iGEM 2020 — Team Ionis_Paris

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# Team:Ionis Paris/Notebook

Notebook

# Notebook

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# Team:Ionis Paris/Engineering

Engineering success

# Engineering success - IGEM IONIS

Engineering success

Research

Build

Imagine

Test

Learn

Improve

BacTail’s iGEM

Engineering Circle

Phase 1

iGEM’s 2020 competition was a particular year. Given the public health situation, our team
faced unexpected circumstances, both positive and negative.
For one, we consider ourselves lucky to have been able to enter a laboratory for a while.
Still, we experienced considerable delays in the reception of our consumables or synthetized
DNA fragments, which slowed BacTail’s iGEM engineering circle drastically.

However, due to the lock-down, we had the opportunity to exchange with other teams more
frequently, which resulted in an unprecedented collaboration with an
iGEM Team from India(IISER TIRUPATI)
for the
phase 3
of our BacTail project.

For more information of our project:
Design Phase 1,
Design Phase 2,
Desgin Phase 3
and
Results.

Notes

**Chassis**: In synthetic biology, chassis are cells which genetic systems can be used for
its basic metabolic pathway to produce desired biological compounds. They should be capable of
essential chemical transformations, such as the production of energy and co-factor regeneration.

***E. coli***: Escherichia coli is a Gram-negative bacteria, meaning that its cell wall is made of
peptidoglycan. In average, E. coli’s are about 2.0 μm long and 0.25–1.0 μm in diameter.

**FITC**: Fluorescein isothiocyanate has excitation and emission spectrum peak wavelengths of
approximately 495 nm/519 nm.

**Gp37**: Gene product 37 is the most distal region of the Long Tail Fiber of the T4
bacteriophage.

**Lpp**: E. coli’s lipoprotein is a alpha-helical protein and numerically the most abundant
protein in E. coli, with more than 1 million copies per cell.

**LTF**: Bacteriophage T4 Long Tail Fibers are composed of four different proteins (gp 34,
gp35, gp36, and gp37) and are segments of approximately 70 nm.13

**Lpp-Ompa**: The co-expressed Lpp-OmpA genes include a lipoprotein signal peptide and the
first nine N-terminal amino acids of the E. coli lipoprotein (Lpp) attached to a transmembrane
domain (amino acids 46–159) from outer membrane protein A (OmpA).3

**OmpA**: Outer membrane protein A family is a group of surface-exposed, porin proteins and
its molecular mass is between 28 kDa to 36 kDa11.

**PCR**: Polymerase chain reaction is a method used to rapidly make millions to billions of
copies of a specific DNA sample.

**RBP**: Receptor Binding Protein, concerning our topic the gp37 is part of the RBPs of the
T4 bacteriophage’s LTFs.

**TEV site**: The TEV cut site is the sequence as follows: ENLYFQ\(G/S) (where “\” denotes
the cleaved site) and is a highly specific sequence of the cysteine protease from Tobacco Etch
Virus (TEV).

## Phase 1 | Targeting and binding to the target bacteria

![]()

The first phase of our project consists in specifically recognizing and binding a target
bacterium. Thus, our very priority was to find a system that would grant recognition and
specific binding to target bacteria which are resistant to antibiotics.

One of our teammates came up with an idea:
**why not exploit the recognition abilities of the "natural" predators of bacteria,
which are bacteriophages?**
Rapidly, as a team, we tried to identified which exact region was responsible for the
bacteriophage binding.

This is where our project BacTail begun.

![]()

Picture of Albane (Ionis Paris Team Leader) with the first draft of the initial idea
of BacTail

Idea came end of April 2020 (Picture was taken after the lock-down in France)

#### Organization – Journal club sessions

From this point on, many questions arose that needed answers and required a substantial
amount of research. Being confined to our respective homes, we started to organize
weekly virtual "journal club" sessions. These sessions entailed presenting scientific
articles that seemed relevant for the questions we each had. Thanks to these sessions,
we were able to start debates and take decisions during the research phase. The
questions and topics we encountered are as follows:

#### Which is the exact region that is responsible for the specific binding of a bacteriophage to its target bacterium?

The first information that came up was that the tip of the long tail fibers (LTF) of a
certain class of bacteriophages recognize specific sites on the outer membranes of
bacteria13. Thus, our goal was to design a system which function is to export
the distal tip of a phage receptor binding protein (RBP) on the outside membrane of our
future chassis.

#### Can we use the distal tip of a phage receptor binding protein on its own?

Once we had found the T4 RBP, specific for recognition, we had to measure the
feasibility of using the RBP of bacteriophages on its own. In fact, the article
published in 2019 by Dunne *et al.* showed that the recognition and binding of
*Salmonella* cells could be accomplished by only using the LTF of the S16 phage,
the natural predator of *Salmonella*1. More information here:
Design PHASE 1.

Eventually, the experiments conducted by Dunne *et al*. successfully proved that
the unique gp37-gp38 protein of the S16 phage is sufficient to bind the target
Salmonella cell. Therefore, we decided to use these receptor binding proteins in our
BioBrick to specifically recognize and bind a target bacterium.

#### How do we export the receptor binding protein at the the surface of our chassis?

For the exportation system, we found the part
[BBa\_K1694002](http://parts.igem.org/Part%3ABBa_K1694002)
designed by iGEM15\_NCTU-Formosa and encoding for the Lpp-OmpA expression
system3.

#### Seeking the appropriate chassis

It demanded a considerable amount of research to decide which target and chassis to use
for our project. The only information we had was that, for safety issues, we were
limited to class 1 micro-organism. For the chassis, we chose an
*Escherichia coli* (*E.coli*) K12 since it is a well-studied benign laboratory
model organism2 and performing experiments, such as transformations, would be
easier.

#### Seeking the appropriate target

For the target, we first needed to evaluate the already characterized bacteriophages in
literature. Many host-associated receptors targeted by bacteriophages have been
researched4,5,6,7,8,9. In Bertozzi *et al.10*, several tables compiles the receptors that have been identified for Gram-positive and
–negative bacteria.

Link table 1 | Gram-positive bacteria:
<https://academic.oup.com/view-large/111286353>

![]()

**Figure 1: Capture of "Table 1. Receptors in the cell wall of Gram-positive bacteria.
Host names are ordered alphabetically."10**

Link table 2 | Gram-negative bacteria:
<https://academic.oup.com/view-large/111286358>

![]()

**Figure 2: Capture of "Table 1. Receptors in the cell wall of Gram-negative bacteria.
Host names are ordered alphabetically."10**

At first, we considered taking *Bacillus subtilis* (Gram-positive) as our
target.
However, gram-positive bacteria have more complex and denser cell walls. According to
Bertozzi *et al.*, this adds some difficulty in the characterization of the phage
receptor's specificity. Additionally, it seemed that the T4 coliphage was a more studied
bacteriophage12. Therefore, for our proof of concept, we decided to target an
 *E. coli* as well. More information on this here:
Design PHASE 1.

![]()

Created with BioRender.com

#### How will we be able to validate the expression of the protein?

Finding how to express the gp37 protein on the surface of our chassis was one challenge
but validating its expression and its specific localization was a real obstacle.
Adding a fluorescence marker, a red fluorescence protein, was a rudimentary idea to
localize the protein or any expression at all, around the bacteria thanks to
microscopy.
Additionally, we wanted to be able to cut the protein once expressed on the outer
membrane of our chassis and purify the expressed protein. Therefore, we added a cleavage
site, the TEV cleavage site, which is a unique cleavage site of the cysteine protease
from the Tobacco Etch Virus (TEV)13. This cleavage site is followed by a
His-tag for the purification step which can be performed with Ni2+
chromatography columns14 , see on **Figure 3**.

![]()

**Figure 3: Validation steps to confirm expression of gp37 on the surface of our chassis**
All icons were created with BioRender.com

![]()

### Imagine

At last, this is a schematic representation of what we have imagined **(Figure 4)**:

![]()

**Figure 4: Schematic representation of the T4 bacteriophage gp37 localization on the
outer membrane of our chassis**
Abbreviations: Co-expressed lipoprotein with the outer membrane protein A (Lpp-OmpA);
Short linker (Gly-Gly-Ser-Gly) to fuse the proteins; TEV peptide signal (ENLYFQ\(G/S)
(where “\” denotes the cleaved site) ; 10xHis-tag; Red fluorescent protein (RFP); Gene
product (gp) 37;

![]()

### Build

After having gathered all scientific knowledge, we translated what we imagined in
**Figure 4** into our first BioBrick:

![]()

**Figure 5: BioBrick 1, expression and localization of the T4 bacteriophage gp37 to
the outer membrane of our chassis**
First line: Promoter (BBa\_J23104), Ribosome binding site (RBS - BBa\_B0030), Co-expressed
lipoprotein with the outer membrane protein A (Lpp-OmpA -
[BBa\_K1694002](http://parts.igem.org/Part%3ABBa_K1694002)), Short linker (Gly-Gly-Ser-Gly) to fuse the proteins (BBa\_K243004), TEV cleavage site
(BBa\_J18918), 10xHis-tag (BBa\_K844000), Red fluorescent protein (RFP - BBa\_E1010), Gene
product (gp) 37 (BBa\_K3409000), Double-terminator (BBa\_B0015) | Second line: Promoter
medium (BBa\_J23107), Ribosome binding site (RBS - BBa\_B0032), chaperone protein gp38
(BBa\_K3409001), chaperone protein 57A (BBa\_K3409002), Terminator (BBa\_B1005).

![]()

### Test

Once our fragments arrived, we tried to assemble them via Hifi Assembly technique to
obtain the BioBrick 1 as depictured in **Figure 5**, more information on this on the
Phase 1 Results page. However, with this BioBrick design we faced different issues.

#### Problem 1 - Our plasmid backbones

We had 4 fragments to assemble, which made it a little bit difficult in the short time
we had. On top of that, the plasmid backbones we had in our possession, already
contained the *RFP*, between the prefix and suffix region.
Even only after amplifying the backbone via PCR, we still obtained too many red colonies
with our ligation control (cut plasmid with no insert but ligase) after transformation.

#### Problem 2 – The verification step

Additionally, for the verification step, we were supposed to reveal the presence of the
BioBrick on the surface of our bacteria with microscopy thanks to the presence of the
*RFP*. However, the microscope we needed was not available on our campus at that
time. Therefore, we needed to urgently adapt our construction to a new validation
technique.

![]()

### Learn / Improve

#### Solutions for Problem 1 - Our plasmid backbones

Solution A: Addition of Alkaline phosphatase

Regarding the plasmid, we obtained too many colonies on the control plates with the
uncut plasmid backbone only.
Our hypothesis is that the plasmid would re-ligate itself instead of ligate with the
inserts. That is why, after some research we realized that this self-ligation process
could be enhanced by the presence of phosphate at the 5' ends of the digested
plasmid.
In a scientific article, we found that adding Alkaline phosphatase15, after
having performed the restriction enzyme digestion, would remove the phosphates group
located on the 5' end of the plasmid DNA and thus prevent self-ligation.

![]()

**Figure 6: Schematic representation of the action of the alkaline phosphatase**

![]()

**Figure 7: Picture of plates with colonies transformed with circular pSB4A5 treated
with and without alkaline phosphatase**
| **1st plate**: The plate is full of red and white colonies; these bacteria were
transformed with plamsids that have not been treated with alkaline phosphatase. |
**2nd plate**: The plate contains few colonies and these bacteria have been
transformed with the plasmid that has been treated with alkaline phosphatase

Solution B: Addition of DpnI

Furthermore, another hypothesis is that some plasmids that were used as DNA template for
our PCR remained intact in our samples until the transformation step.
In that case they would still possess the *RFP* gene, leading to red transformed
bacteria.

DpnI is naturally present in bacteria16 and is naturally used as a defense system
allowing the bacterium to defend itself against bacteriophages that infect it by
inserting their viral DNA .
This DNA is double stranded and methylated while the bacterial DNA is non-methylated.
This is why it has the ability to recognize and cleave only when its recognition site is
methylated and therefore belongs to the bacteriophage.
In our project, the DpnI enzyme will be used on the plasmid after the PCR step, in order
to destroy the original template plasmid that has not been amplified.

We did the test and amplified two pSB1A3 plasmids with the same primers by PCR.
One was treated with DpnI and one not. Both were transformed in bacteria and the next
day a major difference between them was observable, see **Figure 7**.

![]()

### Test

![]()

Figure 8: Picture of the transformation of amplified pSB1A3 plasmid treated with and
without DpnI after PCR amplification

1st plate: The plate is full of red colonies; these
bacteria were transformed with plamsids that have not been treated with DpnI

2nd plate: The plate contains less cells than the 1st
plates and these bacteria have been transformed with the plasmid that has been
treated with DpnI.

![]()

### Learn / Improve

#### Solution for Problem 2 – The verification step

By talking to some of our professors (see
Attributions, section laboratory), we decided to adjust our BioBrick design.
Instead of using the red fluorescence marker to validate the presence of our BioBrick,
we decided to directly target the His-tag with a primary monoclonal anti-his antibody
conjugated to a fluorescence marker that emits wavelength (517 nm) detectable with a
fluorescence spectrophotometer.

![]()

**Figure 9: Schematic representation of the detection technique of gp37 on the surface of
our chassis via His tag immunolabelling**

![]()

### Build

![]()

**Figure 10: BioBrick 1 Version 2 (V2), expression and localization of the T4
bacteriophage gp37 to the outer membrane of our chassis**
First line: Promoter (BBa\_J23104), Ribosome binding site (RBS - BBa\_B0030), Co-expressed
lipoprotein with the outer membrane protein A (Lpp-OmpA -
[BBa\_K1694002](http://parts.igem.org/Part%3ABBa_K1694002)), Short linker (Gly-Gly-Ser-Gly) to fuse the proteins (BBa\_K243004), TEV cleavage site
(BBa\_J18918), 10xHis-tag (BBa\_K844000), Gene product (gp) 37 (BBa\_K3409000),
Double-terminator (BBa\_B0015)
Second line: Promoter medium (BBa\_J23107), Ribosome binding site (RBS - BBa\_B0032),
chaperone protein gp38 (BBa\_K3409001), chaperone protein 57A (BBa\_K3409002), Terminator
(BBa\_B1005).

![]()

### Test

To verify if our bacteria have correctly exported the receptor binding protein gp37 on
their outer membrane, we established an immunofluorescence labelling protocol.
The aim of this protocol is to target bacteria in a liquid culture with an anti-his
antibody conjugated to a FITC fluorophore.
This liquid culture will then be placed into 96-well plates to be put into a
fluorescence plate reader.

A

![]()

B

![]()

**Figure 11: Immunofluorescence labelling** | **A.** Schematic representation of
the expected results after immunofluorescence labelling of our transformed bacteria with
the receptor binding protein gp37, created with BioRender.com | **B. FITC spectra**:
Green dotes line correspond to the excitation spectrum (peak at 498 nm) for FITC and the
green area correspond to the emission spectrum (peak at 517 nm) of FITC. Source: Spectra
Viewer of Thermo Fisher Scientific.

## Phase 2 | Killing the target bacteria via antimicrobial peptides

The conception of phase 2 follows an engineering cycle that is similar to phase 1 as many
questionings were in common. The design of phase 2 was carefully thought through as explained in
[phase 2](window.ROOT_URL%20%2B%20%27/Phases/phase2%27).
For phase 2, we were able to demonstrate the appropriate expression, secretion and activity of
microcin PDI using
*E. coli* DH5 alpha as our chassis. More information can be found in
results
and in the composite part that was created for phase 2:
[BBa\_K3409013](http://parts.igem.org/Part%3ABBa_K3409013)

---

[1] Dunne, M., & Loessner, M. J. (2019). Modified Bacteriophage Tail Fiber Proteins for
Labeling, Immobilization, Capture, and Detection of Bacteria. In Methods in Molecular Biology
(Vol. 1918, pp. 67–86). Humana Press Inc. https://doi.org/10.1007/978-1-4939-9000-9\_6

[2] Nicole T. Perna et. al. (2002). The Genomes of Escherichia coli K-12 and Pathogenic E. coli,
In Escherichia Coli (Chapter 1, pp. 3-53). Academic Press.
https://doi.org/10.1016/B978-012220751-8/50002-1.

[3] Francisco, J. A., Campbell, R., Iverson, B. L., & Georgiou, G. (1993). Production and
fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody
fragment on the external surface. Proceedings of the National Academy of Sciences of the United
States of America, 90(22), 10444–10448. https://doi.org/10.1073/pnas.90.22.10444

[4] Lindberg, A. A., Wollin, R., Gemski, P., & Wohlhieter, J. A. (1978). Interaction between
bacteriophage Sf6 and Shigella flexner. Journal of virology, 27(1), 38–44.
https://doi.org/10.1128/JVI.27.1.38-44.1978

[5] Wright A McConnell M Kanegasaki S Lipopolysaccharide as a bacteriophage receptor Randall LL
Philipson L Virus Receptors—Part 1 London Chapman & Hall 1980 27 5

[6] Heller K. J. (1984). Identification of the phage gene for host receptor specificity by
analyzing hybrid phages of T5 and BF23. Virology, 139(1), 11–21.
https://doi.org/10.1016/0042-6822(84)90325-8

[7] Frost L Conjugative pili and pilus-specific phages Clewell DB Bacterial Conjugation New York
Plenum Press 1993 189 222

[8] Henning U Hashemolhosseini S Receptor recognition by T-even-type coliphages Karam JD
Molecular Biology of Bacteriophage T4 Washington American Society for Microbiology 1994 291 8

[9] Rakhuba, D. V., Kolomiets, E. I., Dey, E. S., & Novik, G. I. (2010). Bacteriophage
receptors, mechanisms of phage adsorption and penetration into host cell. Polish journal of
microbiology, 59(3), 145–155.

[10] Juliano Bertozzi Silva, Zachary Storms, Dominic Sauvageau, Host receptors for bacteriophage
adsorption, FEMS Microbiology Letters, Volume 363, Issue 4, February 2016, fnw002,
https://doi.org/10.1093/femsle/fnw002

[11] Nie, D., Hu, Y., Chen, Z. et al. Outer membrane protein A (OmpA) as a potential therapeutic
target for Acinetobacter baumannii infection. J Biomed Sci 27, 26 (2020).
https://doi.org/10.1186/s12929-020-0617-7

[12] Sergio G. Bartual, et. Al. (2010). Structure of the bacteriophage T4 long tail fiber
receptor-binding tip. Proceedings of the National Academy of Sciences.107 (47) pp. 20287-20292.
DOI: 10.1073/pnas.1011218107

[13] Waugh D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags.
Protein expression and purification, 80(2), 283–293. https://doi.org/10.1016/j.pep.2011.08.005

[14] Crowe, J., Masone, B.S. & Ribbe, J. One-step purification of recombinant proteins with the
6xHis tag and Ni-NTA resin. Mol Biotechnol 4, 247–258 (1995). https://doi.org/10.1007/BF02779018

[15] Ukai, H., Ukai-Tadenuma, M., Ogiu, T., & Tsuji, H. (2002). A new technique to prevent
self-ligation of DNA. Journal of biotechnology, 97(3), 233–242.
https://doi.org/10.1016/s0168-1656(02)00107-4

[16] Johnston, C., Polard, P., & Claverys, J. P. (2013). The DpnI/DpnII pneumococcal system,
defense against foreign attack without compromising genetic exchange. Mobile genetic elements,
3(4), e25582. https://doi.org/10.4161/mge.25582

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# Team:Ionis Paris/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Ionis Paris/Results

Results

Results

The aim of our project BacTail is to create tailor-made and engineered bacteria to specifically
recognize, bind and lyse pathogenic bacteria, then self-destroy, using the powerful tools of
synthetic biology. For the experimental approach, we divided our project into 3 distinct axes for
for the laboratory:

![]()

![]()

**Phase 2**
Lyse the target bacteria using antimicrobial peptides

![]()

**Phase 3**
Incorporate of a Kill-switch system in our chassis to induce its self-destruction

**The properties of our experimental approach are as follows:**

All the steps cited above have been developed as one unique system adapted to our project – yet,
these steps can be used individually and eventually be re-adapted for other application areas.

All our phases are designed for ***Escherichia coli K12*** since we are working under
**Class 1 Biosafety level**. Yet, the ultimate goal is to create a template of this system that
can be specifically applied against different bacterial strains. Specific bacteriophages have
already been identified to target pathogenic bacteria such as ***Salmonella***, ***Listeria***, ***Mycobacterium tuberculosis***, ***Pseudomonas*** or ***Escherichia coli***. For example, the PP01 phage was shown to target the shiga-toxin producing
***E. coli*** strain1.

Therefore, we created a
software to specifically
select and customize a BioBrick to recognize the desired target bacteria for phase 1.

Further detail on our experimental procedures and protocols can be found here:
Procedures and
Protocols.

Phase 1. Targeting and binding to the target bacteria

BioBrick and primer design of the fragments for phase 1

The gene product 37 (gp37) is a critical component for the phase I of the project BacTail. To find
out more about the scientific background and
design of Phase I.

The aim for the first phase is to express the gp37 on the surface of our chassis Escherichia coli
(*E.coli*) K12 thanks to the co-expressed genes Lpp-OmpA. The following BioBrick (**Figure 1**) was created and divided into 4 different fragments to ease the assembly step with the plasmid –
for clarity, the fragments are summarized in **Figure 2B**.

![]()

**Figure 1: DNA sequence of BioBrick 1 of phase 1** | All the parts to express gp37 on the surface of
our chassis: Promoter (BBa\_J23104), Ribosome binding site (RBS - BBa\_B0030), Co-expressed lipoprotein
with the outer membrane protein A (Lpp-OmpA - BBa\_K1694002), Short linker (Gly-Gly-Ser-Gly) to fuse the
proteins (BBa\_K243004), TEV cleavage site (BBa\_J18918), 10xHis-tag (BBa\_K844000), Red fluorescent
protein (RFP - BBa\_E1010), Gene product (gp) 37 (BBa\_K3409000), Double-terminator (BBa\_B0015), Promoter
medium (BBa\_J23107), Ribosome binding site (RBS - BBa\_B0032), chaperone protein gp38 (BBa\_K3409001),
chaperone protein 57A (BBa\_K3409002), Terminator (BBa\_B1005) | Created with Benchling

![]()

**Figure 2: BioBrick 1 divided into four distinct fragments | A.** The expected assembled plasmid
with Fragments 1,2,3,4 and the backbone | **B.** A table giving the detailed composition of the four
fragments of the first phase of BacTail.

Polymer chain reaction (PCR) & gel migrations

Once received, the synthetized fragments are resuspended and amplified via polymerase chain reaction
(PCR) to obtain a higher quantity of DNA. Every PCR sample is verified by gel electrophoresis to
analyze the PCR results. The first time PCR has been performed (**Figure 3A**) for Fragment
(Frag.) 1, Frag. 2, Frag. 3 and Frag. 4, no bands are observable. After some troubleshooting and
verifications, new primers need to be ordered to amplify Fragments 1, 2, 3, and 4 again (**Figure 3B**). However, Fragment 1 required to be amplified in two steps (**Figure 3B** – Frag.1 & Frag. 1
Bis). Due to its complexity, Fragment 1 is synthetized again. Eventually, with the new version of
Fragment 1 and the optimized primers the appropriate bands for Frag. 1 (1218 bp), Frag. 2 (1541 bp),
Frag. 3 (1669 bp) and Frag. 4 (845 bp) are at the expected size (**Figure 3C**).

![]()

**Figure 3: Gel migration results of Fragment 1,2,3, and 4 with optimization. | A.** 1kb+ DNA ladder
was used for our gels **A.** First PCR amplification of Fragment 1,2,3 and 4. **B.** Second round
of PCR amplification of Fragment 1,2,3, and 4 **C.** Third and last round with optimized conditions
of PCR amplification of Fragment 1,2,3, and 4 **D.** Expected results generated after PCR simulation
with SnapGene.

Assembly step

The four fragments purified in the previous step (see above) sequences have been assembled into the
plasmid pSB1A3 using the NEBuilder Hifi Dna Assembly Master Mix. This technique is a molecular
cloning method used to join multiple DNA fragments in a single, isothermal reaction2. The
fragments were designed in such a way that adjacent fragments have identical sequences on the ends,
called overhangs, see **Figure 4**. Overhangs are added during the synthesis of the different
fragments. However, overhang sequences can be added via PCR with primers containing a 5‘ end
identical to the adjacent fragment and a 3‘ end that anneals to the target sequence. In our case,
the primers are 30 base pairs (bp) long, where the first 15 bp match the end of the adjacent
fragment and the last 15 bp annealed to the target sequence.

![]()

**Figure 4: Schema of the HiFi Assembly reaction** | Created with BioRender.com

![]()

**Figure 5: Expected results of the Hifi Assembly of the 4 fragments and the plasmid pSB1A3 | A.**
The expected assembled plasmid with Fragments 1,2,3,4 and the backbone | created with SnapGene

**Conclusion**

Due to difficulties encountered during the experimental procedures and time limitation, we decided
to change our verification technique and ordered a new DNA fragment. More information on this in
Engineering Success.

New BioBrick – Version 2: without RFP

Due to difficulties encountered during the experimental procedures and time limitation, we decided
to change our verification technique and ordered a new DNA fragment. More information on this in
Engineering Success.

![]()

**Figure 6: New DNA sequence of BioBrick 1 version 2 (V2) of phase 1 |** All the parts to express
gp37 on the surface of our chassis: Promoter (BBa\_J23104), Ribosome binding site (RBS - BBa\_B0030),
Co-expressed lipoprotein with the outer membrane protein A (Lpp-OmpA - BBa\_K1694002), Short linker
(Gly-Gly-Ser-Gly) to fuse the proteins (BBa\_K243004), TEV cleavage site (BBa\_J18918), 10xHis-tag
(BBa\_K844000), Gene product (gp) 37 (BBa\_K3409000), Double-terminator (BBa\_B0015),Promoter medium
(BBa\_J23107), Ribosome binding site (RBS - BBa\_B0032), chaperone protein gp38 (BBa\_K3409001), chaperone
protein 57A (BBa\_K3409002), Terminator (BBa\_B1005) | Created with Benchling

Revelation of the presence of gp37 via immunofluorescence labelling

The aim was to detect the receptor binding protein gp37 expressed and directed to the membrane
thanks to the co-expressed Lpp-OmpA genes. This is achieved using fused His-tag placed after the
OmpA and before the gp37. Thus, the His-tag was targeted with a primary monoclonal anti-his antibody
conjugated to a fluorescence marker, fluorescein isothiocyanate (FITC), which excitation and
emission spectrum peak has a wavelengths of approximately 495 nm and 519 nm6, detectable
via fluorescence spectrophotometry.

![]()

**Figure 7: Expected results of the immunofluorescence labelling | 1st possibility:** In the case of
binding of the monoclonal anti-his antibody to the his-tag, a signal is detected at 519 nm.
**2nd possibility:** No attachment of the antibody and therefore no signal is detected at 519 nm |
Created with BioRender.com

**Results of the validation assay of phase I**

The aim of this assay is to target the his-tag fused to the receptor binding protein gp37, which
should be directed to the outer membrane of our transformed bacteria, via a monoclonal anti-his
antibody conjugated to fluorescein isothiocyanate (FITC). Thus, after the immunolabelling, the
fluorescence is measured at excitation wavelength of 497 nm and emission wavelength of 519 nm.

The controls for this assay are bacteria that have been transformed with the pSB1A3 containing the
RFP only and that undergo the same treatment as our samples. The samples are bacteria containing the
plasmid construct of phase I within the his-tag fused to the receptor binding protein gp37.
Additionally, samples and controls are treated with and without anti-his antibodies diluted at
1/1000 and 1/100. Finally, our samples have been done in triplicate (S1-series, B1-series and B2
series).

![]()

**Figure 8: Immunofluorescent labeling of engineered bacteria to verify presence or absence of gp37
receptor binding protein on their outer membrane |**
Controls are bacteria that have been transformed with pSB1A3 containing red fluorescent protein (RFP)
only; Samples are bacteria transformed with the construction of phase I containing the receptor binding
protein gp37 expression system; Three treatment conditions were applied: w/o Ab: without antibody, AB
(1/1000): anti-his antibody diluted 1 to 1000, Ab (1/100): anti-his antibody diluted (1/100).

We can observe that all the negative control samples, which are the samples that are not treated
with the anti-his antibody (S1, B1, B2) and the control (C) emit a similar signal around 27000.
Samples treated with the anti-his antibody 1/1000 dilution (S1 Ab 1/1000, B1 Ab
1/1000, B2 Ab 1/1000 ) and the control that has been treated with the anti-his
antibody 1/1000 dilution (C Ab 1/1000), emit a signal around 27000 as well. Finally,
samples treated with the anti-his antibody 1/100 dilution (S1 Ab 1/100, B1 Ab
1/100 , B2 Ab 1/100 ) emit a signal around 43,000, 38,000, 40,000 restively,
and the control that has been treated with the anti-his antibody 1/100 dilution (C Ab
1/100) emit a signal around 40,000.

Thus, S1 Ab 1/100 (43,000) is the only sample of transformed bacteria with the construct
of phase I that has a higher signal than its controls (C Ab 1/100 = 40,000 and S1 without
antibody = 27,000), which is not concluding enough to suggest that the receptor binding protein is
well expressed on the outer membrane of our chassis. However, the fact that we have similar signals
for each conditions shows that the measured signals are well due to the presence or absence of the
anti-his antibody. However, what this data shows is that the antibody might bind unspecifically to
other regions of the bacteria or other components in the reaction tube. This specificity issue could
not be analysis in more depth because of a lack of time.

After having performed this immunolabelling, we thought that this assay was not enough to confirm
the presence or not of the receptor binding protein gp37 on the surface of our chassis. We should
rather proceed with lysing the cells and differential centrifugation steps to separate the membrane
from the bacterial cytoplasm. Then, both samples should by analyzed by performing a western blot by
targeting again the his-tag with an anti-his antibody. This would show if the protein is present in
the membrane or in the cytoplasm at all.

Experimental perspectives Phase 1 | Targeting and binding to the target bacteria

* Perform a differential centrifuge to extract the fraction with the membrane the fraction with
  the cytoplasm and detect the presence of the gp37 fused to the his-tag via Western blot in
  either fraction.
* The immunolabelling experiments should be repeated to validate or not the presence of gp37 on
  the surface of our chassis by optimizing the unspecific binding (Maybe try to use a higher
  concentration of BSA when performing the blocking step).
* Perform the experiment with the TEV protease and proceed with the protein purification with the
  Ni2+ chromatography columns specific to the his-tag.
* Validate experiments with confocal microscopy to have the possibility to see any signal at the
  surface of the chassis.
* Validate the binding of the chassis to the target.

Phase 2. Lysis of the target bacteria of interest

The aim of this phase is to lyse specific target bacteria. Thus, the microcin MccPDI (Proximity
Dependent Inhibition) is the critical component in phase 2. The MccPDI gene cluster has been
identified in the strain Escherichia coli (E.coli) 25 plasmid (Eberhart et al., 2012 ) for its
ability to kill several strains of E. coli. MccPDI targets pathogenic strains as well as Biosafety
Level 1 E. coli strains. To find out more about the scientific background and
design of Phase II.

For the sake of our project BacTail, it was the necessary to express accurate genes in order to
secrete the antimicrobial peptide to specifically lyse the target bacterium. For the construction of
our BioBrick, we used the MccPDI gene cluster because its genes are all necessary for the
appropriate expression, activity, resistance, and secretion of microcin PDI.

BioBrick and primer design of phase II BioBricks 1,2, and 3

The organization of the native MccPDI gene cluster found in E. coli 25 helped us design our own
BioBricks to enable the engineered bacteria to lyse the bacteria of interest, illustrated below :

![]()

**Figure 9: Our constructs for the expression and secretion of McpM | BioBrick 1** (BBa\_K3409010)
contains the gene mcpM which when expressed, will lead microcin PDI. This microcin has two N-Ter signal
peptides which are cleaved when secreted. The gene mcpA also present in this BioBrick encodes for an
activator of McpM and contains a CaaX protease domain. Finally, the gene mcpI encodes for the immunity
protein of McpM for the producing strain; **BioBrick 2** (BBa\_K3409011) is coding for the McpD
protein which acts as a membrane fusion protein creating a channel through the periplasm and connects to
TolC; **BioBrick 3** (BBa\_K3409012) is coding for the McpB protein which contains transmembrane
domains as well as nucleotide binding domains which have highly conserved motifs characteristic of the
ABC transporter superfamily of proteins | Created with Benchling.

![]()

**Figure 10: The final construct for the expression and secretion of McpM | A.** The expected
assembled plasmid with BioBricks 1,2,3 and the backbone | **B.** A table giving the detailed
composition of the three BioBricks of the second phase of BacTail.

Concerning the design, the three BioBricks were synthetized for further assembly via IDT. Thus, we
decided to add homologous sequences by PCR between the fragments to assemble for
**Hifi Assembly** method (see Hifi Assembly in the Assembly section further down this the page).
Additionally, we added BioBrick prefix and suffix enzyme restriction sites to assemble the entire
cluster via the **BioBrick Assembly** method as well (see BioBrick Assembly in the Assembly
section further down this the page).

![]()

**Figure 11: Schematic representation of the three BioBricks for phase II and their corresponding
length (bp) | A.**
Primers for the BioBrick Assembly method amplifying on the prefix and suffix region **B.** Primers
for the Hifi Assembly method, the primers bind after the prefix and suffix region and add a homologous
short sequence of the fragment it will be assembled with | Created with BioRender.com

Polymer chain reaction (PCR) and gel migration of BioBrick 1, BioBrick 2, and BioBrick 3

Once received, the synthetized fragments are resuspended and amplified via polymerase chain reaction
(PCR) to obtain a higher quantity of DNA. Every PCR sample is verified by gel electrophoresis to
analyze the PCR results. First, the appropriate bands for BioBrick 1 (1.5 kb), BioBrick 2 (1.4 kb),
and BioBrick 3 (2.4 kb) amplified with the primers for Hifi Assembly method are present at their
expected size (**Figure 12A**). Then, the appropriate bands for BioBrick 1 (1.5 kb), BioBrick 2
(1.4 kb), and BioBrick 3 (2.4 kb) amplified with the primers for BioBrick Assembly method, are
present at their expected size on **Figure 12B**.Finally, BioBrick 2 (1.4 kb) and BioBrick 3 (2.4
kb) amplified with the primers for BioBrick Assembly method, are present at their expected size on
**Figure 12C**.

![]()

**Figure 12: Gel migration results of BioBricks 1, 2 and 3 |** A 1kb+ DNA ladder was used for all
gels A. PCR amplification of BioBrick (BB) 1(1.5 kb) , BioBrick 2 (1.4 kb) and BioBrick 3 (2.4 kb) with
primers for Hifi Assembly method B. PCR amplification of BioBrick (BB) 1(1.5 kb), BioBrick 2 (1.4 kb)
and BioBrick 3 (2.4 kb) with primers for BioBrick Assembly method C. Expected results simulated with
SnapGene containing the 1 kb+ DNA ladder, BioBrick (BB) 1(1.5 kb) , BioBrick 2 (1.4 kb) and BioBrick 3
(2.4 kb).

Assembly step

The three fragments purified in the previous step (see above) are assembled into the plasmid pSB1A3,
pSB4A5 or pSB4K5 either using Hifi Assembly or restriction enzyme (RE) digestion and ligation.

A. Hifi DNA Assembly method

Hifi DNA Assembly method is a molecular cloning technique used to join multiple DNA fragments in a
single isothermal reaction2. As depicted in
**Figure 11B**, the three BioBrick fragments were designed to add identical end sequences, or
overhangs to adjacent fragment with specific primers via PCR.

![]()

**Figure 13: Hifi Assembly Method of BioBricks 1,2 and 3 |** 1. BioBrick fragments and backbone are
put together for assembly; 2. The end results is the assembled circular vector containing all the
BioBricks and plasmid backbone; 3. Assembled product is transformed into competent cells | Created with
BioRender.com

![]()

**Figure 14: Transformation after Hifi assembly of Phase 2 – BioBrick 1,2,3 and pSB1A3 | Plate on the
left:**
Escherichia coli DH5 alpha transformed with Hifi assembled product of Phase 2 (BioBrick 1,2,3 and
pSB1A3). Plate on the right: Escherichia coli DH5 alpha transformed with the Hifi Assembly positive
control.

**Conclusion**

After Hifi Assembly for phase 2, we should obtain a plate with a certain number of white colonies.
However, all the colonies we can see on the plate on the left of Figure 6 are red and outnumbered by
small colonies present on the positive control, which can be observed on right plate of Figure 6.
These results show that the Hifi Assembly for the phase 2 did not worked. The red color of the
colonies suggests that the cells were transformed with the plasmid still containing the RFP gene yet
not the inserts. This experiment has been repeated 3 times with no concluding outcome. The second
strategy has been carried out for the assembly, although it is more time-consuming .

B. Restriction enzyme (RE) digestion & ligation assembly method

Different workflows have been tested in parallel for the BioBrick Assembly method. One is the
**2-step workflow** where the the ligation is performed in 2 steps as depicted in
**Figure 16**. The other one is the **1-step workflow** where the ligation is in done in one
step.

The 2-step workflow

BioBrick 2 and 3 need to be assembled first since BioBrick 1 contains the McpM which is toxic for
bacteria (see Design Phase 2). Thus, BioBrick 2 and 3 are not killing bacteria during their
transformation. As a test, BioBrick 1 is inserted into pSB1A3 and transformed into competent cells,
see **Figure 15**.

![]()

**Figure 15: DH5 alpha bacteria transformed with BioBrick 1 in pSB1A3 |** Plates on the left are the
controls (Bacteria transformed only with the plasmid incubated at 30°C and 37°C) and plates on the right
are bacteria that have been transformed with the ligated product (plasmid + BioBrick 1 incubated at 30°C
and 37°C).

Concerning the test of the insertion of BioBrick 1 into the pSB1A3, plates have been incubated at
30°C and 37°C degrees. Both plates with the sample (Figure 14 – on the right) containing only
BioBrick 1 do not show any colony. On the other side, the control plates (Figure 14 – on the left)
displayed a few colonies. This suggests that bacteria was not able to survive when expressing McpM
in BioBrick 1. McpM thus seems to be toxic for the bacteria.

To assemble BioBrick 2, 3, and the backbone, BioBrick 2 is digested with EcorI and SpeI, BioBrick 3
is digested with XbaI and PstI, and the backbone with EcorI and PstI. The cohesive ends of the cut
SpeI and XbaI sites can ligate. This resulted in the incorporation of the BioBrick scar sequence (5'
TACTAGAG 3’) between BioBrick 2 and 3.

![]()

**Figure 16: 2-step BioBrick Assembly method of BioBricks 1,2 and 3 |** 1. Restriction (RE)
digestion: BB2 with EcoRI and SpeI, BB3 with XbaI and PstI, backbone with EcorI and PstI; 2. Ligation;
3. Bacterial transformation with the ligated product; 4. Miniprep of the transformed bacteria to collect
the plasmid; 5. RE digestion of the plasmid construction with EcorI and XbaI and BB1 EcorI and SpeI; 6.
Ligation; 7. Bacterial transformation with the ligated product | Created with BioRender.com

The first ligation product of the **2-step workflow** which is BioBrick 2, BioBrick 3, and the
plasmid pSB1A3 is validated by colony PCR and RE digestion.

**Results of the 2-step workflow**

![]()

**Figure 17: Transformation of assembled BB2, BB3 and pSB1A3 | A. DH5 alpha plates with different
ratio of ligated product of BB2, BB3 and pSB1A3, and the control:**
Plate N°1 is the control (Cut plasmid + ligase without the insert); Plate N°2 Ligation 1:2 ratio; Plate
N°2 Ligation 1:2 ratio; Plate N°3 Ligation ratio 1:5; Plate N°4 Ligation ratio 1:3
**B. Gel migration of verification RE digestions Well N°1, 14:** 1kb+ DNA Ladder //
**Well N°2, 3, 4, 5, 6:** Colonies 1, 2, 3, 4, 5 of Plasmid pSB1A3, BB2, BB3 ( Ratio 1:5) digested
with BglI+ NdeI // **Well N°7, 8, 9, 10, 11, 12:** Colonies 1, 2, 3, 4, 5 of Plasmid pSB1A3, BB2, BB3
( Ratio 1:2) digested with BglI+ NdeI // **Well N°13:** Ligation (20/10/20) pSB1A3, BB2, BB3
**C. Expected results obtained with virtual digestion of plasmid BB2,BB3 construction with
SnapGene:**
Well N°MW: 1kb+ DNA ladder // Well N°1: Assembled plasmid cut with BglI+ NdeI.

**Conclusion of the 2-step workflow**

When compared to different ligation ratios, a proportional amount of white colonies can be observed
on the plates (Figure 17A) with no colonies counted on the negative control plate. Thus, the
transformation seems to have worked. To verify the successful ligation of the plasmid with BB2 and
BB3, colonies are tested after miniprep DNA isolation to purify the plasmid of interest. The
plasmids purified from the bacterial colonies have been cut with restriction enzymes (RE) BgII and
NdeI. The expected bands are depicted on **Figure 17C**. Sample of lane 2, 4, and 6 ,
corresponding to colony N°1, colony N°3, and colony N°5 respectively, seem to have bands at 3.5 kb
and 2.4 kb which correspond to the expected size.

This workflow was stopped at this step for a matter of time. However, the next step would have been
to assembly the obtained plasmid of colonies 1, 3, and 5 with the BioBrick and after confirmation to
continue with the verification procedures (Via agar diffusion assays among others).

The 1-step workflow

To assemble BioBrick 1, BioBrick 2, BioBrick 3, and the backbone, BioBrick 1 is digested with EcoRI
and SpeI, BioBrick 2 is digested with XbaI and SpeI, BioBrick 3 is digested with XbaI and PstI, and
the backbone with EcorI and PstI. The cohesive ends of the cut SpeI and XbaI sites can ligate
together and as a result a BioBrick scar sequence (5' TACTAGAG 3’) will be between both BioBrick 1
and 2, and BioBrick 2 and 3.

![]()

**Figure 18: 1-step BioBrick Assembly method of BioBricks 1,2 and 3 |** All in the same tube: 1. 1st
Ligation with BioBrick 1 and BioBrick 2; 2. Addition of BioBrick 3 for 2nd Ligation; Addition of the
plasmid for 3rd ligation. Assembled plasmid can be transformed| Created with BioRender.com

**Results of the 1-step workflow**

![]()

**Figure 19: 1-step workflow - Transformation of assembled BB1, BB2, BB3 and pSB1A3 | A. DH5 alpha
plates with ligated product of BB1, BB2 BB2 and pSB1A3, and the control:**
Plate N°1 One-step workflow ligated product with plasmid 39; Plate N°2 One-step workflow ligated product
with plasmid 31 **B. Gel migration of the RE digestions: Well N°1,2,3**: 1kb DNA Ladder //
**Well N°2, 3, 4**: Colony C1, C2, C3 of plasmid 39 Phase II (miniprep) digested by BglI // Well N°5
-> 14 : Colony 1 ->10 plasmid 39 Phase II (miniprep) digested by BglI // **Well N°15, 16, 17, 18**:
Colony C1, C2, C3, C4 of plasmid 31 phase II (miniprep) digested by NdeI //
**Well N°19, 20, 21, 22**: Colony C1, C2, C3, C4 of plasmid 31 phase II (miniprep) digested by SapI
**C. Expected resultsimulated with SnapGene**: Well N°MW : 1kb+ DNA Ladder //Well N°1: plasmid 39
Phase II digested by BglI expected //Well N°2: plasmid 31 Phase II digested by SapI expected.

**Conclusion of the 1-step workflow**

Several white colonies are obtained on plate N°1 and N°2 (**Figure 19A**) and no colonies are
counted on the control (plate not present). Thus, the transformation seems to have worked. Regarding
the verification of the ligation, colonies needed to be tested after having completed a miniprep
procedure to obtain the plasmid. The extracted plasmids of the colonies have been cut with
restriction enzymes (RE) other than the one used for the BioBrick Assembly method. Then, NdeI is
used to linearize the plasmid 31. SapI is used to cut twice plasmid 31 to obtain 2 distinct bands at
10 kb and 3.3 kb as depicted on Well N°2 **Figure 11C**. For plasmid 39 we did not have any RE
available in our inventory to linearize it. Thus, we only used BglI to cut twice plasmid 39 to
obtain 2 distinct bands at 6.7 kb and 1.6 kb as depicted on Well N°1 **Figure 19C**.
**For colony 3 of plasmid 39**: On lane 4, three bands can be observed: one around 6.7 kb and one
around 1.6 kb and a last one unexpected around 3.5 kb. The two first bands correspond to the
expected results. Nevertheless, we proceeded with the verification step for the colony 3 of plasmid
39.

Verification of the McpM expression

The aim of this phase is to lyse specific target bacteria through the expression of the microcin
McpM. After the assembly of the three BioBricks, the presence of McpM in the introduced plasmid need
to be confirmed.

A. Extraction, purification and validation of McpM production

To validate the production of McpM, the protein need to be extracted and purified from the cellular
culture via Ni2+ chromatography column and further revelation via SDS-Page and staining technique.

![]()

**Figure 20: Extraction, purification and validation of McpM production |** 1. Transformation of the
competent bacteria with our final construction containing the McpM and its secretion system; 2.
Overnight growth of the cells; 3. Overnight inoculation of the appropriate clone; 4. Cell lysis; 5.
Purification with the his-tag with a Ni2+ IMAC column; 6. SDS-Page, analysis of purification via
staining | Created with BioRender.com

**Expected results**

The McpM size is 8.2 kDa. Therefore, we expect that in the lane of the purified cell lysate
(elutions) a band appears at the size of 8.2 kDa. In the first lane, this band should be more
intense than lanes where Elution 2 and 3 have been loaded. We also expect to have few bands in the
flow-through, corresponding to potential waste material. Regarding the lysate, the same bands as the
flow through might appear, plus one at 8.2 kDa corresponding to McpM. Finally, in the lane of the
Laemmli no bands should appear.

![]()

**Figure 21: Expected results of extraction, purification and validation of McpM production |** Well
1: Ladder, Well 2: Elution tube N°1, Well 3: Elution tube N°2, Well 4: Elution tube N°3, Well 5: Flow
through, Well 6: Total lysate, Well 7: Laemmli

![]()

**Figure 22: SDS-PAGE and Coomassie staining of the purified cell lysate, flow through and total
lysate to validate the presence of McpM |**
Well 1: Ladder, Well 2: Elution tube N°1, Well 3: Elution tube N°2, Well 4: Elution tube N°3, Well 5:
Flow through, Well 6: Total lysate.

**Conclusion**

The chromatography was not 100% efficient as the elution bands still contain many proteins (even
though the band are less intense than for the total lysate). The molecular weight marker did not
migrate very well as the well was a little blocked by polymerized acrylamide towards the top. The
10kDa did not migrate correctly and is not horizontal. The bottom of the gel is not perfect, and it
is difficult to visualize proteins. A light band can be observed at a weight inferior to 10kDa in
the elution 2 lane. This band is expected to be the McpM which is 8.2kDa and which is not
necessarily supposed to be present in large amount. However, we cannot be sure of the presence of
McpM in well 3 (elution 2).

To completely validate the presence of McpM in the purified cell lysate, a Western blot and
immunodetection using an anti-His antibody should be performed. This is done by transferring the
proteins from the gel to a nitrocellulose membrane and then performing the immunodetection, with a
direct rabbit anti-His antibody conjugated to Horseradish Peroxidase, then detecting color using the
HRP color detecting reagent. This will allow us to fully confirm that the low-intense band present
10kDa corresponds to McpM.

For a matter of time and available equipment, we were not able to perform the Western Blot.

Instead, we performed an agar diffusion assay to validate the production and activity of McpM (see
below) by the clone containing plasmid 39.

B. Agar diffusion assay

![]()

**Figure 23: Agar diffusion assay to validate the secretion of McpM | 1. The day before:** Overnight
inoculation of the Escherichia coli (E.coli) transformed only with the pSB1A3, E.coli DH5 alpha
transformed with our construction (BB1,BB2,BB3), E.coli DH10B containing the native MccpDI cluster.
**2. The day of the experiment:** The E.coli only with the pSB1A3 are lawn on the plate (spread
culture onto the agar) and 5-10 ul of supernatant of E.coli DH10B, E.coli DH5 alpha (BB1,BB2,BB3) and LB
only are added on 6-mm ter discs on the plate and placed at 37°C overnight. **3. The day after:** A
little circle should appear around the disc of the cultures that expressed mcpM | Created with
BioRender.com

**Expected results**

The E.coli DH5 alpha (BB1,BB2,BB3) should kill the surrounding plated E.coli DH5 alpha when it
expresses McpM. Therefore, they are forming little radiuses around their colony of the white 6-mm
filter discs. This observation confirms their ability to kill E.coli DH5 alpha bacteria. This
antimicrobial activity is what we are aiming for phase 2 of the BacTail project.

**Results**

We tested in duplicate colony with plasmid 39 assembled with BioBrick 1,2, and 3 (Disc N°2 and N°4),
in duplicate colony with plasmid 31 assembled with BioBrick 1,2, and 3 (Disc N°1 and 3), in
duplicate E.coli DH10B strain containing the native MccpDI cluster (Disc N°5 and 7) and as a control
only LB medium (Disc 6).

![]()

**Figure 24: Results of the agar diffusion assay |** Plate on the left side: Assay N°1 and plate on
the right: Assay n°2; For both plates: E.coli DH5 alpha transformed with pSB1A3 containing RFP bacterial
lawn; Disc 1: E.coli DH5 alpha colonies from plasmid 31; Disc 2: E.coli DH5 alpha colonies from plasmid
39; Disc 3: E.coli DH5 alpha colonies from plasmid 31; Disc 4: E.coli DH5 alpha colonies from plasmid
39; Disc 5: Positive control - E.coli DH10B strain containing the native MccpDI cluster; Disc 6:
Negative control- LB medium; Disc 7: Positive control - E.coli DH10B strain containing the native MccpDI
cluster.

To discuss the results, we zoomed on each disc of both assays, summarized in the table below. Two
characteristics are taken into consideration during observation: presence or absence of a radius
around the disc (due to the excretion of McppPDI microcin) and if some colonies have grown onto the
disc or if the disc remains white.

| Disc N° and compound | Assay N°1 | Assay N°2 | Disc observations |
| --- | --- | --- | --- |
| 1 – plasmid 31 | ![](https://static.igem.org/mediawiki/2020/6/60/T--Ionis_Paris--1_1.png) | ![](https://static.igem.org/mediawiki/2020/b/b6/T--Ionis_Paris--1_2.png) | - Disc 1 Assay N°1: red colonies have grown on the disc and no radius around the disc - Disc 1 Assay N°2: red colonies have grown on the disc and no radius around the disc |
| 2 – plasmid 39 | ![](https://static.igem.org/mediawiki/2020/2/2a/T--Ionis_Paris--2_1.png) | ![](https://static.igem.org/mediawiki/2020/b/bd/T--Ionis_Paris--2_2.png) | - Disc 2 Assay N°1: no red colonies have grown on the disc and radius around the disc - Disc 2 Assay N°2 (40 uL instead of 20 uL): no red colonies have grown on the disc and   radius around the disc |
| 3 – plasmid 31 | ![](https://static.igem.org/mediawiki/2020/1/1a/T--Ionis_Paris--3_1.png) | ![](https://static.igem.org/mediawiki/2020/a/aa/T--Ionis_Paris--3_2.png) | - Disc 3 Assay N°1: red colonies grown on the perimeter of the disc and no a radius around   the disc - Disc 3 Assay N°2: red colonies have grown on the perimeter of the disc and no a radius   around the disc |
| 4 – plasmid 39 | ![](https://static.igem.org/mediawiki/2020/6/6b/T--Ionis_Paris--4_1.png) | ![](https://static.igem.org/mediawiki/2020/8/8a/T--Ionis_Paris--4_2.png) | - Disc 4 Assay N°1: red colonies have grown on the perimeter of the disc and no a radius   around the disc - Disc 4 Assay N°2: red colonies have grown on the perimeter of the disc and no a radius   around the disc |
| 5 – Positive control (plasmid original cluster) | ![](https://static.igem.org/mediawiki/2020/c/c9/T--Ionis_Paris--5_1.png) | ![](https://static.igem.org/mediawiki/2020/3/32/T--Ionis_Paris--5_2.png) | - Disc 5 Assay N°1: no red colonies have grown on the perimeter of the disc and a radius   around the disc - Disc 5 Assay N°2: a radius around the disc |
| 6 – Negative control (LB media only) | ![](https://static.igem.org/mediawiki/2020/1/1b/T--Ionis_Paris--6_1.png) | ![](https://static.igem.org/mediawiki/2020/9/96/T--Ionis_Paris--6_2.png) | - Disc 5 Assay N°1: no red colonies have grown on the perimeter of the disc and a radius   around the disc - Disc 5 Assay N°2: a radius around the disc |
| 7 – Positive control (plasmid original cluster) | ![](https://static.igem.org/mediawiki/2020/8/84/T--Ionis_Paris--7_1.png) | ![](https://static.igem.org/mediawiki/2020/e/e8/T--Ionis_Paris--7_2.png) | - Disc 7 Asssay N°1 fell –no results - Disc 7 Asssay N°2: No red colonies have grown on the disc perimeter and a radius around   the disc |

**Conclusion**

Around the original strains (Disc 5 and 7 - positive controls) a small radius (1mm) is observed, and
no red colonies have grown on the disc perimeters, which suggests that the E.coli DH5 alpha of the
bacterial lawn have been killed or could not grow in this area of the plate due to the presence of
the original MccPDI cluster. On the contrary, around the negative control (Dis 6 – LB medium only),
no radius can be observed, and red colonies could grow on the disc, which shows that nothing hinders
the E.coli DH5 alpha of the bacterial lawn to grow on this region. Regarding our samples, we
performed the agar diffusion assay with the candidates kept after the construction validation (**Figure 11B**). We could observe on Disc N°2 Assay N°1 (the construction of plasmid 39), that no red colonies
have grown on the disc perimeters and a small radius (1 mm) appeared on the disc perimeters.
Additionally, a three-time bigger radius (3mm) on the disc N°2 Assay N°1 (construction of plasmid
39) could be observed. This difference in radius size can be explained by the higher volume that has
been used for Disc N°2 Assay N°2. Finally, this observations shows that the construction of plasmid
39 has the same effect than the one of the original MccpDI cluster, meaning to induce the growth of
the E.coli DH5 alpha of the bacterial lawn. This suggest that the construction of plasmid 39
expresses the McpM responsible for killing E.coli DH5 alpha.

Experimental perspectives of Phase 2 | The lysis of the target bacteria

For the 1-step workflow:

* A Western BLOT should be performed to further the validation of McpM production and
  secretion.
* Regarding the agar diffusion assay, the experiment should be repeated and E.coli ∆ OmpF should
  be used as bacterial lawn as a control plate.
* We should continue with the 2-step workflow to add BioBrick 1 to the already assembled BioBrick
  2, 3 fragments with the plasmid and transform it into competent cells. After transformation, the
  plasmid needs to be purified to proceed with the verification of the assembly of BB1, BB2, and
  BB3, which means digestion of the plasmid with an enzyme that cuts it once to linearize it and
  another pair of enzymes that cut it twice in the region on the insert. Additionally, the plasmid
  should be cut with XbaI and PstI to perform an insert release. Furthermore, a colony PCR can be
  performed. After having verified that the plasmid was well assembled, the selected clone will
  continue with the further verification steps. These steps include the extraction, purification,
  and validation of McpM production. Finally, an agar diffusion assay should be performed as well
  for this 2-step workflow.
* For future step phase 1/phase 2: insert the plasmid of phase I and phase II into the same
  bacteria to enable recognition and lysis at the same time and see if the expression of gp37
  enhances the lysis of the bacteria of interest (it should enable our chassis to approach the
  target and enhance the antimicrobial concentration in proximity of the target).

Results of Phase 3. The Kill-Switch

In Phase III, the arabinose operon is the key parameter for the regulation of the death inducting
gene DNASE I. To find out more about the scientific background and
design of Phase III.

This phase aims at controlling the life cycle of our engineered bacteria through a kill switch
system. In presence of L-arabinose enables the synthesis of the endonuclease bpDNaseI I that will
destroy the producer (i.e. the bacteria). For this phase, two constructs were designed. Each of them
are divided into 2 different fragments (for a matter of cost and assembly).

1. The first fragment (BBa\_K3519010) contains, among others, the gene coding for the araC regulatory
protein and a minimal pBAD promoter (or araBAD promoter) upstream of the second fragment. Through
the binding of araC to L-arabinose, araC enables the arrival of the RNA polymerase to the pBAD
promoter so the transcription of the gene of the following fragment can occur. To this fragment, the
second or third fragment can be attached (Figure 24).

2. The second fragment is the one containing the DNASE I gene coding for the bpDNaseI enzyme.
Additionally, a his-tag is added for further purifications and analysis.

3. The third fragment possesses a gene coding for the mRFP. This fluorescent protein enables the
detection of the expression of the gene following pBAD via fluorescence assays. Furthermore, its
quantification will enable the validation of this construct.

The fragments are summarized in **Table 1** for clarity.

![](https://static.igem.org/mediawiki/2020/7/7a/T--Ionis_Paris--resultsPhase3img1.png)

**Figure 25: Representation of the different fragment assembly and the BioBricks they can form |**
Minimal araBAD promoter (BBa\_K2442101), Promoter medium (BBa\_J23118), Strong RBS (BBa\_BB0034), E. coli
araC coding region (BBa\_K2442103), mRFP (BBa\_E1010), DNASE I + poly his-tag (BBa\_K3519004), Spacer
(BBa\_0040), Terminator (BBa\_0015), araC+araBADpromoter (BBa\_K3519010), araC+araBADpromoter+mRFP
(BBa\_K3519011), araC+araBADpromoter+DNASE I (BBa\_K3519012).

![](https://static.igem.org/mediawiki/2020/3/32/T--Ionis_Paris--resultsPhase3img2.png)

**Table 1: List of the fragments composing the BioBricks and the different backbones used**

PCR & Gel Migrations

For each fragments, BioBrick prefix and suffix sequences are added on both sides. This is done to
assemble the fragments and the backbone all together by restriction enzyme digestion followed by
ligation (see restriction enzyme digestion and ligation assembly method further down this the page).
However, restriction enzymes need more nucleotides to specifically bind and cut. As the restriction
sites are located on both ends of the fragment, few nucleotides are added to the primers.([Restriction Enzyme Digest Protocol: Cutting Close to DNA End. *New England Biolabs*](https://international.neb.com/tools-and-resources/video-library/restriction-enzyme-digest-protocol-cutting-close-to-dna-end))

The primers and the fragments are synthetized by IDT. Once received, the synthetized fragments are
resuspended and amplified via Polymerase Chain Reaction (PCR) to obtain a higher quantity of DNA.
The same process is realized for purified circular pSB1A3, pSB4A5 and pSB4K5 plasmids (already in
our possession) to amplify their backbones. Each PCR samples is verified on a 1% agarose gel through
electrophoresis to verify its size.

After some trouble-shouting, we determined the parameters for an optimal PCR (Figure 26). Concerning
the amplification of the fragments, a two-steps PCR is performed as the primers used anneal at 72°C.
The annealing temperature for the backbone amplification is about 68°C with another set of primer.
While a 50 seconds elongation step is sufficient for the amplification of the fragments, a 2 minutes
elongation step is required for the backbones as the longest plasmid, pSB4K5 is 3 378 bp long (Table
1).

![](https://static.igem.org/mediawiki/2020/2/24/T--Ionis_Paris--resultsPhase3img3.png)

**Figure 26: PCR Program used for the amplification of fragments and backbones**

The previously presented PCR parameters enabled the amplification of each fragments and backbones as
shown in Figure X. Throughout our repetitive PCR, we observe that the amplification of the DNASE I
is always leading to double bands. However, one of the two bands correspond to the expected size
(1061 bp). Therefore, this band is cut and purified to obtain the fragment bpDNaseI. The same
phenomenon is observed for the backbones. One band corresponds to the amplified backbone size
(pSB1A3: 2155 bp, pSB4A5: 3395 bp, pSB4K5: 4489 bp) and the other to the size of the circular
purified plasmid used as DNA template (pSB1A3: 3225 bp, pSB4A5: 4465 bp, pSB4K5: 3419 bp). Thus, two
decisions have been taken:

First, a gel purification is realized to discard the circular plasmid template.

Second, following this purification step, DpnI is used so it can digest the non-PCR products of this
amplification: the template DNA. This way, we reduce the chance of transforming our chassis with the
original circular plasmid used for backbone amplifications (cf. Engineering success.).

![](https://static.igem.org/mediawiki/2020/0/07/T--Ionis_Paris--resultsPhase3img4.png)

**Figure 27: Gel migration results following a PCR of the fragments “araC+araBAD promoter”, “mRFP” and
“bpDNaseI” and the plasmids pSB1A3, pSB4A5 and pSB4K5 | A.**
Expected results with SnapGene Gel simulation **B.** Gel Caption after migration through UV
transilluminator **C.** Wells contents and expected sizes.

Restriction enzyme (RE) digestion ligation assembly method

The three fragments purified in the previous step (see above) are assembled into plasmid pSB1A3,
pSB4A5 and pSB4K5 using restriction enzyme (RE) digestion and ligation (Figure. X). For this
assembly:

The fragment “araC+araBAD promoter” is digested with EcoRI and SpeI.

The fragment “DNASE I” or “mRFP”, depending on the construct, is digested with XbaI and PstI.

The backbone is digested with EcoRI and PstI.

The cohesive ends of the cut SpeI and XbaI sites can ligate together but a BioBrick scar sequence
(5' TACTAGAG 3’) will result from it between the two fragments. Between the digestion and the
ligation step, an alkaline phosphatase was added to the digested backbones to dephosphorylate their
cuts so they can not re-ligate (For more information see paragraph Alkaline Phosphatase in Engineering Success). Once assembled, the recombinant plasmid is used to transform the chassis.

![](https://static.igem.org/mediawiki/2020/5/5f/T--Ionis_Paris--resultsPhase3img5.png)

**Figure 28: BioBrick Assembly method to assemble the possible constructs to the backbone for Phase
III |**
1. Restriction digestion (RE) : araC+araBAD promoter with EcoRI and SpeI, DNASE I or mRFP with XbaI and
PstI, backbone with EcoRI and PstI; 2. Ligation; 3. Bacterial transformation with the ligated product |
Created with BioRender.com

According to the plasmid backbones which are used for the ligation, six different recombinant
plasmids can be obtained (Table. 2). The different constructs can be obtained with pSB1A3, pSB4A5
and pSB4K5 backbones associated to BBa\_K3519012 (araC+araBADpromoter+DNASE I) and BBa\_K3519011
(araC+araBADpromoter+mRFP).

![](https://static.igem.org/mediawiki/2020/b/b1/T--Ionis_Paris--resultsPhase3img6.png)

**Table 2. Different recombinant plasmids that can be obtained after BioBrick assembly method for
Phase III.**

Transformation

The assembled constructs (see above) are transformed into NEB DH5α chemically competent
*E. coli*. The results are depicted in the following table (Table .3).

![](https://static.igem.org/mediawiki/2020/b/b9/T--Ionis_Paris--resultsPhase3img7.png)

**Table 3: Results of transformations with the final constructions for Phase III**

Verification Steps

To verify that clones obtained after transformation contained the well assembled construction,
several validation assays are realized: Colony PCR, Linearization, Insert release, additional double
digestion and *DNASE I* activation.

1. Colony PCR

The presence or the absence and the size of the inserts can be verified by Colony PCR followed by an
electrophoresis on agarose gel. Additionally, each clone used for this technique are plated on petri
dishes (Table .4).

![](https://static.igem.org/mediawiki/2020/1/1f/T--Ionis_Paris--resultsPhase3img8.png)

**Table 4: Pictures of Colony PCR plates.**

Through several trouble shouting, the optimal program for Colony PCR was found to be the one
presented in
**Figure 30**. There, the elongation time is about 2 minutes as the longest construct,
araC+araBADpromoter+DNASEI, is 2538bp long. The set of primer used, VF2 and VR, anneal at 62,5°C.
They are commonly used primers for Colony PCR.

![](https://static.igem.org/mediawiki/2020/5/53/T--Ionis_Paris--resultsPhase3img9.png)

**Figure 29: PCR Program used for the Colony PCR**

![](https://static.igem.org/mediawiki/2020/7/7f/T--Ionis_Paris--resultsPhase3img10.png)

**Figure 30: Gel migration results following a Colony PCR on the fragments “araC+araBAD promoter+mRFP”
and “araC+araBAD promoter+DNaseI” assembled with pSB1A3, pSB4A5 and pSB4K5 backbones | A.**
Expected results with SnapGene Gel simulation **B.** Gel Caption after migration through UV
transilluminator **C.** Wells contents and expected sizes.

The results of this amplification are shown in **Figure 31**. For the two controls (circular
pSB4A5 and circular pSB4K5 plasmids), bands with the expected size can be observed (1350 bp both).
Concerning the samples, only wells N°1 and 4 present bands in adequation with the gel simulation
(bands at 2759 bp). They respectively correspond to clones 1 and 4 of the pSB1A3+DNASE I plate.
These colonies both seem to have a well constructed recombinant plasmid with the
araC+araBADpromoter+DNASEI insert but additional verifications are required.

2. Linearization (Single Digestion)

A second way to verify if the transformed bacteria contain our recombinant plasmids is by digesting
them with restriction enzymes. First, we chose to linearize the purified plasmids by choosing an
enzyme that cuts only once. We decided to go with BglI for bacteria with pSB1A3 backbone and NdeI
for pSB4A5 and pSB4K5. Both of their restriction sites are situated on the backbone. The results are
presented in **Figure 32**.

![](https://static.igem.org/mediawiki/2020/f/f6/T--Ionis_Paris--resultsPhase3img11.png)

**Figure 31: Gel migration results following a single restriction enzyme digestion | A.** Expected
results with SnapGene Gel simulation **B.** Gel Caption after migration through UV transilluminator
**C.** Wells contents and expected sizes.

The three controls (circular pSB1A3, circular pSB4A5 and circular pSB4K5 digested) gave the expected
bands (respectively at 3225 bp, 4465 bp and 5712 bp). Concerning the samples, the observed bands for
wells N°2, 4, 5, 9, 11, 16, 19 & 21 correspond to the expected bands on the gel simulation. They
respectively correspond to:

Colonies 1, 3, 4 of the pSB1A3 + DNASE I plate

Colony 4 of the pSB1A3 + mRFP plate

Colony 2 of the pSB4A5 + DNASE I plate

Colony 3 of the pSB4A5 + mRFP plate

Colony 1 of the pSB4K5 + DNASE I plate

Colony 1 of the pSB4K5 + mRFP plate

3. Insert release (Double Digestion)

The insert release assay consists in a double digestion of the plasmid at both ends of the insert.
It enables the separation of the backbone and the insert. Several enzymes possess a restriction
sites in the prefix and suffix region of the assembled fragment. But, due to the restriction enzyme
digestion and ligation assembly method, most of them present scares preventing another digestion.
XbaI and SpeI being only used for the assembly of the two fragments, their associated restriction
sites remain intact at both end of the construct. Thus they represent ideal enzymes for this step,
whose results are presented in **Figure 32**.

![](https://static.igem.org/mediawiki/2020/b/b7/T--Ionis_Paris--resultsPhase3img12.png)

**Figure 32: Gel migration results following an insert release of the clones of Phase III**|
**A.** Expected results with SnapGene Gel simulation **B.** Gel Caption after migration through UV
transilluminator **C.** Wells contents and expected sizes.

The three controls (circular pSB1A3, circular pSB4A5 and circular pSB4K5 digested) gave the expected
bands. Concerning the samples, the observed bands for wells N°4, 16, 19 & 21 correspond to the
expected bands on the gel simulation. They respectively correspond to:

- Colonies 3 of the pSB1A3 + DNASE I plate
- Colony 3 of the pSB4A5 + mRFP plate
- Colony 1 of the pSB4K5 + DNASE I plate
- Colony 1 of the pSB4K5 + mRFP plate

4. Additional Double Digestion

An additional double digestion is performed to validate all the previous verification assays. While
the pSB1A3 recombinant plasmids are digested with XbaI and BglI, pSB4A5 and pSB4K5 backbones are
digested with XbaI and NdeI (**Figure. 33**)

![](https://static.igem.org/mediawiki/2020/1/13/T--Ionis_Paris--resultsPhase3img13.png)

**Figure 33: Gel migration results following the additional insert release of the selected clones of
Phase III**| **A.** Expected results with SnapGene Gel simulation **B.** Gel Caption after migration through
UV transilluminator **C.** Wells contents and expected sizes

The three controls (circular pSB1A3, circular pSB4A5 and circular pSB4K5 digested) gave the expected
bands. Concerning the samples, the observed bands for wells N°6, 7, 9 & 10 correspond to the
expected bands on the gel simulation. They respectively correspond to:

- Colonies 2 of the pSB4A5 + DNASE I plate
- Colony 3 of the pSB4A5 + mRFP plate
- Colony 1 of the pSB4K5 + DNASE I plate
- Colony 1 of the pSB4K5 + mRFP plate

5. DNASE I Activation

Now that we have identified few possible recombinant plasmids containing the *DNASEI* gene, its
expression can be validated via addition of L-arabinose. As the presence of L-arabinose is supposed
to activate the kill switch system, bacteria are supposed to produce bpDNaseI, leading to their own
destruction (see
Design Phase III). Thus, liquid cultures with antibiotics were incubated with clones presenting at least one good
result among the previously described verification assays. In a first step, initial OD600
of each sample is determined through spectrophotometry. Then, L-arabinose is added and variations of
the OD600 is studied 1h30 later. Following this, even more L-arabinose is added and the
OD600 is measured one last time 1h30 after. A negative control corresponding to
untransformed DH5α *E. coli* was realized but without antibiotic. The results are presented in
the following table:

![](https://static.igem.org/mediawiki/2020/3/30/T--Ionis_Paris--resultsPhase3img14.png)

**Table 5: OD600 variations following addition of L-arabinose in potential
*DNASEI* recombinant bacteria**

Globally, OD600 of each sample was increasing through the experiment similarly to the
negative control except for the clone 1 of the pSB4K5 + DNASE I plate. This sample showed a
decreased OD following the addition of 100mg of L-arabinose. This information strongly suggest that
this clone has the well constructed plasmid.

Conclusion

![](https://static.igem.org/mediawiki/2020/4/4f/T--Ionis_Paris--resultsPhase3img15.png)

**Table 6**: Summary table of clones presenting good results for at least on verification experiment

Several clones present good results for the Colony PCR, Linearization, Insert release, Additional
double digestion and DNASEI Activation assays. None of them is good for each of them. Nevertheless,
the clone 1 of the pSB4K5 + DNASE I plate presents good results for the three digestion assays in
addition to the *DNASE I* Activation assay. These data suggest that this clone possess the
right recombinant plasmid. Due to a lack of time, we can not proceed to further investigations. But
in the future, we would like to send the clones showing good results for at least two experiments to
be sequenced. This will enable us to get the precise DNA sequence of their plasmids.

Perspectives

Once the recombinant plasmids identified and purified, we will transform those containing the
*mRFP*
gene into BL21 cells. This will help us determine the optimum arabinose concentration required for
maximum induction by the promoter using fluorescence.

Next, recombinant *DNASEI plasmid* will be transformed into BL21 cells. These cells will be
stimulated with the determined appropriate arabinose concentration and cell death will be measured
by monitoring the OD of the culture which is supposed to diminish. In addition, the culture will be
plated at regular intervals to determine the CFU. Then, DNA from such cultures will be isolated and
run on an agarose gel to check for any intact DNA prevailing.

Finally, the bpDNaseI will be overexpressed and will be purified, thanks to its poly his tag
sequence, and will be incubated with several DNA concentrations for different time periods for its
in-vitro characterization. Thus, DNA degrading efficiency of the enzyme will be determined.

---

PHASE1

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