABBa_K5061216), respectively)
using as template genomic DNA extracted from *E. coli* MG1655\* ∆*flu* ∆*pyrF* ∆*ung* ∆*nfi* strain **[Álvarez *et
al.*, 2020]** that was kindly provided by Dr. Luis Ángel Fernández, at Centro Nacional de Biotecnología
(CNB-CSIC) in Spain.

#### II.1.2.3. Test

*E. coli* S2060 cells were first transformed with the pCrepe plasmid carrying both the λ Red and Cas9 genes
**[Choudhury *et al.*, 2020a, b]** and grown in LB media supplemented with 35 µg/mL chloramphenicol.
Once the OD600 reaches 0.4, the culture was transferred to a water bath at 42°C for 15 minutes with shaking at 200 rpm
to activate the expression of the λ Red recombination system proteins. The cells were then placed on ice for 15-20
minutes, then made electrocompetent. Subsequently, they were transformed by electroporation using 0.5 to 2 μg of
plasmid DNA (expressing the gRNA) and 0.5 to 2 μg linear repair DNA in an electroporation cuvette at 1.8 kV and
immediately resuspended in 1 mL of LB medium. After 3 hours of incubation at 30°C with shaking at 200 rpm, aliquots
were spreaded on LB agar supplemented with chloramphenicol and ampicillin. The cultures were incubated overnight at
30°C. Isolated colonies were streaked on LB agar plates (without antibiotics) and cultured at 37°C to get cured from
both plasmids (pCrepe and gRNA).

#### II.1.2.4. Learn

Unfortunately, the CRISPR/Cas9 and λ Red recombineering technique proved inefficient in our attempts to construct *E. coli* S2060 ∆*ung* ∆*nfi*.
Although we have successfully assabled the gRNA expressing plasmids and readily obtained the repair templated by PCR,
we were unable to obtain colonies upon their transformation in electrocompetent S2060 cells containing the pCrepe
plasmid.
The process was particularly challenging due to the slow growth rate of the S2060 cells with the pCrepe plasmid, which
had an estimated doubling time of 2 hours, making it difficult to align with the working hours of our host lab.
After multiple unsuccessful attempts, we opted to explore alternative methods for constructing *E. coli* S2060 ∆*ung*
∆*nfi*.

#### II.1.3 Construction of *E. coli* S2060 ∆*ung* ∆*nfi* through λ Red mediated recombineering

#### II.1.3.1. Design

As mentioned above, phage-derived recombination systems are commonly used to enable targeted genetic modifications in
*E. coli*, with the λ Red system being one of the most effective tool. However, since recombination events are quite
rare, a selection system is essential to identify successful modifications. The method developed by **Datsenko &
Wanner, 2000** addresses this need, and its effectiveness was demonstrated in the construction of the Keio gene deletion
collection **[Baba *et al.*, 2006]**.

The method relies on the use of a linear donor DNA fragment containing 5′ and 3′ "homology arms" (H1/H2), matching
the sequences immediately upstream and downstream of the target site. Upon expression of the λ Red genes carried on a
thermosensitive plasmid like pKD46, the sequence between the homology arms in the donor DNA replaces the corresponding
sequence in the target DNA. If this sequence includes an antibiotic resistance gene, successfully transformed cells
become resistant to the antibiotic. Additionally, if the resistance gene is flanked by FRT sites, it can be excised by
expressing the Flp recombinase from a second plasmid (pCP20 for instance **[Cherepanov &
Wackernagel, 1995]**).

To use this method, we designed primers with H1 and H2 "homology arms" of 50 nt specific to the 5’ and 3’ sequences
immediately upstream and downstream of both *ung* and *nfi* genes (**Table 2**).

**Table 2**. *ung* and *nfi* specific primers designed to amplify the Apramycin (ApraR) and Spectinomycin (SpecR) resistance
genes carried on the pEVL408 **[Trottier, 2019]** and pEVL410 **[Schulz, 2023]** plasmids
respectively. Nucleotides in capital letters are specific to the 5’ and 3’ sequences immediately upstream and
downstream of *ung* or *nfi* genes, while those in lowercase letters are specific to the template DNA plasmids.

| Primer name | Sequence (5'->3') |
| --- | --- |
| delta-ung-F | TAGAAAGAAGCAGTTAAGCTAGGCGGATTGAAGATTCGCAGGAGAGCGAGagagcggccgccaccgcggg |
| delta-ung-R | TGATAAATCAGCCGGGTGGCAACTCTGCCATCCGGCATTTCCCCGCAAATgcatatgctgcgtgcatgcg |
| delta-nfi-F | TGGAGGCAGTGCATCGACTGTCTGAACAGTATCACCGCTAAGGAGTGATTagagcggccgccaccgcggg |
| delta-nfi-R | TGTAACATGTTGAGTTCTCAAATACGGAAATTATCCGCAGTTTACCTGAAgcatatgctgcgtgcatgcg |

#### II.1.3.2. Build

No genetic constructs were necessary: all required plasmids were kindly provided by our host lab: pKD46
**[Datsenko & Wanner, 2000]**, pCP20 **[Cherepanov & Wackernagel, 1995]**, pEVL408
**[Trottier, 2019]** and pEVL410 **[Schulz, 2023]**.

#### II.1.3.3. Test

*E. coli* S2060 cells were first transformed with the pKD46 plasmid carrying the λ Red genes **[Datsenko &
Wanner, 2000]** and grown in LB media supplemented with 100 µg/mL ampicillin. Once the OD600 reached 0.6, the
expression of the λ Red recombination system proteins was induced by adding L-arabinose to a final concentration of 10
mM. After 2 hours of incubation at 30°C with shaking at 200 rpm, cells were made electrocompetent and transformed with
0.5 µg PCR product amplified as described above. After 3 hours of incubation at 37°C with shaking at 200 rpm, aliquots
were spreaded on LB agar supplemented with either 50 µg/mL aparamycin or 100 µg/mL spectinomycin (depending on which
plasmid pEVL408 or pEVL410 respectively was used as template for the PCR reaction). The cultures were incubated
overnight at 37°C to eliminate the pKD46 plasmid.
∆*ung* ApraR colonies were selected on apramycin and the correct insertion confirmed by colony PCR with external primers
binding the *grcA* gene (GAACTTCTGGTTTCACTTCTACTGGAACTTCACG) and *yfiF* gene (CAGGATTGCGTTCTGGCACTGG).
∆*nfi* SpecR colonies were selected on spectinomycin and the correct insertion confirmed by colony PCR with external primers
binding the *hemE* gene (GCGTCACTTTGTATCTGAATGCGCAGATTAAAGC) and *yjaG* gene (CTCATTTCGCGACCAGCCTGAGTCATTTCC).
In a subsequent step, antibiotic resistant cells were transformed with the pCP20 plasmid and selected on LB agar
plates supplemented with 100 µg/mL carbenicillin. An isolated colony was then grown at 42°C to activate the expression
of the Flp recombinase, then streaked on LB agar plates containing no antibiotic. The pCP20 plasmid was eliminated by
growing the cells at 37°C.
The correct excision of the antibiotic was confirmed first by colony PCR with the external primers mentioned above,
then by Sanger sequencing of the PCR product at Eurofins Genomics.

Finally, genomic DNA of the *E. coli* S2060 ∆*ung* ∆*nfi* strain was extracted using the PureLink™ Genomic DNA Mini Kit
(Invitrogen) from 1 mL overnight culture following the supplier’s Gram Negative Bacterial Cell Lysate Protocol. The
concentration of dsDNA was evaluated with the Qubit™ 1X dsDNA BR (Broad Range) Assay (Invitrogen), then sequenced at
Eurofins Genomics using Oxford Nanopore Technologies for bacterial genome sequencing.

#### II.1.3.4. Learn

λ Red recombineering technique following the protocol described by **Datsenko & Wanner, 2000** proved very efficient
and we have successfully constructed the *E. coli* S2060 ∆*ung* ∆*nfi* strain.
It was a multi step process in which we first inserted the ApraR resistance cassette at the *ung* locus, then used the
Flp recombinase to excise it. Afterwards, we inserted the SpecR resistance cassette at the *nfi* locus and finally we
used the Flp recombinase to excise it too.
At each step, several clones were isolated through multiple passages on appropriate antibiotics and analyzed by colony
PCR and Sanger sequencing. Regularly, antibiotic resistance profiles were checked on many antibiotics in parallel, to
avoid any carry over of the ‘previous’ strain and sudden reappearance of the wild type phenotype.

Finally, we used the bacterial genome sequencing service provided by Eurofins Genomics that employs state-of-the-art
Oxford Nanopore Technologies long-read sequencing technology, and completely sequenced the genome of our *E. coli* S2060
∆*ung* ∆*nfi* strain.

The first analysis of the NGS data was performed by Eurofins Genomics, using their established pipeline:
“Raw nanopore sequencing reads are assessed for quality and filtering. Filtlong v0.2.1 **[<https://github.com/rrwick/Filtlong>]** is used to
remove short and
low-quality reads from the raw nanopore sequencing data. The high-quality nanopore sequencing reads are then used
for de novo assembly of the bacterial genome. The assembly is performed using Flye v2.9.3 **[Kolmogorov
*et al.*, 2019]** with parameters optimized for bacterial genomes. The resulting contigs are further
polished using Medaka v1.8 **[<https://github.com/nanoporetech/medaka>]** to
improve base
accuracy. The assembled genomes are annotated using Bakta v.1.8.2 **[Schwengers *et al.*, 2021]**.
The annotation includes the prediction of coding sequences, tRNAs, rRNAs, and other genomic features based on
published databases such as RefSeq and UniProt. The quality of the assembled genomes is assessed using various quality
assessment tools (QUAST v5.2 **[Gurevich *et al.*, 2013]**, CheckM2 v1.0.1 **[Chklovski
*et al.*, 2023]**, Mash v2.3 **[Ondov *et al.*, 2016]**). Genome completeness,
contiguity, and accuracy are evaluated to ensure the reliability of the assemblies. A final QC step is included to
ensure the purity of samples, which uses minimap2 v2.24 **[Li, 2018]** to map the sequence-cleaned reads
onto the assembly and finally employs Clair3 v1.0.4 **[Zheng *et al.*, 2022]** to call variations
(SNPs and INDELS) within the assembled genome. Any variants detected along with the location in the bacterial assembly
are reported.” (text provided by Eurofins Genomics and edited to adapt the style of the bibliography of this page)

Through this professional analysis, 6 contigs were assembled for a total of 4827035 nucleotides (nt), with an average
coverage depth of the assembled genome of 42 and an overall GC content of 50.67%. The machine learning algorithm used
to assess the assembly completeness estimated it at 100% and revealed a 1.78% contamination rate. Importantly, no
variants were detected in the ‘Purity Check’ of the assembled genome during which the sequenced reads are mapped
against the assembled genome and the variants (SNPs, insertions & deletions) are determined and reported if having
at least 0.3 minor allele frequency(MAF) and >30x read support.

The 6 contigs assembled by Eurofins Genomics have, in descendent order: 4168152 nt, 266411 nt, 198664 nt, 112058 nt,
79301 nt and 2449 nt.

We performed further sequence analysis and compared them with the published genome of *E. coli* DH10B **[Durfee
*et al.*, 2008]**, the parental strain of S2060 (**Table 1**).
The longest contig of 4168152 nt really aligned on DH10B (GenBank NC\_010473), albeit with a 507224 nt gap at position
233634..740857. Contigs 2, 4, 5 and 6 partially covered this gap as they all aligned in this region (**Figure
9**). It is to be noted that contig 6, the smallest one carries the *proA* and *proB* genes that in S2060 are no
longer in the chromosome, but on the F’ plasmid.

Contig 3, of 198664 nt, only partially and to a very limited extent aligned to the DH10B genome. A blast analysis
revealed that it’s the F’ plasmid, with the F'Iq (GenBank CP053608) being the top best hit (Score 2.968e+05, 95% query
cover and 100% identity over 160718 nt). The F’ plasmid of S2060 was also engineered starting from the one carried by
*E. coli* ER2738, it is thus not unexpected to observe differences.

This comparative genomics analysis allowed us to investigate and map the genetic differences compared to DH10B and
thus confirm the genetic modifications made to the S2060 strain. The alignments confirmed that the *ung* and *nfi* genes
are no longer present in our strain and that the sequence is as expected and exactly the same as the one Sanger
sequencing of the colony PCR products.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/dh10b-aligned-contigs.png)

**Figure 9**. Alignment of the contigs assembled by Eurofins Genomics on the genome of *E. coli* DH10B. The
alignment was generated using SnapGene (to circumvent le 1000000 nt limit, the largest contig was splitted in 5, then
the image was edited to remove the artifacts). Mutations indicated on top are expected according to the strain
genotype, while those indicated in the bottom were unexpected and mapped following the breseq analysis.

Moreover, to understand why this gap was present in the assembled contigs by the sequencing company, we performed
further analysis using minimap2 **[Li, 2018, 2021]** and breseq **[​​Deatherage & Barrick,
2014]**.

Minimap2 quickly showed that Nanopore reads were aligned in the gap region from position 233634 to 740857 on DH10B
genome (**Figure 10**). The alignments in this part showed differences in depth of coverage within the
gap, but also with the rest of the alignment outside this region. This may explain the failure of *de novo* assembly
algorithms to properly assemble this region in a contig that meets the quality standards.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/alignement-igv-all-gap.png)

**Figure 10**. Alignment of the Nanopore reads on the genome of *E. coli* DH10B with a zoom on the 233634..740857 position. The alignment was generated using minimap2 and the visualization was made with Integrative
Genomics Viewer (IGV, v2.18.2) **[Robinson *et al.*, 2011]**.

The analysis performed with breseq allowed us to map and identify the differences between S2060 and DH10B. The full
results are available [HERE](https://2024.igem.wiki/evry-paris-saclay/static/breseq/index.html). Notably, no reads mapped the *ung* and *nfi* locus which undoubtedly confirmed their deletion
(**Figures 11 and 12**).

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/ngs-ung.png)

**Figure 11**. Alignment of the Nanopore reads on the genome of *E. coli* DH10B with a zoom on the *ung*
locus. The alignment was generated using breseq.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/ngs-nfi.png)

**Figure 12**. Alignment of the Nanopore reads on the genome of *E. coli* DH10B with a zoom on the *nfi*
locus. The alignment was generated using breseq.

---

### II.2. Selection Phages (SP) for XylS evolution

In PACE / PANCE / PRANCE and PHAGEVO, the production of selection phages (SP) is a preliminary, but essential step
without which evolution cannot be started.

#### II.2.1. Design

As described above, the SP is a modified M13 phage lacking a gene responsible for its infectivity which is replaced
by the gene of interest to be evolved (GOI).
Traditionally, it's the gIII that was chosen as a selection marker **[Esvelt *et al.*, 2011]**,
but, as its low expression makes the host cell resistant to infection **[Boeke *et al.*, 1982]**
which hinders phage auto propagation and the auto screening of the best variants of GOI, gVI was developed as an
alternative **[Brödel *et al.*, 2016; Miller *et al.*, 2020b]**. Based on this, in our
setup we choose to implement in PHAGEVO two different approaches: one using gVI and the other using both gIII and gVI
as selection markers.
Genetically engineering phages is not as easy as for plasmids. For this reason, helper phages (HP) were developed.
M13KO7 is one known example of such HP that includes the origin of replication from p15A and a kanamycin resistance
gene, both inserted into M13 origin of replication **[Vieira & Messing, 1987]**. The presence of these
two elements allows it to replicate as a plasmid in *E. coli* cells, but does not prevent it to replicate, pack and
secrete single-stranded phage DNA. M13KO7 is commercially available from New England Biolabs (Cat#N0315S), and we
decided to use it as a platform for the PHAGEVO HP constructs.
To allow the evolution of XylS, we designed a total of 10 helper phages (**Table 3**), two of which are Golden Gate
platforms to facilitate the insertion of the GOI and eight of them are XylS-specific HP. Our set of HP is composed of
half of HP designed for using gVI as a selection marker and the other half for the gIII-gVI.
The XylS-specific HP are of several types:

* Half are expressing XylS-K38R-L224Q and the other half the XylS-W88C-L224Q variants reported to have the ability
  to weakly detect the plastic degradation products PA and TPA, respectively **[Li *et al.*,
  2022]**.
* Half are expressing XylS in the PHAGEVO mutational region, while the other half as in the classical PANCE.

In PANCE, the GOI is equipped with an RBS and inserted in the HP in place of the selection marker (gIII and/or gVI).
This disrupts the expression of the downstream gI gene in the M13 genome, thus the GOI is followed by the J23107
constitutive promoter and an RBS.
In PHAGEVO, we keep this general architecture and added upstream and downstream of our GOI the T7 promoters and
terminators as illustrated in **Figure 8**.

Insertion of the GOI in the HP leads to the disruption of the infectious cycle of the M13 phage as at least an
important gene in this process is missing. To complete this cycle, its expression *in trans* from the accessory plasmid
(AP) is required.
In our project we designed a total of 6 AP (**Table 4**):

* 2 are Golden Gate platforms to facilitate the insertion of the promoter dependent on the GOI’s activity
* 2 that express the missing phage gene under the control of the PspA phage shock promoter, allowing for phage
  production independent of GOI’s activity
* 2 that express the missing phage gene under the control of the Pm promoter, allowing for phage production
  dependent on XylS activity

These APs express in an operon not only the missing gene from the HP (gVI or gIII-gVI), but also the LuxAB, which is
a fusion protein between LuxA and LuxB from *Photorhabdus luminescens* (*Xenorhabdus luminescens*) via a 21 amino acids
flexible linker. LuxA, the alkanal monooxygenase alpha chain (Uniprot P19839) is a 360 amino acids protein involved in
light emission by luminous bacteria together with LuxB, the alkanal monooxygenase beta chain of 327 amino acids
(Uniprot P19840) **[Johnston *et al.*, 1990, Xi *et al.*, 1991]**. The LuxAB complex is an
FMN bound enzyme (EC:1.14.14.3) catalyzing the oxidation of an aliphatic long-chain-aldehyde to the corresponding
fatty acid. The enzyme exhibits high specificity for reduced FMN and long-chain aliphatic aldehydes containing eight
or more carbon atoms, with maximum efficiency observed using tetradecanal (aka myristyl aldehyde) **[Li &
Meighen, 1994]**. The aldehyde is synthesized *in vivo* by the action of the LuxCDE operon, expressed from the
F’ plasmid carried by the host cells of *E. coli* S2060 strain, as described above. The expression of luciferase
(LuxABCDE) allows monitoring the phage propagation through luminescence monitoring experiments **[Badran *et
al.*, 2016; DeBenedictis *et al.*, 2022]**.

**Table 3**. Helper phages (HP) designed and built for the evolution of XylS through PANCE and PHAGEVO.

| Usage | HP Description | HP Part Number |
| --- | --- | --- |
| Golden Gate cloning platform | Helper phage (HP) M13KO7-∆gVI Golden Gate cloning platform | [BBa\_K5061031](https://parts.igem.org/Part%3ABBa_K5061031) |
| Golden Gate cloning platform | Helper phage (HP) M13KO7-∆gIII-∆gVI Golden Gate cloning platform | [BBa\_K5061131](https://parts.igem.org/Part%3ABBa_K5061131) |
| PACE / PANCE | Helper phage (HP) M13KO7-∆gVI carrying XylS-K38R-L224Q | [BBa\_K5061051](https://parts.igem.org/Part%3ABBa_K5061051) |
| PACE / PANCE | Helper phage (HP) M13KO7-∆gIII-∆gVI carrying XylS-K38R-L224Q | [BBa\_K5061151](https://parts.igem.org/Part%3ABBa_K5061151) |
| PHAGEVO | Helper phage (HP) M13KO7-∆gVI carrying XylS-K38R-L224Q in the PHAGEVO mutational region | [BBa\_K5061052](https://parts.igem.org/Part%3ABBa_K5061052) |
| PHAGEVO | Helper phage (HP) M13KO7-∆gIII-∆gVI carrying XylS-K38R-L224Q in the PHAGEVO mutational region | [BBa\_K5061152](https://parts.igem.org/Part%3ABBa_K5061152) |
| PACE / PANCE | Helper phage (HP) M13KO7-∆gVI carrying XylS-W88C-L224Q | [BBa\_K5061053](https://parts.igem.org/Part%3ABBa_K5061053) |
| PACE / PANCE | Helper phage (HP) M13KO7-∆gIII-∆gVI carrying XylS-W88C-L224Q | [BBa\_K5061153](https://parts.igem.org/Part%3ABBa_K5061153) |
| PHAGEVO | Helper phage (HP) M13KO7-∆gVI carrying XylS-W88C-L224Q in the PHAGEVO mutational region | [BBa\_K5061054](https://parts.igem.org/Part%3ABBa_K5061054) |
| PHAGEVO | Helper phage (HP) M13KO7-∆gIII-∆gVI carrying XylS-W88C-L224Q in the PHAGEVO mutational region | [BBa\_K5061154](https://parts.igem.org/Part%3ABBa_K5061154) |

**Table 4**. Accessory Plasmids (AP) designed and built for the evolution of XylS through PANCE and PHAGEVO.

| Usage | AP Description | AP Part Number |
| --- | --- | --- |
| Golden Gate cloning platform | Accessory Plasmid (AP) Golden Gate cloning platform for M13 gVI expression | [BBa\_K5061032](https://parts.igem.org/Part%3ABBa_K5061032) |
| Golden Gate cloning platform | Accessory Plasmid (AP) Golden Gate cloning platform for M13 gIII and gVI expression | [BBa\_K5061132](https://parts.igem.org/Part%3ABBa_K5061132) |
| activity-independent phage growth | Accessory Plasmid (AP) expressing the M13 gVI and LuxAB from the PspA promoter | [BBa\_K5061045](https://parts.igem.org/Part%3ABBa_K5061045) |
| activity-independent phage growth | Accessory Plasmid (AP) expressing the M13 gIII, M13 gVI and LuxAB from the PspA promoter | [BBa\_K5061145](https://parts.igem.org/Part%3ABBa_K5061145) |
| XylS-dependent phage growth | Accessory Plasmid (AP) expressing the M13 gVI and LuxAB from the Pm promoter | [BBa\_K5061055](https://parts.igem.org/Part%3ABBa_K5061055) |
| XylS-dependent phage growth | Accessory Plasmid (AP) expressing the M13 gIII, M13 gVI and LuxAB from the Pm promoter | [BBa\_K5061155](https://parts.igem.org/Part%3ABBa_K5061155) |

#### II.2.2. Build

All HP and AP plasmids (**Tables 3 and 4**) were assembled by Golden Gate using either BsaI or BsmBI type IIS restriction
enzymes.
First we constructed the HP and AP Golden Gate cloning platforms from DNA fragments obtained either by PCR from
plasmid templates purchased from New England Biolabs (M13KO7) or from Addgene (pJC175e) or synthesized. During this
process, care was taken to remove BsaI and BsmBI sites from M13KO7 and pJC175e. Upon sequencing, differences compared
to the M13KO7 HP sequence available on New England Biolabs website were revealed, the reason for which we added this
M13KO7 to the Parts Registry ([BBa\_K5061000](https://parts.igem.org/Part%3ABBa_K5061000)). Some of these differences are present in another M13 helper phage VCSM13
(GenBank AY598820). A complete list is presented on the Parts Registry page.
Using these ‘universal’ Golden Gate cloning platforms for HP and AP, we readily built all the other HP and AP
plasmids.

#### II.2.3. Test

Selection phage (SP) production was carried out in *E. coli* S2060 cells in three different conditions in a
XylS-dependent or independent manner, with or without antibiotics selection as described by **Miller *et al.*,
2020a**. The thus produced phages were analyzed by plaque assay and luminescence monitoring.

**XylS-independent SP production without antibiotics selection**
The *E. coli* S2060 cells were first transformed with the Accessory Plasmid (AP) expressing either the M13 gVI or
gIII-gVI and LuxAB from the PspA promoter ([BBa\_K5061045](https://parts.igem.org/Part%3ABBa_K5061045) and [BBa\_K5061145](https://parts.igem.org/Part%3ABBa_K5061145), respectively). Transformed cells were
made competent and then transformed with the various HP constructs (**Table 3**). Care was taken for matching the
selection markers (gVI and gIII-gVI) between the HP and the AP. As controls, the HP Golden Gate cloning platform as
well as the M13KO7 were included. At the end of the transformation protocol, cells were cultured in 10 mL liquid LB
media (without any antibiotic) at 37°C with shaking at 200 rpm. After this overnight incubation, cells were pelleted
by centrifugation (4000 rpm for 10 minutes at 4°C), and the supernatant filtered using a 10 mL syringe fitted with a
13-mm, 0.22 µm PVDF or PES syringe filter to remove residual cells. Filtered phages were stored at 4°C.

**XylS-independent SP production with antibiotics selection**
As the transformation procedure is not always very efficient especially when using home-made prepared competent cells,
we modified the above described protocol and cultured the transformed cells in LB media supplemented with 5 µg/mL
Tetracycline (to select for F’ containing S2060 cells), 12.5 µL kanamycin (to select for HP containing cells) and 50
µg/ml ampicillin (to select for AP containing cells).

**XylS-dependent SP production**
The XylS-dependent SP production was carried out slightly differently depending on whether the PANCE of PHAGEVO HP
were used:

* for HP constructs designed for PANCE, *E. coli* S2060 cells were used.
* for HP construct designed for PHAGEVO, *E. coli* S2060 cells were first transformed with a plasmid expressing the T7
  RNA polymerase from the pBad promoter inducible by L-Arabinose provided by the hosting lab to allow expression of
  XylS from the T7 promoter.

These *E. coli* cells were then co-transformed with the various HP constructs (**Table 3**) and the corresponding AP
expressing either the M13 gVI or gIII-gVI and LuxAB from the Pm promoter ([BBa\_K5061055](https://parts.igem.org/Part%3ABBa_K5061055) and [BBa\_K5061155](https://parts.igem.org/Part%3ABBa_K5061155),
respectively). Care was taken for matching the selection markers (gVI and gIII-gVI) between the HP and the AP. As
controls, the HP Golden Gate cloning platform as well as the M13KO7 were included. At the end of the transformation
protocol, cells were cultured in 10 mL liquid LB media supplemented with 1 mM m-toluate to activate XylS that induces
the expression of the M13 phage protein expressed from the AP plasmid. After an overnight incubation at 37°C with
shaking at 200 rpm, cells were pelleted by centrifugation (4000 rpm for 10 minutes at 4°C), and the supernatant
filtered using a 10 mL syringe fitted with a 13-mm, 0.22 µm PVDF or PES syringe filter to remove residual cells.
Filtered phages were stored at 4°C.

**Plaque assay**
Plaque assay were performed in an activity-independent manner using *E. coli* S2060 cells carrying the AP expressing
either the M13 gVI or gIII-gVI and LuxAB from the PspA promoter ([BBa\_K5061045](https://parts.igem.org/Part%3ABBa_K5061045) and [BBa\_K5061145](https://parts.igem.org/Part%3ABBa_K5061145),
respectively).
For this 150 µL of these recipient cells at an OD600 between 0.6 and 1 were mixed with 10 µL of SP phages prepared as
described above, 10 mL of liquid Soft LB Agar (10 g/L Tryptone, 5 g/L Yeast Extract, 5 g/L NaCl, 7.5 g/L Agar, melted
and cooled down at 55°C), and 40 µL of Bluo-Gal 20 mg/mL, then rapidly spread on LBagar plates (without antibiotics).
After an overnight incubation at 37°C, lysis plaques were observed with the naked eye.

**Luminescence monitoring**
Luminescence monitoring of phage propagation was performed in an activity-independent manner using *E. coli* S2060 cells
carrying the AP expressing either the M13 gVI or gIII-gVI and LuxAB from the PspA promoter ([BBa\_K5061045](https://parts.igem.org/Part%3ABBa_K5061045) and
[BBa\_K5061145](https://parts.igem.org/Part%3ABBa_K5061145), respectively).
For this 10 µL of these recipient cells at an OD600 between 0.6 and 1 were mixed with 10 µL of SP phages prepared as
described above in 200 µL of LB medium supplemented with 5 µg/mL Tetracycline (to select for F’ containing S2060
cells) and 50 µg/mL ampicillin (to select for AP containing cells) and 400 ng/µL anhydrotetracycline in an opaque
96-well polystyrene microplate (COSTAR 3603, Corning). The plate was then incubated overnight at 37°C with shaking at
200 rpm and the luminescence and optical density at 600 nm (OD600) were taken every 15 minutes using a CLARIOstar
(BMGLabtech) plate reader. Luminescence values were normalized by OD600 to account for variations in cell density.

#### II.2.4. Learn

Both luminescence and plaque assays demonstrated the successful production of selection phages (SP) in both
XylS-dependent and independent manner (**Figures 12, 13 and 14**).

Luminescence monitoring in 96-well plate proved to be an easy way to monitor phage propagation, the reason for which
the *E. coli* S1030 strain was initially developed **[Carlson *et al.*, 2014]**. Typical bacterial
growth curves and the luminescence output are presented in **Figure 12**, where we can observe a shift
between the 2 curves indicating that the phages are produced mainly in the stationary growth phase.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/luminescence-monitoring.png)

**Figure 12**. *In vivo* characterization of the Selection Phage (SP) propagation in *E. coli* S2060 cells
carrying an Accessory Plasmid (AP) expressing LuxAB from the PspA promoter. Example of a growth curve (A) and of the
luminescence produced during phage multiplication (B) and the corresponding Luminescence/OD600 values (C).

Using this approach, we evaluated the SP phages produced in three different conditions in a XylS-dependent or
independent manner, with or without antibiotics selection, along with the controls (**Figure 13**). When
available, we tested several clones obtained upon HP assembly by Golden Gate.
Analyzing the results presented in figure **Figure 13**, we can observe that high Luminescence/OD600
values were obtained as expected for the positive controls performed with the M13KO7 taken directly from the tube
purchased from New England Biolabs (NEB) or from several preparations performed by us starting from this commercial
HP.
When phages were prepared in an activity-independent manner (**Figures 13A and 13B**), we can observe variable degrees of
Luminescence/OD600 values indicating efficient and less efficient phage preparations or testing conditions.
Transformation efficiency during phage preparation may account for these variations which may lead to higher or lower
phage titer. Testing conditions are also important, the output being dependent on the proper complementation by the AP
of the missing phage protein or the differences in cell densities of the host cells. Indeed, we can observe that lower
Luminescence/OD600 values were obtained in general when the gIII-gVI selection marker was used compared to gVI alone,
including for the M13KO7 positive control. This experiment was carried out in parallel for all samples, with two
different host cells, an inherent variable to this type of experiment.
Variable degrees of Luminescence/OD600 values were also observed when phages were prepared in a XylS
activity-dependent manner (**Figure 13C**). In this case, XylS production and induction of Pm promoter may be responsible
for these differences. It is remarkable the highest Luminescence/OD600 value was obtained with a phage prepared in a
XylS activity-dependent manner, demonstrating the efficiency of our experiment setup.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/luminescence-sppreps.png)

**Figure 13**. *In vivo* characterization of our various Selection Phage (SP) preparation through
luminescence monitoring in *E. coli* S2060 cells carrying an Accessory Plasmid (AP) expressing LuxAB from the PspA
promoter. (A) SP prepared in an activity-independent manner without antibiotics selection. (B) SP prepared in an
activity-independent manner with antibiotics selection. (C) SP prepared in a XylS activity-dependent manner. PANCE and
PHAGEVO denote the absence of presence of T7 promoters and terminators flanking the XylS gene. SP phages carry either
the XylS-K38R-L224Q or the XylS-W88C-L224Q, the two variants reported in the literature as being able to weakly detect
PA and TPA, respectively **[Li *et al.*, 2022]**.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/plaque-assay.png)

**Figure 14**. *In vivo* characterization of the Selection Phage (SP) propagation in *E. coli* S2060 cells
carrying an Accessory Plasmid (AP) expressing LuxAB from the PspA promoter. Example of lysis plaques obtained with two
different SP preparations.

---

### II.3. XylS evolution: experimental setup and phage library analysis

In PACE / PANCE / PRANCE and PHAGEVO, the evolution of the GOI happens in a reaction vessel called lagoon, where
phages infect the bacteria, replicate, and then go on to infect new bacterial cells, as illustrated in **Figures 4 and
8**. Setting up this evolution involves mixing the selection phages with the host cells, applying a selection pressure,
monitoring the phage propagation before harvesting them and analyzing the activity of the GOI.

#### II.3.1. Design

Performing XylS evolution experiments required not only the preparation of selection phages described above, but also
the preparation of the appropriate lagoon host cells.
Selection phages are lacking either the gVI or both gIII and gVI. As for the experiments performed during the phage
production, host cells need to carry the AP plasmids expressing these missing genes. We used the same AP plasmids
listed above (**Table 4**) that allowed us, when using the Pm promoter ones, to apply a selection pressure depending on
the level of XylS induction by either PA or TPA. The existing AP plasmids also allowed us to have positive and
negative controls over phage infection when the PspA phage shock or no promoter conditions were used, respectively.
Moreover, these AP plasmids allow real-time monitoring of phage propagation by luminescence in *E. coli* S2060.
In addition, to increase mutation rates, host bacterial cells carry a mutagenesis plasmid (MP in **Figures 4 and 8**). The
most potent one described in the literature is MP6 which expresses 6 genes (dnaQ926, dam, seqA, emrR, ugi and PmCDA1)
that insert mutations, block the DNA repair system and are involved in the DNA methylation system **[Badran &
Liu, 2015]**. The drawback of this system is that mutations are inserted all over the genome and not only on
our gene of interest, which can result in loss of interesting variants if the system becomes non-functional due to
mutations in essential genes.
In contrast, in Evolution.T7 **[[https://2021.igem.org/Team:Evry\_Paris-Saclay](https://2021.igem.org/Team%3AEvry_Paris-Saclay%5D)]**,
mutations are focused in the GOI, thanks to the specificity of the T7 RNA polymerase (T7RNAP) for its cognate promoter
and terminators flanking the GOI as illustrated in **Figure 5**. The Evolution.T7 constructs expressing the
T7RNAP linked to a base deaminase (BD) either a cytosine or an adenosine deaminase (respectively CD and AD)
are carried by low copy plasmids (pSEVA221 and pSEVA471) and the expression is inducible by anhydrotetracycline for
BD-T7RNAP and by L-arabinose for BD-T7RNAPCGG-R12-KIRV.
In PHAGEVO we decided to construct 4 mutagenic plasmids to express the most effective of the CD and AD
deaminases of Evolution.T7, evoCDA1-BE4max and ABE8.20-m respectively, each fused to the T7RNAP and T7RNAPCGG-R12-KIRV
for forward and reverse sense, respectively. These constructs aimed also to harmonize the expression system by using
the same pBad promoter and the same backbone as the MP6 plasmid. The list of all mutagenic plasmids and their controls
is presented in **Table 5**.

**Table 5**. Mutagenesis plasmids (MP) designed, built or purchased and used for the evolution of XylS through PANCE and
PHAGEVO.

| Usage | MP Description | MP Part Number / Reference |
| --- | --- | --- |
| PA(N)CE | MP6, Mutagenesis Plasmid expressing dnaQ926, dam, seqA, emrR, ugi and PmCDA1 proteins from the pBad promoter | **[Badran & Liu, 2015]**, Addgene #69669 |
| Negative control | Expression Plasmid (EP) Golden Gate cloning platform, used here as a negative control as it is an empty backbone with the same origin of replication and the same antibiotic resistance marker as the MP constructs | [BBa\_K5061033](https://parts.igem.org/Part%3ABBa_K5061033) |
| PHAGEVO | Mutagenesis Plasmid (MP) expressing the evoCDA1-BE4max-T7RNAP fusion protein from the pBad promoter | [BBa\_K5061041](https://parts.igem.org/Part%3ABBa_K5061041) |
| PHAGEVO | Mutagenesis Plasmid (MP) expressing the ABE8.20-m-T7RNAP fusion protein from the pBad promoter | [BBa\_K5061042](https://parts.igem.org/Part%3ABBa_K5061042) |
| PHAGEVO | Mutagenesis Plasmid (MP) expressing the evoCDA1-BE4max-T7RNAPCGG-R12-KIRV fusion protein from the pBad promoter | [BBa\_K5061043](https://parts.igem.org/Part%3ABBa_K5061043) |
| PHAGEVO | Mutagenesis Plasmid (MP) expressing the ABE8.20-m-T7RNAPCGG-R12-KIRV fusion protein from the pBad promoter | [BBa\_K5061044](https://parts.igem.org/Part%3ABBa_K5061044) |

As a bacterial host cell, we decided to construct the *E. coli* S2060 ∆*ung* ∆*nfi* that combines the advantages of the
PACE strain (the S2060) with those of the Evolution.T7 strain (∆*ung* ∆*nfi*), as described above.

Several host cells carrying combinations of various AP and MP plasmids are thus possible. Considering also the number
of selection phages we prepared and tested (**Figure 13**), the number of XylS evolution experiments to
be conducted is not a negligible one. This combinatorial explosion was noticed early on in the development of our
project, and, to be able to test many conditions in parallel, we decided to adopt the non continuous PANCE strategy to
implement the PHAGEVO system.

#### II.3.2. Build

All MP plasmids (**Table 5**) were assembled by classical restriction digestion between MP6 and the Evolution.T7 plasmids
build by the iGEM Evry Paris-Saclay 2021 team ([BBa\_K3766109](https://parts.igem.org/Part%3ABBa_K3766109), [BBa\_K3766110](https://parts.igem.org/Part%3ABBa_K3766110), [BBa\_K3766106](https://parts.igem.org/Part%3ABBa_K3766106), [BBa\_K3766107](https://parts.igem.org/Part%3ABBa_K3766107)).

The EP Golden Gate cloning platform, used here as a negative control as it is an empty backbone with the same origin
of replication and the same antibiotic resistance marker as the MP construct, was assembled by Golden Gate using the
BsmBI type IIS restriction enzymes and DNA fragments obtained either by PCR from plasmid templates purchased from
Addgene (MP6) or synthesized.
The construction of AP plasmids is presented above, as well as that of the *E. coli* S2060 ∆*ung* ∆*nfi* strain.

#### II.3.3. Test

**XylS evolution experiments**
XylS evolution experiments were performed in 96-well microplates and were monitored through luminescence measurements as
described above.
The *E. coli* S2060 ∆*ung* ∆*nfi* cells were first co-transformed with combinations of the various AP and MP plasmids listed
in **Tables 4 and 5**, respectively. Transformed cells were selected and propagated in LB media supplemented with 5 µg/mL
tetracycline (to select for F’ containing S2060 ∆*ung* ∆*nfi* cells), 50 µg/ml ampicillin (to select for AP containing
cells), 17.5 µL chloramphenicol (to select for MP containing cells) and 100 mM Glucose to catabolically inhibit any
potential leaky expression form the pBad promoter and thus the expression of the mutagenic genes.
Then, 10 µL of these recipient cells at an OD600 between 0.6 and 1 were mixed with 10 µL of SP phages prepared as
described above in 200 µL of LB medium supplemented with 5 µg/mL tetracycline, 50 µg/mL ampicillin, 17.5 µg/mL
chloramphenicol, 400 ng/µL anhydrotetracycline, 0.2% L-arabinose (to induce the expression of the mutagenic genes from
the pBad promoter) and 100 µM of either PA or TPA. Control experiments without PA / TPA were also performed.
The opaque 96-well polystyrene microplate (COSTAR 3603, Corning) was then incubated overnight at 37°C with shaking at
200 rpm and the luminescence and optical density at 600 nm (OD600) were taken every 15 minutes using a CLARIOstar
(BMGLabtech) plate reader. Luminescence values were normalized by OD600 to account for variations in cell
density.
Cultures displaying Luminescence/OD600 values higher in the presence of MP plasmids compared to the values obtained in
their absence were selected, transferred in 1.5 mL microtubes and centrifuged (7000 rpm for 3 minutes at 4°C) and the
supernatant filtered using a MultiScreen-GV Sterile, clear 96-well filter plate with 0.22 um pore size Hydrophilic
PVDF membrane (Millipore) fixed on a DeepWell plate. Filtered phages were stored at 4°C.

**Analysis of evolved phages**
Evolved phages were analyzed by plaque assay and luminescence monitoring following the same protocols as described
above.
As their volume is low (
<200 µL), an aliquot was used to re-infect host cells and were thus multiplied in a XylS-independent manner as
described above.
Phage DNA was prepared using the E.Z.N.A. M13 DNA Kit (Omega Bio-Tek) and analyzed by spectrophotometry.
To produce dsDNA for sequencing purposes, we used the Illustra TemphiPhi amplification kit (Cytiva) and performed
rolling circle amplification (RCA) on 0.5 µL of ether the evolved phages directly recovered from the evolution 96-well
plate, or on the phage DNA extracted with the E.Z.N.A. M13 DNA Kit, following the supplier's protocol. RCA products
were further digested with the AvrII/XmaJI restriction enzyme, then purified using the Monarch DNA CleanUp kit (New
England Biolabs). The concentration of dsDNA was evaluated with the Qubit™ 1X dsDNA BR (Broad Range) Assay
(Invitrogen), then the samples having a concentration above 30 ng/µL were sequenced at Eurofins Genomics using Oxford
Nanopore Technologies for clonal / linear amplicons.

Evolved phages were also used as templates to amplify the XylS coding sequence as detailed on the [Part Collection](https://2024.igem.wiki/evry-paris-saclay/part-collection/)
page on this wiki.

#### II.3.4. Learn

96 XylS evolution experiments were performed in parallel in 96-well plates using all phage preparations described
above. As the number of possible combinations between the SP, AP, MP and PA/TPA inducers was high, we only assessed
MP6 as a mutagenic plasmid for SP phages having the XylS gene not flanked by the T7 promoters and terminators
(classical PANCE), and all 5 mutagenic plasmids on constructs having them (PHAGEVO). Moreover, evolution experiments
toward PA detection were initiated only with SP phages carrying the XylS-K38R-L224Q gene while the evolution toward
TPA detection were initiated only with SP phages carrying the XylS-W88C-L224Q gene as these variants were reported to
have the ability to weakly detect the plastic degradation products PA and TPA, respectively **[Li *et
al.*, 2022]**.
Luminescence monitoring of these evolution experiments allowed us to identify 30 phage populations showing increased
Luminescence/OD600 values in the presence of a given MP plasmids compared to the values obtained with no mutagen
(**Figure 15**).

These 30 phage populations were isolated and the presence of phages was confirmed by plaques assays (data not shown)
and through luminescence monitoring (**Figure 16**). Data show that some phages are present in higher
titer than others in the various preps.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/luminescence-evolution-ratios.png)

**Figure 15**. Luminescence monitoring of *in vivo* evolution experiments in *E. coli* S2060 ∆*ung* ∆*nfi* cells
carrying an Accessory Plasmid (AP) expressing LuxAB from the Pm promoter, various mutagenic plasmids and (A) SP
prepared in an activity-independent manner without antibiotics selection, (B) SP prepared in an activity-independent
manner with antibiotics selection, (C) SP prepared in a XylS activity-dependent manner. PANCE and PHAGEVO denote the
absence or presence of T7 promoters and terminators flanking the XylS gene, respectively. SP phages carry either the
XylS-K38R-L224Q or the XylS-W88C-L224Q, the two variants reported in the literature as being able to weakly detect PA
and TPA, respectively **[Li *et al.*, 2022]**. Data represent the ratio between the
Luminescence/OD600 values observed in the presence of a given MP plasmid compared to the values obtained with no
mutagen.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/luminescence-evolvedsp.png)

**Figure 16**. *In vivo* characterization of the 30 selected Evolved Phages through luminescence monitoring
in *E. coli* S2060 cells carrying an Accessory Plasmid (AP) expressing LuxAB from the PspA promoter. PANCE and PHAGEVO
denote the absence or presence of T7 promoters and terminators flanking the XylS gene. PA and TPA denote the target
compound for which the phages were evolved for starting from XylS-K38R-L224Q or XylS-W88C-L224Q, respectively.

To further characterize them, M13 ‘minipreps’ were performed and the single stranded DNA (ssDNA) amount was estimated
by spectrophotometry. The results presented in **Figure 17** show that the amount of purified ssDNA is
around 10 ng/µL (for an elution volume of 50 µL). The 260/280 ratios are, for the majority of the samples, around 1.8,
a value generally accepted as an indication of ‘pure’ for DNA. However, the 260/230 ratios are low, indicating the
presence of contaminants which absorb at 230 nm like EDTA, guanidine, trizol, phenol, carbohydrates.

![](https://static.igem.wiki/teams/5061/wiki/figures/engineering-success/nanodrop.png)

**Figure 17**. Spectrophotometric analysis on M13 ssDNA preparations of evolved phages. (A) ssDNA
concentration estimated based on the absorbance values at 260 nm and an extension coefficient of 33 ng/µL-1 (B)
260/280 and 260/230 Ratios.

Furthermore, in order to analyze the sequence variability of the evolved phages, the phage ssDNA was subject to
rolling circle amplification (RCA), and the thus obtained produced digested with AvrII/XmaJI restriction enzyme (that
has a unique site in all SP in the J23107 promoter driving the expression the gI M13 gene).
3 samples only had a dsDNA concentration compatible with the Eurofins Genomics requirements for NGS sequencing using
Oxford Nanopore Technologies for clonal / linear amplicons, but, unfortunalely, the analysis did not yield a result.

Finally, the 30 evolved phage preparations were used in PCR reactions that readily allowed the amplification of the
XylS gene with specific primers. All the experimental details and the subsequent results obtained for this experiment
are detailed in the [Part Collection](https://2024.igem.wiki/evry-paris-saclay/part-collection/) page on this wiki.

---

## CONCLUSIONS

The goal of this year iGEM Evry Paris-Saclay team was to develop a new tool for *in vivo* continuous evolution of
proteins, with improved properties compared to previous technologies. We came up with this idea and decided to create
PHAGEVO: a new system that takes advantage of the best aspects of two powerful directed evolution technologies: the
Phage-Assisted (Non) Continuous Evolution, or PA(N)CE, and Evolution.T7. The former has the big advantage to
continuously evolve and select, in the same reaction medium, for the variants of the gene of interest with improved
activity. The latter is a targeted *in vivo* mutagenesis system based on a modified T7 RNA polymerase fused with base
deaminase (BD-T7RNAP) that mutates a region of the genome flanked on both 3’ and 5’ end by specific promoters of the
BD-T7RNAP fusions, enabling mutation on both DNA strands.

Our team successfully designed and implemented the new PHAGEVO system. For this purpose, we first engineered the *E. coli* S2060 ∆*ung* ∆*nfi* strain, also known as the PHAGEVO *E. coli* strain, and confirmed its genotype by whole bacterial
sequencing using Nanopore technology. Then, we engineered bacteriophage M13 to insert our gene of interest, XylS with
the Evolution.T7 cassette in the phage genome. We also constructed several different plasmids constructs for both
mutagenesis (mutagenesis plasmids) and selection (accessory plasmids) based on Evolution.T7 and the latest improvements of
the PA(N)CE technology. Different combinations of these plasmids where used in the subsequent evolution experiments.

We used the newly developed PHAGEVO system to run evolution experiments on the promiscuous benzoate derivative
sensitive XylS transcription factor to make it more sensitive and specific toward either Phthalic Acid (PA) or
Terephthalic Acid (TPA). Phage production in both XylS-dependent and independent manner was achieved, demonstrating
the complementation between XylS and its cognate promoter in the PHAGEVO setup.

Finally, evolved phages were used to isolate a collection of XylS variants that were further analyzed as described on
the [Part Collection](https://2024.igem.wiki/evry-paris-saclay/part-collection/) page on this wiki.

---

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