E x p e r i m e n t s

Introduction

In this section, we present the experimental strategies developed to engineer E. coli strains for melanin production, enhanced tyrosine biosynthesis, and biocontainment. We detail the construction of the pAIDA-tyr1 plasmid to express tyrosinase, the optimization of tyrosine availability via aroF mutagenesis, and the development of a ΔmurI biocontainment system complemented by the pMurI plasmid. These workflows combine molecular cloning, plasmid validation, enzymatic assays, and chromosomal engineering techniques, reflecting a multi-level synthetic biology approach to construct functional and safe microbial platforms.

For detailed protocols of all experimental procedures, please visit our Protocols page.

Construction of the Melanin Production System pAIDA-tyr

Plasmid design

Cloning Strategy : clonage PCR of tyr1 into pAIDA

Here we use :

• 35 µL of H2O
• 1 µL of Template DNA (pAIDA)
• 5 µL of Tyr1
• PFU in separate composants that contains :
  • 5 µL of 10X PFU Buffer
  • 4 µL of dNTPs
  • 2 µL of DMSO
  • 0,8µL of PFU enzyme

Thermocycling Conditions for a Routine PCR:

• Initial Denaturation: 95°C for 5 min
• 29 Cycles:
  • 95°C for 1 min
  • 56°C for 1 min
  • 68°C for 13 min
• Final Extension: 68°C for 20 minutes
• Hold: 4–10°C

We hold it at 16°C

Clonage PCR of Tyr1 in pAIDA : DpnI treatment

• Add 1 µL of DpnI in the PCR cloning products
• Incubate at 37°C for 3h

Clonage PCR of Tyr1 in pAIDA : transformation into DH5 alpha super-competent

Preparation of LB + Cm plates.

Under sterile conditions (Bunsen burner flame):

• Add 5 µL of the Dpn1-digested product in 50 µL of DH5 alpha
• Incubate in ice 30 min
• Apply heat shock : 1 min at 42 °C
• Incubate in ice 1 min
• Add 1 mL of LB
• Incubate at 37 °C with shaking for 45 min and 1 hour max
• Centrifuge at 1100 rpm for 1 min
• Remove 900 µL of supernatant and resuspend the remaining 100 µL by pipeting
• Spread 100 µL of bacteria an LB + cm

Colony PCR Protocol with oLS 4.1, oLS 4.2 and pAIDA Tyr 1 cells

• We do this for 6 colonies of pAIDA Tyr1 and 1 colony of pAIDA (negative control)
• Pick a well-isolated colony with a toothpick of pAIDA Tyr 1
• Transfer the colony into a Chloramphenicol + LB plate, than into a PCR tube
• Prepare a PCR mix in an Eppendorf tube:
  • 5 µL of EconoTaq (2X)
  • 0.5 µL of Forward Primer (10 µM)
  • 0.5 µL of Reverse Primer (10µM)
  • H2O to complete until final volume
• For 6 PCR tubes, we prepare xµL of PCR mix so everything x + x µL of H2O
• To finish, vortex the PCR mix
• Add 10 µL of PCR Mix into every PCR tube and vortex a little
• Transfer PCR tubes to a PCR machine and begin thermocycling
• Thermocycling Conditions for a Routine PCR: This time we do 60°C and 1 minute

Pre-culture preparation of pAIDA Tyr1 1, 5 and 6 to do a Miniprep

• Inoculate colony 1 of pAIDA Tyr1 into a 5 mL Erlenmeyer flask of LB with Chloramphenicol
• Same for pAIDA Tyr1 colonies 5 and 6
• Shake overnight at 37°C

Miniprep of pAIDA Tyr1 1,5 and 6

Follow the NEB protocol for T1110S (50-prep) as described:

For detailed protocols of miniprep procedures, please visit our Protocols page.

• Use a Nanodrop Spectrophotometer to measure pKNG DNA
• Store the DNA at -20°C

After that, we obtained the plasmid concentrations of each colony, which we then sent for sequencing. We then continued with colonies 1 and 5, confirming through sequencing that tyr1 is indeed present in pAIDA. However, here we have chosen to discuss only the results from colony 1.

Plasmid Validation of Construction

After obtaining the plasmid concentrations of each colony, which we then sent for sequencing, we continued with colonies 1 and 5, confirming through sequencing that tyr1 is indeed present in pAIDA. However, here we have chosen to discuss only the results from colony 1.

We performed various Western blots to confirm the expression of tyrosinase, which enables melanin production. First, we used the anti-His tag antibody, and second, the anti-Myc antibody.

Western Blot with Anti-His Antibody

Pre-culture preparation:

• Pre-culture of pAIDA-tyr1 into W3110 ΔompT, W3110 and the control His tag of the Laetitia team (abbreviated as CHI)
• Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C
• For the control His tag of the Laetitia team, inoculated 1 colony into a 5 mL LB + Km Erlenmeyer flask. Shake overnight at 37°C

Preculture Dilution: pAIDA-tyr1 W3110 ΔompT, pAIDA-tyr1 W3110, pAIDA W3110ΔompT, pAIDA W3110, W3110 ΔompT, W3110 and control tag His of the Laetitia team.

Procedure under sterile conditions (Bunsen Burner flame):

1. Collect the precultures of pAIDA-tyr1 into pAIDA-tyr1 W3110 ΔompT, pAIDA-tyr1 W3110, pAIDA W3110ΔompT, pAIDA W3110, W3110 ΔompT, W3110 and control tag His of the Laetitia team
2. Add 500μL of preculture to a 250 mL flask containing 50 mL of LB for each sample
3. Incubate for 3h to observe the stationary phase and perform the Western blot
4. We performed 2 dilutions in two different Erlenmeyer flasks: one that will be induced with 100μL of IPTG, and the other without IPTG

At OD=0.7, induction with IPTG was performed.

Western Blot with Anti-Myc Antibody

Preculture Dilution: colonies pAIDA W3110, pAIDA-tyr1 W3110 and pAIDA-tyr1 W3110ΔompT

Procedure under sterile conditions (Bunsen Burner flame):

1. Collect the precultures of pAIDA-tyr1 in W3110 and pAIDA-tyr1 in W3110ΔompT and pAIDA W3110 (the control)
2. Add 500μL of preculture to a 250 mL flask containing 50 mL of LB for each sample
3. Incubate for 3h to observe the stationary phase and perform the Western blot

At OD=0.6, induction with 100μL of IPTG (200μM) was performed.

Optimization of IPTG Concentration

We performed additional Western blots with colonies 1 and 5 at different IPTG concentrations to determine the optimal concentration for tyrosinase expression.

Preculture dilution of colony PCR 1 transformed in W3110Δ ompT:

1. Collect the precultures of colonies PCR 1 pAIDA W3110ΔompT
2. Add 200 μL of preculture to a 100mL flask containing 20mL of LB for each sample + Cm
3. Incubate for 3h to observe the stationary phase and perform the Western blot

For detailed protocols of western blot, please visit our Protocols page.

Plasmid Visual Test

This test allowed us to see if tyrosinase is properly expressed and therefore observe melanin production.

Preparation plates for plasmid pAIDA induced by IPTG:

• 10ml LB
• 20μL of 10mM Cu²⁺
• 20μL IPTG (200μM)
• 13μL Chloramphenicol
• 100mg L-tyrosine

Preparation plates for plasmid pAIDA induced by rhamnose:

• 10 mL LB
• 20μL of 10mM Cu²⁺
• 100μL rhamnose (1g/L)
• 13μL Chloramphenicol
• 100mg L-tyrosine

Copper (Cu²⁺) plays an essential role in the visual test for melanin production, as it is an indispensable cofactor of the enzyme tyrosinase, enabling the activation of tyrosinase.

Enzymatic Activity Tests

Activity test with pAIDA Tyr-1 Sweden:

• We adjust the OD at 0.05 for a final volume of 25ml for colony with plasmid induced by rhamnose and OD at 0.5 for colony with plasmid induced by IPTG
• Delta ompT pAIDATyr-1: 4.6mL of preculture (OD: 0.27)
• W3110 pAIDATyr1: 4.3mL of preculture (OD: 0.29)

Procedure:

1. Add in each Erlenmeyer 25mL of LB and 200μL of Rhamnose with the appropriate volume of preculture
2. For IPTG-induced samples, add 50 μL of IPTG
3. Incubate at 30°C and 180rpm for 3 hours
4. Centrifuge at 3270g for 10 minutes
5. Resuspend the pellet in 10mL of buffer

Buffer preparation:

• 40μL of copper
• 5mL of Tris-HCL pH 7.2
• 100mg of tyrosine
• Add water to a final volume of 100mL
• Put the solution in the microwave until it's clear

6. Incubate at 30°C, 220rpm for 15 hours
7. Measurement of Melanin in the supernatant: Centrifuge at 13,000 rpm for 5min
8. Measure the supernatant at 400nm, using the prepared buffer as blank

This procedure was inspired by: Hörnström, David, et al. "Molecular optimization of autotransporter-based tyrosinase surface display". Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 1861, no 2, February 2019, p. 486-94.

Enhancement of Tyrosine Biosynthesis (aroF)

Slic AroF using pkng101 and pKO3

Things to know about these two plasmids:

• pkng101 is replicative only in CC118. For it to be integrated into the chromosome, you must transform a strain that doesn't recognize the origin of replication. Its resistance is to streptomycin. It can be linearized by using BAMH1
• pkO3 is heat-sensitive. It's replicative at 30° C. You can do the first transformation into the DH5 alpha cells. Put it at 42°C for integration into the chromosome of your strain. The resistance is to chlorophenicol. We used oligos to linearized it

Materials:

• Buffer R CutSmart (NEB)
• T4 DNA Polymerase
• Buffer R.2.1 (for SLIC)
• Sterile water / MilliQ
• LB agar + streptomycin (for pKNG101)
• LB agar + chloramphenicol (for pKO3)
• LB + 6% sucrose (LB + 50% filtered sucrose, adjusted to 6%)

1-Plasmid Linearisation:

Digestion of pKNG101 with BAMH1:

1. In a 1.5 mL Eppendorf tubes add (for a final volume of 30µL):
   • 20µL pkng101
   • 5 µL H20
   • 3µL Buffer R CutSmart
   • 2 µL BAMHI
2. Incubate for 3 hours at 37°C

Since our strain was resistant to streptomycin we used another plasmid PKO3

PCR for linearized PKO3 using Q5® High-Fidelity 2X Master Mix following NEB Protocol with oLS 4.3 and oLS 4.4:

• For the 72°C step we did 2:50
• You can see the plasmid on gel use a control do determine if it's linearized
• Do a Clean up of the PCR

2-Amplification of AroF fragments to induce the mutation S101A:

PCR and Electrophoresis of genomic DNA of W3110 with oLS 4.5 + 2.2 and oLS 2.3 + 4.6 using Q5® High-Fidelity 2X Master Mix following NEB Protocol

• We did 2 PCR tubes. One for each fragments
• For W3110 delta ompT, we pick an isolated colony with a toothpick and put it in 30 µL of H2O. Heat at 92°C for 10 min to lyse cells
• F.1 (aroF mut): primers = oLS 4.5, oLS 2.2 ; Template DNA = W3110 Delta ompT
• F.2: primers = oLS 4.6, oLS 2.4 ; Template DNA = W3110 Delta ompT
• For the 25-35 cycles, we put the 2nd step at 72°C
• The fragments should be at 500 PB

3-Assembling the fragments in pkO3 or pKng101:

• It has to be performed on ice
• For the ratio you can do as you like depending on the concentration of your fragments and plasmid
• Exemple: Ratio 2/2 = 2µL of Plasmid for 2µL of Fragments

1. Add in order for a final volume of 10µL:
   • 2,5µL H2O
   • 2µL PCR Fragment 1
   • 2µL PCR Fragment 2
   • 2µL The linearized vector
   • 1µL Buffer (for us it was R.2.1)
   • 0,4µL T4 polymerase
2. Lastly, T4 polymerase is added to the tubes on ice
3. Incubate for 2min45 at room temperature
4. Return to ice
5. Transform the 10µL of your tube into 100µL of the right competent cell. If the plasmid is pkng101 use CC118 (it can only recognize the origin of replication)
6. For the last step, spread (using glass beads) 100µl of bacteria onto a LB with the right antibiotic
7. Incubate for one or two nights at the right temperature

4-Colony PCR for verification of fragments insertion into the plasmid:

• Follow the protocol as described
• Modify the hybridization and the elongation to suit your oligos
• For example we put the hybridization at 60°C and the elongation's time at 1min30
• The next day do a miniprep for the rights colony

5-Transformation into the strain chosen (First recombination event):

• Follow the protocol described
• For pko3 incubate 1H30 at 30°C and put the plate at 42°C overnight

6-Preparation of LB + Sucrose petri dish for the second recombination event:

• For 5 petri dishes, one for each colony add:
  • 12 mL of Sucrose 50% (it must be filtered)
  • 88 mL of LB
• We want a concentration final of 6% Sucrose
• Spread the colonies into the petri LB+Sucrose dish and put it at 12°C for 48H-72H

7-Colony PCR for the verification of the second recombination event:

• Use the designed oligos to verify the insertion of the mutation or deletion in your gene

The Biocontainment Strategy

Construction of W3110 Δ ompT ΔmurI for auxotrophy

Chromosomal Deletion-Insertion (λ-Red System)

For detailed protocols of chromosomal deletion, please visit our Protocols page.

DAY 1

• Order primers

DAY 2

• Streak the target strain onto an agar plate for isolation

DAY 3

• Start an overnight culture of the target strain in 2YT medium
• Place a 150 mL bottle of sterile water at 4°C

DAY 4

1. Preparation of Electrocompetent Cells

• Dilute the target strain in 20 mL of LB medium
• Pre-cool the centrifuge for Falcon tubes to 4°C
• Prepare cold 10% glycerol using the bottle of sterile water
• Grow the strain at 37°C to an OD600 of approximately 0.6
• Place the culture on ice for at least 20 minutes
• Centrifuge for 8 minutes at 5000 g (Falcon tube centrifuge)
• Discard the supernatant
• Gently resuspend the pellet in 1 mL of cold 10% glycerol, then fill up to 20 mL with cold glycerol
• Incubate on ice for 5 minutes
• Repeat the last four steps (centrifugation, washing) two more times
• After the last centrifugation, resuspend the pellet in 100 µL of cold 10% glycerol
• Distribute into 2 aliquots of 60 µL in cold 1.5 mL Eppendorf tubes
• Cells can be used within 24 hours (on ice) or frozen in liquid nitrogen and stored at -80°C

2. Preparation of Agar Media

• Prepare two LB-agar plates supplemented with chloramphenicol

3. Electroporation of Plasmid pKOBEG

• Add 1 µL of pKOBEG plasmid to one tube of electrocompetent cells. Use the second tube as a control (add nothing)
• Incubate on ice for 15 minutes
• Transfer the mixture to a cold electroporation cuvette. Tap to distribute the liquid and remove air bubbles
• Apply the electric shock (program EC1 on the electroporator)
• As quickly as possible, add 900 µL of 2YT medium. Transfer the suspension back to the original Eppendorf tube
• Incubate for 1 hour 20 minutes at 30°C
• Centrifuge for 5 minutes at 5000 g, remove 800 µL of supernatant
• Resuspend the pellet in the remaining supernatant and spread onto an LB-agar / chloramphenicol plate
• Incubate overnight at 30°C

DAY 5

• Start an overnight culture of the transformed strain (pKOBEG) in 2YT, at 30°C
• Place a bottle of sterile water at 4°C (volume = 50 mL per strain to be constructed)

DAY 6

1. Induction of λ-red Genes

• Dilute the overnight culture 1/100 in LB with chloramphenicol (volume = 12.5 mL per strain)
• Incubate at 30°C
• At OD600 = 0.2-0.4, add sterile L-arabinose to 0.05% (1/400 dilution from a 20% stock)
• Continue incubation at 30°C until OD600 = 0.6-0.8

2. Preparation of Electrocompetent Cells

• Pre-cool the centrifuge for Falcon tubes to 4°C
• Prepare cold 10% glycerol
• Place the induced culture on ice for at least 20 minutes
• Centrifuge for 10 minutes at 5000 g (Falcon tube centrifuge)
• Discard the supernatant
• Gently resuspend the pellet in 1 mL of cold 10% glycerol, then fill back to the initial volume with cold glycerol
• Incubate on ice for 5 minutes
• Repeat the last four steps (centrifugation, washing) two more times
• After the last centrifugation, resuspend the pellet in 1/200 of the initial volume of cold 10% glycerol
• Aliquot 60 µL into cold 1.5 mL Eppendorf tubes
• Cells can be used within 24 hours (on ice) or frozen

3. Preparation of Agar Media

• Prepare LB-agar plates supplemented with kanamycin (one plate per strain + one extra)

4. Cassette Preparation

• Dilute primers 1/10

5. Cassette Amplification by PCR

Prepare the following PCR reaction mix (make two tubes per cassette):

• H₂O: 30 µL
• Q5 Buffer: 10 µL
• dNTPs (2.5 mM): 5 µL
• Primer #1: 1 µL
• Primer #2: 1 µL
• Vector (diluted 1/10): 2 µL
• Q5 polymerase: 0.6 µL

• Vortex, then brief centrifugation
• PCR Program (thermocycler): "Eric - Phusion". Extension time = 30 sec per kb
• Usable Vectors: pKD4 (deletion, ~1.6 kb), pKD4-Nter-GFP, pKD4-Cter-GFP, etc. (FP/APEX insertion, ~2.7 kb)

6. Cassette Verification and Purification

• Pool the two PCR tubes corresponding to the same cassette
• Check amplification on a 1% agarose gel (load 6 µL). Include a molecular weight marker (Smart Ladder)
• If amplification is correct, purify the PCR product using a PCR purification kit. Elute with 50 µL of water (preferably in two steps: 20 then 30 µL)

7. Cassette Electroporation

• Add 10-20 µL of the purified cassette to one tube of electrocompetent cells. Use a second tube as a control (add nothing)
• Incubate on ice for 15 minutes
• Transfer to a cold electroporation cuvette. Tap to distribute and remove bubbles
• Apply the electric shock (program EC1)
• As quickly as possible, add 900 µL of 2YT medium. Transfer back to the Eppendorf tube
• Incubate for 1 hour at 37°C
• Centrifuge for 5 minutes at 5000 g, remove 800 µL of supernatant
• Resuspend the pellet in the remaining supernatant and spread onto an LB-agar / kanamycin plate
• Incubate overnight at 37°C

DAY 7

1. Clone Verification by Colony PCR

• Pick 7 colonies
• Prepare a PCR Master Mix:
  • Econotaq Green: 90 µL
  • Primer #1: 3.6 µL
  • Primer #2: 3.6 µL
  • H₂O: 83 µL
  • Total: ~180 µL
• Vortex
• Aliquot into 8 PCR tubes (8 x 20 µL)
• Pick a small amount of each selected colony and add to a tube (first 7 tubes). To the 8th tube (negative control), add a bit of a colony from the starting strain
• Primers to use: one in the adjacent gene (upstream/downstream) and one in the kanamycin cassette
• PCR Program: "Eric - EconoTaq". Extension time = 1 min per kb
• Run the entire samples on a 1% agarose gel. Do not forget the molecular weight marker (Smart Ladder)

SUPPLEMENTARY PART: CASSETTE FLIPPING (if necessary)

DAY 8

1. Preparation of Electrocompetent Cells

• Dilute a PCR-positive clone in 20 mL of LB + kanamycin
• Pre-cool the centrifuge for Falcon tubes to 4°C
• Prepare 10% glycerol using the water bottle
• Grow at 37°C to OD ~0.6
• Place the culture on ice for at least 20 minutes
• Centrifuge for 8 minutes at 5000 g
• Discard the supernatant
• Gently resuspend in 1 mL of cold 10% glycerol, then fill up to 20 mL with cold glycerol
• Incubate on ice for 5 minutes
• Repeat the last four steps (centrifugation, washing) two more times
• After the last centrifugation, resuspend the pellet in 100 µL of cold 10% glycerol
• Distribute into 2 aliquots of 60 µL in cold 1.5 mL Eppendorf tubes

2. Preparation of Agar Media

• Prepare two LB-agar plates supplemented with chloramphenicol + ampicillin

3. Electroporation of Plasmid pCP20

• Add 1 µL of pCP20 plasmid to one tube of cells. Use the second tube as a control
• Incubate on ice for 15 minutes
• Transfer to a cold electroporation cuvette. Tap
• Apply the electric shock (program EC1)
• Quickly add 900 µL of 2YT. Transfer back to the Eppendorf tube
• Incubate for 1 hour 20 minutes at 30°C
• Centrifuge for 5 minutes at 5000 g, remove 800 µL of supernatant
• Resuspend the pellet and spread onto an LB-agar / chloramphenicol+ampicillin plate
• Incubate overnight at 30°C

DAY 9

• Pick several colonies and resuspend them in 1 mL of LB without antibiotics
• Incubate at 30°C until OD ~0.5
• Shift the culture to 37°C for 2 hours
• Dilute 1/100 in 1 mL of LB without antibiotics and incubate at 37°C until OD ~0.8
• Streak for isolation onto an LB-agar plate (without antibiotics) using a sterile loop
• Incubate overnight at 37°C

DAY 10

Verification of cassette and plasmid loss: Pick 6 colonies and patch them onto 3 types of plates:

• LB-agar (no antibiotic)
• LB-agar + kanamycin
• LB-agar + chloramphenicol + ampicillin

DAY 11

1. Clone Verification by Colony PCR

• For KanS (cassette excision) and CmS/AmpS (pCP20 plasmid loss) clones, perform colony PCR
• Prepare a PCR Master Mix (volume per reaction):
  • Econotaq Green: 10 µL
  • Primer #1: 0.4 µL
  • Primer #2: 0.4 µL
  • H₂O: 9.2 µL
• Vortex
• Aliquot 20 µL into PCR tubes
• Add a small amount of each selected colony. Use as controls: the starting strain and the strain with the inserted cassette
• Primers to use: one primer in the upstream gene and one primer in the downstream gene (to verify the junction)
• PCR Program: "Eric - EconoTaq". Extension time = 1 min per kb
• Run the entire samples on a 1% agarose gel. Load 5 µL of the corresponding PCR product from step D7 into a 9th well (positive control). Do not forget the molecular weight marker

Figure explaining the strategy for deleting the murI gene via lambda red recombination and FLP recombinase

Preparation of DL-glutamic acid Stock Solution

For detailed protocols of the preparation of DL-glutamate solution, please visit our Protocols page.

Protocol

1. Weigh 441.5 mg of DL-glutamic acid
2. Add QS to 50 mL with milli-Q water
3. Sterile filtration
4. Store at 4°C

Stock concentration: 53 mM

For 20 mM plates: Dilution factor 0.38

Hydrogel Encapsulation and Functionality

For detailed protocols of the encapsulation, survival test and escape test, please visit our Protocols page.

To physically contain our modified cells, we tested a 1.5% alginate hydrogel capable of encapsulating E. coli. We characterized this hydrogel by assessing both the ability of the cells to escape the matrix and their survival rate.

For these experiments, we used two E. coli strains available in our lab: W3110 ΔompT (the final strain used in our project) and W3110. Both strains had been used in previous experiments. We selected one motile and one non-motile strain to evaluate the containment capability of the hydrogel.

First, we performed a motility test to confirm the differences between the two strains. Once this difference was established, we developed our own protocols for hydrogel synthesis. After optimizing this protocol, we created additional procedures to assess bacterial escape and survival rates, analyzing the results by plotting escape curves and survival histograms.

We also used scanning electron microscopy (SEM) to examine the hydrogel matrix and the spatial distribution of the cells. Additionally, we performed fluorescence microscopy to confirm that the encapsulated cells were indeed our engineered strains and that they were able to produce molecules such as GFP.

1) Motility Test of W3110 ΔompT compared to W3110 motile

Dessert-like LB agar petri dish preparation (final LB agar concentration should be 0.3%)

• LB agar final volume = 20mL
• LB agar initial concentration = 1.5%

Calculation:

• Vi = (0.3 × 20mL)/1.5 = 4mL of LB agar
• Vf - V_{LB agar} = V_{LB liquid} = 20mL - 4mL = 16mL LB liquid

Methods:

1. Once the LB agar kind of solidifies (the idea is for it not to be completely solid), we pick one W3110 colony to transfer as a single point on the petri dish
2. We do the same for a W3110 ΔompT colony
3. Incubate at 37°C for 3h (carefully, since the LB agar is not completely solid for this test)

2) Hydrogel synthesis encapsulating W3110 ΔompT and W3110 non-motile cells

A) Bacterial pellet preparation

Materials:

• Overnight W3110 non motile + pBAD33 preculture and W3110 ΔompT + pBAD33 preculture
• LB + Chloramphenicol broth
• Spectrophotometer cuvettes
• Centrifuge
• Spectrophotometer
• 1 mL Eppendorf tubes
• Vortex

Methods:

1. Using LB as a blank, measure OD of overnight cultures
2. Dilute the overnight cultures with LB (+ antibiotic) until OD₆₀₀ = 1.0 (final volume 1mL)
3. NOTE: Consider preparing a LB + Chloramphenicol (CM) solution to use in each step we need LB
4. Prepare 2 Eppendorf tubes:
   • One containing 1mL OD 1.0 W3110 non motile + pBAD33 bacterial O.N preculture
   • One containing 1mL OD 1.0 W3110 ΔompT + pBAD33 O.N bacterial preculture
5. Centrifuge the cultures at 4,000 rpm for 10 min
6. Remove supernatant and obtain bacterial pellets

B) Bacterial encapsulation

Materials:

• Bacterial pellets
• Alginate
• 1% CaCl₂ solution
• DI water
• Analytical balance
• Mixing spatula
• 2 mL Eppendorf tubes
• 14mL Falcon tubes

Methods:

1. Weight hydrogel reagent: 192 mg alginate
2. Dissolve the bacterial pellets into 12.8 mL of DI water. For practicality:
   • Start by dissolving the pellets with 1mL of DI water
   • Transfer the solution to a 14mL falcon tube containing 11.8 mL of DI water
3. Dissolve 192 mg alginate in the mixture
4. Mix until alginate is fully dissolved to obtain a 1.5% bacterial alginate hydrogel
5. Carefully pipette 200 µL of the alginate mix into a sterile petri dish in order to create 200µL hydrogel drops
6. Add a 1% CaCl₂ solution to obtain hydrogel marbles

3) Hydrogel Bacterial Escape Test using W3110 ΔompT and W3110 non-motile cells

Materials:

• Sterile starter tubes
• Spectrophotometer
• Incubator set to 37°C
• LB + Chloramphenicol broth
• Bacterial hydrogel marbles

Methods:

1. Add 3 bacterial hydrogel marbles to the bottom of a starter tube
2. Repeat the procedure 5 more times for each cell type and name the tubes 1, 2, 3 and *1, *2, *3 for each cell type
3. In our case we have two cell types: W3110 XM and W3110 ΔompT. So in the end we should have 12 tubes:
   • XM1, XM2, XM3, XM*1, XM*2, XM*3
   • Δ1, Δ2, Δ3, Δ*1, Δ*2, Δ*3
4. Place the tubes in the incubator at 37°C for 20 min
5. Add 3.6 mL of LB+CM (Chloramphenicol) to each tube
6. For tubes *, break the pellet under sterile conditions then vortex the broken pellet to let the encapsulated bacteria escape the gel
7. NOTE: Consider the working concentration of chloramphenicol and dilute the stock accordingly into the LB
8. Measure OD after filling the tubes (save a LB+CM starter tube as a blank). Record the results, then leave to incubate at 37°C
9. Repeat the OD measuring process after:
   • t=0, t=30min, t=1h, t=1h30, t=2h30, t=17h, t=24h

OD measurements table structure:

4) Hydrogel Bacterial Survival Test

Tube Preparation:

Prepare 36 sterile Eppendorf tubes and name them as follows:

Sample preparation:

1. For each mX tube, add one 200µL non-motile hydrogel marble
2. For each Δ tube, add one 200µL ΔompT hydrogel marble
3. Add 1200µL of sterile 1X PBS solution to each tube
4. Prepare an additional control tube containing only PBS
5. Incubate all tubes at 37°C, except for the t=0 tubes, which will be processed immediately

Processing t=0 tubes:

1. Under sterile conditions, mechanically disrupt (crash) the hydrogel marble in each t=0 tube
2. Centrifuge for 3 min at 3000 rpm
3. Collect 110µL of supernatant from each tube and transfer to column A (rows 12A–7A) of a sterile 96-well ELISA plate, following the layout diagram

Fill in of the first ELISA well plate:

• A12 → 110µL MX1 t=0
• A11 → 110µL MX2 t=0
• A10 → 110µL MX3 t=0
• A9 → 110µL Δ1 t=0
• A8 → 110µL Δ2 t=0
• A7 → 110µL Δ3 t=0

Serial dilution:

1. Add 90µL of sterile 1X PBS to all remaining wells of the same row (B to H)
2. Perform 7 serial 1:10 dilutions by transferring 10µL from column A to column B, mixing well, and repeating sequentially until column H
3. This achieves a final dilution factor of 10⁻⁷ for each sample

ELISA Plate Layouts:

First ELISA well plate (t=0):

• A12 → 110µL MX1 t=0
• A11 → 110µL MX2 t=0
• A10 → 110µL MX3 t=0
• A9 → 110µL Δ1 t=0
• A8 → 110µL Δ2 t=0
• A7 → 110µL Δ3 t=0

Figure 1: ELISA plate layout for t=0 time point

Second ELISA well plate (t=30 and t=1h):

• A12 → 110µL MX1 t=30
• A11 → 110µL MX2 t=30
• A10 → 110µL MX3 t=30
• A9 → 110µL Δ1 t=30
• A8 → 110µL Δ2 t=30
• A7 → 110µL Δ3 t=30
• A6 → 110µL MX1 t=1h
• A5 → 110µL MX2 t=1h
• A4 → 110µL MX3 t=1h
• A3 → 110µL Δ1 t=1h
• A2 → 110µL Δ2 t=1h
• A1 → 110µL Δ3 t=1h

Figure 2: ELISA plate layout for bacterial survival test - Plate 2 configuration

Third ELISA well plate (t=3h and t=6h):

• A12 → 110µL MX1 t=3h
• A11 → 110µL MX2 t=3h
• A10 → 110µL MX3 t=3h
• A9 → 110µL Δ1 t=3h
• A8 → 110µL Δ2 t=3h
• A7 → 110µL Δ3 t=3h
• A6 → 110µL MX1 t=6h
• A5 → 110µL MX2 t=6h
• A4 → 110µL MX3 t=6h
• A3 → 110µL Δ1 t=6h
• A2 → 110µL Δ2 t=6h
• A1 → 110µL Δ3 t=6h

Figure 3: ELISA plate layout for bacterial survival test - Plate 3 configuration

Bacterial culture spotting:

1. Collect 4µL of each of the samples on the well plate and spot them onto a LB + CM (Chloramphenicol) + arabinose agar plate
2. Draw out a grid on the petri dish beforehand to facilitate the spotting
3. Try to maintain the same layout as the ELISA well plate for consistency

Subsequent Time Points:

1. After 30 minutes of incubation of the rest of the Eppendorf tubes at 37°C, process the t=30 tubes as described above (crush, centrifuge, transfer supernatant, serial dilution, spotting)
2. Repeat the same procedure for tubes t=1h after 1 hour of incubation, t=3h after 3 hours, and so on
3. Incubate the spotted agar plates overnight (ON) at 37°C

5) Scanning Electron Microscopy (SEM)

Samples:

Three alginate beads per condition were prepared: 3 × XM (W3110 non-motile), 3 × Δ (W3110 ΔompT), and one negative control bead.

Methods:

1. Fixation: Fix samples in 2.5% glutaraldehyde prepared in PBS 1X. Incubate for 1 h at room temperature
2. Rinsing: Rinse samples 3 × 10 min in PBS 1X
3. Post-fixation: Incubate samples in 0.5% osmium tetroxide in PBS 1X for 1 h at 4°C. Store at 4°C until dehydration
4. Dehydration: Sequentially dehydrate samples in ethanol:
   • 25% (15 min)
   • 50% (15 min)
   • 70% (15 min)
   • 100% (15 min)
   Leave overnight to evaporate
5. Mounting: Cut beads and mount on support stubs using conductive carbon tape
6. Coating and observation: Carbon coat the mounted samples and observe by SEM

6) Fluorescence Microscopy

Methods:

1. Mechanically disrupt (crash) the hydrogel marble in each tube
2. Centrifuge for 3 min at 3000 rpm
3. Collect 110µL of supernatant from each tube
4. Look under the microscope for fluorescence detection

Conclusion

Overall, the success of these experiments would be the first step to demonstrate the feasibility of combining surface-display systems, metabolic pathway optimization, and genetic biocontainment in E. coli. The pAIDA-tyr1 plasmid enabled measurable melanin production, with tyrosinase expression confirmed by Western blot and visual tests. Tyrosine biosynthesis would be enhanced through aroF mutagenesis, providing a more robust metabolic background for pigment production. Finally, the ΔmurI deletion and its complementation with pMurI illustrate a controlled strategy for biocontainment, ensuring that engineered strains maintain functionality while limiting environmental risk. Together, these results showcase the integration of molecular design, precise genetic engineering, and functional testing in building safe and productive microbial systems.

Combining genetic engineering with biomaterials (hydrogel) can create functional cellular systems capable of producing target molecules, opening possibilities for biotechnological applications, antioxidant protection, and controlled delivery of bioactive compounds.