iGEM 2025 — Team aix-marseille

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block header\_image %}
![Bannière de la page](https://static.igem.wiki/teams/5932/footer/wet-lab-notebook-nadia.webp)
{% endblock %}
{% block page\_content %}

![Chat qui dort](https://static.igem.wiki/teams/5932/photos/image-1-1.webp)

### Notebook Sections

* [Oligo names](#amorce)
* [The beginning of the wet lab](#introduction)
* [Construction of a hydrogel](#hydrogel)
* [Construction of pAIDA-tyr1](#AIDA)
* [Shikimate pathway amplification: Mutation AroF](#aroF)
* [Construction of isogenic mutant murI of W3110 Δ ompT](#murI)
* [Construction of complementation plasmid of Δ murI : pMurI](#pmurI)

## Oligo table names

▼

### Oligo Table

| Code | Oligo Name | Sequence |
| --- | --- | --- |
| oLS 1.1 | murcheckup | TGTCAGTGGTGGCGCATTAT |
| oLS 1.2 | murcheckdo | CCGTTAAAATGGGCGGTGTG |
| oLS 1.3 | Del-murI-W3110-5-DW | accaaactgcaggacgggaatacaccttgtctggcagctacaccttctgaTGTGTAGGCTGGAGCTGCTTCG |
| oLS 1.4 | Del-murI-W3110-3-DW | gacaagaggaaatttaaaataattttctgaccgcgcaacattcaaccaaaCATATGAATATCCTCCTTAGTTC |
| oLS 2.1 | 5'pkng AroF Slic1 | CCCTGCAGGTCGACGGATCCACTCCGGAACAACTGAAGGC |
| oLS 2.2 | 3' AroF Slic2 | Aagcccggaggccatttcac |
| oLS 2.3 | 5' aroF S181A slic3 | Atggcctccgggcttgcgatgccggttggttttaaaaacggc |
| oLS 2.4 | 3'pkng aroF Slic4 | GACCCTCCGCCGGCCGCTGCCAGCTAATGCAGGCATCGGT |
| oLS 2.5 | 5B pkng | CTGGTTGAACCGGATGCTGG |
| oLS 2.6 | 6 pkng | CATATCACAACGTGCGTGGA |
| oLS 2.7 | 5'Verif S181A aroF | Tggcctccgggcttgcg |
| oLS 3.4 | pA12-UP | CTTGGAACACCTGCTTTTCGC |
| oLS 3.5 | pA12-DO | cttcgaagacaactccctgcag |
| LS 4 | Try1 CA085 | AGCGCTGTTCCAGGGTCCGGGTACCGGTAATAAATATCGCGTTCGTAAAAATGTGTTGCATCTGACCGACACCGAAAAACGCGATTTCGTGCGTACCGTTCTGATTCTCAAAGAGAAAGGTATCTACGACCGCTATATCGCCTGGCATGGCGCAGCGGGTAAATTTCATACTCCTCCTGGCAGCGATCGCAACGCCGCGCATATGAGCAGCGCCTTCCTGCCGTGGCACCGCGAATATCTGCTGCGTTTTGAACGCGATCTGCAAAGCATCAACCCAGAAGTGACCCTGCCGTACTGGGAGTGGGAAACCGATGCGCAAATGCAGGATCCGAGTCAGTCGCAGATCTGGTCGGCGGACTTTATGGGCGGCAACGGTAACCCGATCAAAGATTTTATTGTCGATACCGGTCCGTTCGCCGCAGGCCGCTGGACCACCATTGATGAACAGGGCAATCCGAGCGGCGGCCTGAAGCGCAATTTTGGCGCCACCAAAGAAGCGCCGACGCTGCCGACCCGAGATGACGTGCTGAACGCGCTGAAAATTACCCAGTATGACACGCCGCCGTGGGATATGACCAGCCAGAATAGCTTTCGTAACCAACTGGAAGGTTTTATCAATGGCCCGCAGCTGCACAACCGCGTGCATCGTTGGGTTGGTGGTCAAATGGGCGTAGTCCCAACTGCGCCGAACGATCCAGTATTTTTCCTGCATCACGCCAACGTGGATCGAATCTGGGCGGTCTGGCAGATTATTCACAGGAATCAGAATTATCAGCCAATGAAAAACGGTCCGTTCGGTCAGAACTTCCGCGACCCGATGTATCCGTGGAACACCACCCCAGAAGATGTTATGAACCATCGCAAACTGGGCTATGTCTACGATATCGAACTGCGCAAAAGCAAACGTTCCAGCGAGCTCGAAAACCTGTACTTCCAGGGTGAAC |
| oLS 4.1 | tyr1UP | AGCGCTGTTCCAGGGTCCGGGTACC |
| oLS 4.2 | tyr1DO | GTTCACCCTGGAAGTACAGGTTTTCGA |
| oLS 4.3 | 5-PKO3lin | CCGCTTATTATCACTTATTCAGGCGTAGC |
| olS 4.4 | 3-PKO3lin | GGTCGTTAAATAGCCGCTTATGTCTATTGC |
| olS 4.5 | 5'Pko3 AroF Slic1 | gaataagtgataataagcggACTCCGGAACAACTGAAGGC |
| olS 4.6 | 3'pko3 aroF Slic4 | taagcggctatttaacgaccCAGCTAATGCAGGCATCGGT |
| olS 4.7 | 5'VerifPko3 | Ggcttccatgtcggcagaatg |
| olS 4.8 | 3'Verifpko3 | Gcgtcccattcgccattctccg |

## The beginning of the wet lab

▼

This section contains all the initial steps and preparations necessary for our wet lab experiments and training in laboratory procedures.

### **2 June 2025**

**Pre-culture preparation of E.coli DH5𝛂 and E.coli BL21 to make them super-competent**

Inoculate 1 colony of E. coli DH5𝛂 into a 10 mL Erlenmeyer flask of LB and 1 colony of E. coli BL21 into another 10 mL Erlenmeyer flask of LB. Shake overnight at 37°C

### **3 June 2025**

**Preculture Dilution**

Under sterile conditions (Bunsen burner flame)

* 1. Retrieve the E. coli DH5 alpha and BL21 preculture
* 2. Add 1 mL of preculture to a 500 mL Erlenmeyer flask containing 100 mL of LB (separately for DH5 alpha and BL21)
* 3. Incubating for 2 hours at 37°C with shaking at 180 rpm

**OD Measurement by Spectrophotometer**

Measure the OD of the cells in 1 mL cuvettes to reach an approximate OD600 = 0.5.

**Preparation of Supercompetent Cells (DH5 alpha and BL21)**

Preparation of Solutions 1 and 2 (used during centrifugation steps) :

Solution 1 (40 mL):

* 1,2 mL KoAc 1 M
* 4 mL MnCl2 0,5 M
* 4 mL Kcl 1 M
* 4 mL CaCl2 0,1 M
* 7,5 mL Glycérol 80%
* 19,3 mL sterile H2O

Solution 2 (40 mL):

* 200 µL MOPS 0,2 M
* 40 µL KCl 1 M
* 3 mL CaCl2 0,1 M
* 750 µL glycérol 80%
* 10µL sterile H2O

* 1. Transfer 80 mL from the Erlenmeyer flasks into two 40 mL Falcon tubes (for DH5 alpha and BL21)
* 2. Centrifuge cells for 10 min at 3500 rpm, 4°C
* 3. Discard the supernatant and resuspend the pellet in 400 µL of Solution 1 (first wash). Combine pellets into one Falcon tube and add 39.6 mL of Solution 1.
* 4. Centrifuge for 5 min at 3500 rpm, 4°C.
* 5. Discard the supernatant and resuspend the pellet in 200 µL of Solution 2 (second wash). Combine pellets into one tube and add 3.8 mL of Solution 2.
* 6. Freeze aliquots (100 µL) in liquid nitrogen, then store at -80°C. Retain one aliquot for supercompetency testing.

**Test : Transformation with Supercompetent Cells**

Preparation of LB + Ampicillin plates (final concentration : 1µg/µL)

Under sterile conditions (Busen burner flame) :

* 1. Prepare 2 tubes with 20 µL of bacteria
* 2. Add 1 µL of each plasmid (10 ng/µL) in 1 tube
* 3. Incubate on ice for 15 min
* 4. Apply heat shock : 45 sec at 42°C (preheat block in advance)
* 5. Return to ice for 2 min
* 6. Add 1 mL of LB.
* 7. Incubate at 37°C with shaking for 45 min and 1 hour max
* 8. Centrifuge at 8000 rpm for 30 sec
* 9. Remove ~900 µL of supernatant and resuspend the remaining 100 µL by pipetting
* 10. Spread 3µl of bacteria onto half of an LB + ampicillin plate for each plasmid (half DH5 alpha / half BL21) (using a spreader/pasteur pipette)

**Contamination Test**

Spread 3 µL of E. coli DH5 alpha on half of 3 plates and 3 µL of E. coli BL21 on the other half of each plate (on LB agar + ampicillin, LB agar + kanamycin and LB agar + chloramphenicol)

### **4 June 2025**

Tuesday’s transformation failed; repeating the procedure.

**Preparation of Supercompetent Cells (DH5 alpha and BL21)**

Follow the same protocol as Monday 03/06/2025

### **10 June 2025**

**Preparation of pre-cultures for E. coli DH5𝛂 and E. coli BL21 to make them super-competent**

Inoculated 1 colony of E. coli DH5𝛂 into a 5 mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

Inoculated 1 colony of E. coli BL21 into a 5 mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

### **11 June 2025**

The LB was contaminated, repeated the pre culture

**Preparation of solutions for super-competent cell transformation**

* MnCl2 0,5M ; 197,9 g/mol
* Kcl 1M ; 74,555 g/mol
* MOPS 0,2M ; 209,26 g/mol
* KoAc 1M ; 98,17 g/mol
* Prepared by dissolving:
* 9,9 g MnCl2 and 7,455g Kcl in 100mL d’H20
* 2,09 g in 50 mL d’H20
* 4,9 g KoAc in 50 mL d’H20

### **12 June 2025**

**Preparation of pre-culture for E.coli DH5alpha and E.coli W3110 to make them super-competent:**

Inoculated 1 colony of E.coli DH5alpha into a 5mL LB Erlenmeyer flask. Inoculated 1 colony of E.coli W3110 into a separate 5mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

### **13 June 2025**

**Dilution of pre-culture**

(Performed under sterile conditions using a Bunsen burner flame)

* Added:* 1 mL of pre-culture into a 500 mL Erlenmeyer flask containing 100 mL LB (for DH5𝛂).
  * 250 µL of pre-culture into a 125 mL Erlenmeyer flask containing 25 mL LB (for W3110).
* Incubated with shaking for 2 hours at 37°C.

**OD measurement using a spectrophotometer**

Measured cell OD in 1 mL cuvettes to target OD₆₀₀ ≈ 0.5.

W3110 : 0,72

DH5 alpha : 0,47

**Preparation of Supercompetent Cells DH5 alpha cells**

Prepared Solution 1 and Solution 2 for use during centrifugation steps.

40 mL of Solution 1 :

* 1,2 mL KoAc 1 M
* 4 mL MnCl2 0,5 M
* 4 mL Kcl 1 M
* 4 mL CaCl2 0,1 M
* 7,5 mL Glycérol 80%
* 19,3 mL H2O stérile

4 mL of Solution 2 :

* 200 µL MOPS 0,2 M
* 40 µL KCl 1 M
* 3 mL CaCl2 0,1 M
* 750 µL glycérol 80%
* 10µL H2O stérile

Preparation of Supercompetent Cells DH5 alpha cells

* 1. Transferred 80 mL from the Erlenmeyer flask into two 40 mL Falcon tubes (for DH5𝛂).
* 2. Centrifuged cells at 3500 rpm for 10 min at 4°C.
* 3. Discarded supernatant and resuspended pellet in 20 mL Solution 1 per tube (first wash).
* 4. Centrifuged again at 3500 rpm for 10 min at 4°C.
* 5. Discarded supernatant and resuspended pellet in 2 mL Solution 2 (second wash), combining pellets into one tube and adding 3.8 mL Solution 2.
* 6. Aliquoted into 100 µL samples, flash-frozen in liquid nitrogen, and stored at -80°C.
* 7. Kept one aliquot for super-competency testing.

**Contamination test**

Plated 3 µL of E. coli DH5𝛂 on:

* LB agar + Ampicillin
* LB agar + Kanamycin
* LB agar + Chloramphenicol

For W3110 :

Prepared 10% glycerol solution:

* Vi = 10% (Cf) \* 200 mL (Vf) / 80% (Ci) = 25 mL glycérol 80%
* Added H2O to final volume of 200 mL

**Preparation of Electrocompetent Cells W3110 cells**

Two washes with 35 mL of 10% glycerol (perform on ice)

* 1. Transfer 35 mL from the Erlenmeyer flask into a 50 mL Flacon tube for W3110
* 2. Wait in ice 20 minutes
* 3. Centrifuge at 5000 rpm, 10 min, 4°C
* 4. Discard the supernatant
* 5. Resuspend in 35 mL of 10% glycerol
* 6. Wait in ice 5 minutes
* 7. Centrifuge at 5000 rpm, 10 min, 4°C
* 8. Discard the supernatant
* 9. Resuspend in 35 mL of 10% glycerol
* 10. Centrifuge at 5000 rpm, 10 min, 4°C
* 11. Discard the supernatant
* 12. Resuspend in 250 µL of 10% glycerol

**Electroporation of pKOBEG Vector into W3110**

Prepare two 1.5 mL Eppendorf tubes :

* 1.
  + CONTROL : 60 µL W3110 culture in 10% glycerol
  + pKOBEG : 60 µL W3110 culture in 10% glycerol + 1 µL pKOBEG vector
* 2. Incubate on ice for 15 min
* 3. Transfer the 60 µL mixtures into electroporation cuvettes
* 4. Electroporate using the appropriate machine
* 5. Immediately add 900 µL of LB broth to each cuvette (control and vector) and resuspend
* 6. Transfer into 1.5 mL Eppendorfs tubes
* 7. Shake at 30°C, 750 rpm, 1 hour
* 8. Centrifuge 5000 rpm, 5 min
* 9. Remove 800 µL of supernatant
* 10. Resuspend the pellet in the remaining 100 µL
* 11. Streak into LB agar + chloramphenicol plates for selection

### **16 June 2025**

**Preparation of E.coli DH5𝛂 and E.coli W3110 precultures to make them super-competent.**

Inoculated 1 colony of E.coli DH5𝛂 into a 5mL LB Erlenmeyer flask.

Inoculated 1 colony of E.coli W3110 into a 5mL LB Erlenmeyer flask.

Shake overnight at 37°C.

### **17 June 2025**

**Preparation of antibiotic petri dishes**Make a stock of 10 plates !

Cm : (chloramphenicol) :

* Working concentration Ci = 30 µL/mL
* 1,3 µL/mL culture.
* 1 plate = 20 mL for 10 plates = 200 mL final volume
* 1,3 \* 200 = 260 µL Cm in 200 mL de LB agar

Km : (kanamycin) :

* Working concentration Ci = 10,5 µL/mL
* 1 plate = 20 mL for 5 \* 20 = 100 µL Km in 20 mL de LB agar → only made 1 plate.

Amp : Ampicillin

* Working concentration Ci = 25,4 µL/mL
* 1 plate = 20 mL, for 100 plates = 200 mL final volume
* 4 \* 200 = 800 µL Amp in 200 mL de LB agar

**Preculture Dilution**

Follow the same procedure as made the 13 june

**OD Measurement**

**Preparation of Competent Cells**

* 1. Transfer 80 mL from Erlenmeyer flask into two 40 mL Flacon tubes for DH5 alpha.
* 2. Centrifugate cells at 3500 rpm à for 10 minute 4°C
* 3. The supernatant is discarded and the pellet is resuspended in 20 mL of solution 1 for each of two tubes ( first wash). The pellet is resuspended gently using two sticks to avoid damaging it.
* 4. Centrifugate again at 3500rpm for 10 minutes at 4°C.
* 5. The supernatant is discarded and the pellet is resuspended in 2 mL of Solution 2 (second wash). The pellets are then combined into a single flask, and 3.8 mL of Solution 2 is added.
* 6. The mixture is left to rest for 1 hour on ice.
* 7. The solutions are aliquoted into 100 µL portions, flash-frozen in liquid nitrogen and stored at -80°C. One aliquot is kept for the supercompetency test

**Contamination Test** 6 µL of E. coli DH5 alpha bacteria were spread on each plate :

* LB agar Ampicillin
* LB agar Kanamycin
* LB agar Chloramphenicol

**Preparation of Supercompetent DH5 alpha Cells**Preparation of Petri plates with LB + Kanamycin (1 µG/µL final concentration)

* 1. Prepare 1 tube with 100 µL of bacteria
* 2. Add 1 µl of plasmid pRSF (10ng/µl) into a tube (one tube per plasmid)
* 3. Incubate the tubes on ice for 15 min.
* 4. Apply heat shock : 42°C for 45 sec (pre-warm the heat block in advance)
* 5. Return the tubes to ice for 2 min
* 6. Add 1 mL of LB medium
* 7. Incubate the tubes at 37°C with shaking for 45 min to 1 hour (max)
* 8. Centrifuge at 8000 rpm for 30 sec
* 9. Remove ~900 µL of supernatant and resuspend the remaining 100 µL by pipetting
* 10. Spread 6µL of bacteria onto an LB + kanamycin plate (one plate per plasmid) using a spreader/pasteur pipette.

### **18 June 2025**

**Dilution of pre-culture**Follow the same procedure as described on Tuesday, 03/06 but this time with E.coli CC118

**OD Measurement by Spectrophotometer**

## Construction of a hydrogel

▼

This section contains all the steps involved in constructing a hydrogel.

### **22 July 2025**

**1st Hydrogel Synthesis (Test) → Hydrogel Marbles**

Hydrogel Preparation (1.5% Alginate Solution):

* 1. Weigh 150 mg of sodium alginate powder and transfer it into a Falcon tube.
* 2. Add 10 mL of distilled water and vortex thoroughly until a homogeneous viscous solution is obtained.

Gelation:

* 3. For a 1% CaCl₂ solution (prepared from a 1 M stock) into a clean glass container.
* 4. Using a pipette, carefully drop the alginate solution into the CaCl₂ bath drop by drop, allowing each droplet to solidify into a mini hydrogel sphere (this will be our hydrogel marbles) via ionic crosslinking.

**Results:**

![hydrogel-marbles](https://static.igem.wiki/teams/5932/wiki/notebook/hydrogel-marbles.png)

Preculture W3110 non-motile pBAD33 and W3110ΔompT pBAD33 + arabinose to test the motility of the strains.

* Inoculate 1 colony of the strain into 5 mL LB + 0.1% arabinose (with cloraphenicol) in a sterile 25 mL flask. Shake overnight at 37°C

### **23 July 2025**

**Magic Box**

We transferred 500 µL of our pBAD33 + W3110 non-motile preculture and 500 µL of our pBAD33 + W3110ΔompT preculture into two eppendorf tubes, then centrifuged them in order to obtain cell pellets. We took a look at our pellets at the magic box to check the GFP expression → we decided to redo our precultures with a 0.2% arabinose induction to obtain better results during our tests

**Preculture** pBAD33 + W3110 non-motile + arabinose :

Inoculate 1 colony of non-motile E. coli W3110 (pBAD33) into 20 mL LB + 0.2% arabinose (with cloraphenicol) in a sterile 100 mL flask. Shake overnight at 37°C

Preculture pBAD33 + W3110ΔompT + arabinose

Inoculate 1 colony of E. coli W3110ΔompT (pBAD33) into 20 mL LB + 02% arabinose (with cloraphenicol) in a sterile 100 mL flask. Shake overnight at 37°C

**Magic Box**We transferred 1mL of our pBAD33 + W3110 non-motile preculture and 1mL of our pBAD33 + W3110ΔompT preculture into two eppendorf tubes, then centrifuged them in order to obtain cell pellets. We took a look at our pellets at the magic box to check the GFP expression → our 0.2% arabinose induced precultures expressed better our GFP protein.

**Hydrogel bacterial escape test**

1. Bacterial pellet preparation:

* Materials: Overnight w3110 non motile + pBAD33 preculture and W3110 ΔompT + pBAD33 preculture, LB + Cloraphenicol broth, Spectrophotometer cuvettes, Centrifuge, Spectrophotometer, 1 mL Eppendorf tubes, Vortex
* 1. Using LB as a blank, measure OD of overnight cultures.
* 2. Dilute the overnight cultures with LB (+antibiotic) until the value of its OD600 is equal 1.0 (Final volume 900 µL)
* NOTE: Consider preparing a LB + Cloramphenicol (CM) solution to use in each step we need LB.
* 3. Prepare 2 Eppendorf tubes, one containing 900 µL OD 1.0 W3110 non motile + pBAD33 bacterial culture and the other one containing 900 µL OD 1.0 W3110ΔompT + pBAD33 bacterial culture.
* 4. Centrifuge the cultures at 4,000 rpm for 10 min
* 5. Remove supernatant and obtain bacterial pellets

2. Bacterial encapsulation:

* Materials: Bacterial pellets, Alginate, 1% CaCl2 solution, DI water, Analytical balance, Mixing spatula, 2 mL Eppendorf tubes, 14mL Falcon tubes
* 6. Weight hydrogel reagent: 96 mg alginate
* 7. Dissolve the bacterial pellets into 6,4 mL of DI water. For practicity, start by dissolving the pellets with 1mL of DI water then transfer the solution to a 14mL falcon tube containing 5,4mL of di water.
* 8. Dissolve 96 mg alginate in the mixture. Mix until alginate is fully dissolved to obtain a 1.5% bacterial alginate hydrogel
* 9. Carefully pipette 200 µL of the alginate mix in order to create 200µL hydrogel drops and add a 1% CaCl2 solution to obtain hydrogel marbles

3. Leakage Test

* Materials: 2 mL Eppendorf tubes, Spectrophotometer, Incubator set to 37°C, LB + Chloramphenicol broth, Bacterial hydrogel marbles
* 10. Add 1 bacterial hydrogel marble to the bottom an eppendorf tube (repeat the procedure 3 times for each of the two pellets and name the tubes 1,2,3)
* 11. Place the tubes in the incubator at 37°C for 20 min.
* 12. Add 1200 µL of LB+CM to tubes number 1
* 13. For tubes number 2, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 5.6 mL LB+CM. Transfer back 1,4mL of the new mix into tube number 2
* 14. For tubes number 3, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 8 mL LB+CM. Transfer back 2 mL of the new mix into tube number 3.
* NOTE: Consider the working concentration of chloramphenicol and dilute the stock accordingly into the LB
* 15. Measure OD after filling the tubes. Record the results, then recharge each tube with the sample measured and leave to incubate at 37 degrees.
* 16. Repeat the OD measuring process after 30 min, 1 hour, 1.5 hours, etc

### **30 July 2025**

**Hydrogel bacterial escape test**

1. Bacterial pellet preparation:

* Materials: Overnight w3110 non motile + pBAD33 preculture and W3110 ΔompT + pBAD33 preculture, LB + Cloraphenicol broth, Spectrophotometer cuvettes, Centrifuge, Spectrophotometer, 1 mL Eppendorf tubes, Vortex
* 1. Using LB as a blank, measure OD of overnight cultures.
* 2. Dilute the overnight cultures with LB (+antibiotic) until the value of its OD600 is equal 1.0 (Final volume 900 µL)
* NOTE: Consider preparing a LB + Cloramphenicol (CM) solution to use in each step we need LB.
* 3. Prepare 2 Eppendorf tubes, one containing 900 µL OD 1.0 W3110 non motile + pBAD33 bacterial culture and the other one containing 900 µL OD 1.0 W3110ΔompT + pBAD33 bacterial culture.
* 4. Centrifuge the cultures at 4,000 rpm for 10 min
* 5. Remove supernatant and obtain bacterial pellets

2. Bacterial encapsulation:

* Materials: Bacterial pellets, Alginate, 1% CaCl2 solution, DI water, Analytical balance, Mixing spatula, 2 mL Eppendorf tubes, 14mL Falcon tubes
* 6. Weight hydrogel reagent: 96 mg alginate
* 7. Dissolve the bacterial pellets into 6,4 mL of DI water. For practicity, start by dissolving the pellets with 1mL of DI water then transfer the solution to a 14mL falcon tube containing 5,4mL of di water.
* 8. Dissolve 96 mg alginate in the mixture. Mix until alginate is fully dissolved to obtain a 1.5% bacterial alginate hydrogel
* 9. Carefully pipette 200 µL of the alginate mix in order to create 200µL hydrogel drops and add a 1% CaCl2 solution to obtain hydrogel marbles

3. Leakage Test

* Materials: 2 mL Eppendorf tubes, Spectrophotometer, Incubator set to 37°C, LB + Chloramphenicol broth, Bacterial hydrogel marbles
* 10. Add 1 bacterial hydrogel marble to the bottom an eppendorf tube (repeat the procedure 3 times for each of the two pellets and name the tubes 1,2,3)
* 11. Place the tubes in the incubator at 37°C for 20 min.
* 12. Add 1200 µL of LB+CM to tubes number 1
* 13. For tubes number 2, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 5.6 mL LB+CM. Transfer back 1,4mL of the new mix into tube number 2
* 14. For tubes number 3, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 8 mL LB+CM. Transfer back 2 mL of the new mix into tube number 3.
* NOTE: Consider the working concentration of chloramphenicol and dilute the stock accordingly into the LB
* 15. Measure OD after filling the tubes. Record the results, then recharge each tube with the sample measured and leave to incubate at 37 degrees.
* 16. Repeat the OD measuring process after 30 min, 1 hour, 1.5 hours, etc

**Results:**

![od](https://static.igem.wiki/teams/5932/wiki/notebook/od.png)

### **31 July 2025**

**Hydrogel bacterial escape test**

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

**Résultats:** Results interpretation: Although we did not observe a significant difference between our intact and mechanically disrupted hydrogel marbles, as initially expected, we did notice a clear difference between motile and non-motile strains. The OD of our motile E. coli W3110ΔompT cells increased significantly more than that of the non-motile W3110 strain. This suggests that, in order to effectively contain bacteria within the hydrogel matrix, it may be necessary to eliminate motility in the final cell line. A possible strategy could involve deleting flagellar genes such as fliC.

### **07 August 2025**

**Hydrogel bacterial survival test**

Bacterial pellet preparation:

* Follow the same protocol as Wednesday 30/07/2025

Bacterial encapsulation:

* Follow the same protocol as Wednesday 30/07/2025 and Wash the marbles 3 times with a 1XPBS solution before first incubation at 37 degrees.

Survival Test

* 1. Prepare 36 sterile eppendorf tubes and name them as followed:
![naming](https://static.igem.wiki/teams/5932/wiki/notebook/naming.png)* References: 1, 2, 3 corresponds to the triplicate of each cell sample mX corresponds to W3110 non motile + pBAD33 Δ corresponds to W3110 ΔompT + pBAD33 t = time of measurement
* 2. Sample preparation: For each mX tube, add one 200 µL non-motile hydrogel marble. For each Δ tube, add one 200 µL ΔompT hydrogel marble. Add 1200 µL of sterile 1X PBS solution to each tube. Prepare an additional control tube containing only PBS. Incubate all tubes at 37 °C, except for the t=0 tubes, which will be processed immediately.
* 3. Processing t=0 tubes: Under sterile conditions, mechanically disrupt (crash) the hydrogel marble in each t=0 tube. Centrifuge for 3 min at 3000 rpm, then collect 110 µL of supernatant from each tube and transfer to column A (rows 12A–7A) of a sterile 96-well ELISA plate, following the layout diagram.
![layout](https://static.igem.wiki/teams/5932/wiki/notebook/layout.png)* 4. Serial dilution: Add 90 µL of sterile 1X PBS to all remaining wells of the same row (B to H). Perform 7 serial 1:10 dilutions by transferring 10 µL from column A to column B, mixing well, and repeating sequentially until column H. This achieves a final dilution factor of 10⁻⁷ for each sample.
![dilution](https://static.igem.wiki/teams/5932/wiki/notebook/dilution.png)* 5. Bacterial culture spotting: collect 4µL of each of the samples on the well plate and spot them onto a LB + CM (Cloramphenicol) + arabinose agar plate. (draw out a grid on the petri dish beforehand in order to facilitate the spotting, and try to maintain the same layout as the ELISA well plate for consistency)
* 6. Subsequent Time Points: 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). Repeat the same procedure for tubes t=1h after 1 hour of incubation, t=3h after 3 hours, and so on.
* 7. Incubation: Incubate the spotted agar plates overnight (ON) at 37 °C.

**Résultats:** Identification des sites d'interaction clés pour la liaison.

### **08 August 2025**

**Hydrogel bacterial survival test results**

Only the t=30 min and t=1h samples grew. We think it is due to a problem during the manipulation of the samples, and we will redo our test next week

![results](https://static.igem.wiki/teams/5932/wiki/notebook/results.png)

### **12 August 2025**

**Hydrogel bacterial survival test**

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

![naming2](https://static.igem.wiki/teams/5932/wiki/notebook/naming2.png)

References: 1, 2, 3 corresponds to the triplicate of each cell sample, xM corresponds to W3110 non motile + pBAD33, Δ corresponds to W3110 ΔompT + pBAD33, \* corresponds to the tubes in which the marbles have been crushed, t = time

**Résultats:** Results interpretation: We observed growth for all samples except for the t=0h samples, which is expected since they were processed immediately after encapsulation. The growth of both motile and non-motile strains over time indicates that our hydrogel matrix supports bacterial viability. However, we did not observe a significant difference in growth between the motile and non-motile strains, suggesting that motility may not play a crucial role in bacterial escape from the hydrogel under these conditions. Further experiments could involve testing different hydrogel compositions or crosslinking densities to assess their impact on bacterial containment.

### **13 August 2025**

**Hydrogel bacterial survival test results**

![results2](https://static.igem.wiki/teams/5932/wiki/notebook/results2.png)

Conclusion: Our bacteries are still alive inside our hydrogel after 24h. We shall repeat our test during a longer period of time

### **28 August 2025**

**Hydrogel bacterial escape test**

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

![naming3](https://static.igem.wiki/teams/5932/wiki/notebook/naming3.png)

References: 1, 2, 3 corresponds to the triplicate of each cell sample, xM corresponds to W3110 non motile + pBAD33, Δ corresponds to W3110 ΔompT + pBAD33, \* corresponds to the tubes in which the marbles have been crushed, t = time

Results interpretation: We compared our current results with those obtained from the same test conducted on Thursday 31/07/2025 to ensure that our measurements were both representative and accurate. After refining our technique for producing alginate marbles, we achieved improved results. To minimize operator error, we combined data from both the July 31 test and the current experiments for our final results graph.

**Résultats:** Results interpretation: We observed growth for all samples except for the t=0h samples, which is expected since they were processed immediately after encapsulation. The growth of both motile and non-motile strains over time indicates that our hydrogel matrix supports bacterial viability. However, we did not observe a significant difference in growth between the motile and non-motile strains, suggesting that motility may not play a crucial role in bacterial escape from the hydrogel under these conditions. Further experiments could involve testing different hydrogel compositions or crosslinking densities to assess their impact on bacterial containment.

## Construction of pAIDA-tyr1

▼

This section contains all the steps involved in constructing a plasmid pAIDA-Tyr1 and all the tests to test the melanin activity

### **27 June 2025**

**pAIDA-I from AddGene**

Pick cells forms the AddGene tube with a toothpick than transfer into a Cm + LB plate and incubate overnight at ambient temperature

### **30 June 2025**

**pAIDA-I stock**

**Preculture**:Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

### **01 July 2025**

**Plasmid Miniprep Kit for pAIDA-I**

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 18 ng/µL

**Preculture**:Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

### **02 July 2025**

**pAIDA-I stock**

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 24,9 ng/µL

**Preculture**: Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

### **03 July 2025**

**pAIDA-I stock**

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 28,8 ng/µL

### **08 July 2025**

**Clonage PCR of Tyr1 in pAIDA**

Here we use :

* 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 minutes
* Denaturation: 95°C for 1 min
* Annealing: 56°C for 1 min
* Extension: 68°C for 13 minute
* 29 cycles of Denaturation - Annealing - Extension
* Final Extension: 68°C for 20 minutes
* Hold: 4°C indefinitely

### **09 July 2025**

**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 on LB + cm plates

### **10 July 2025**

**Result of Clonage PCR of Tyr1 in pAIDA :**

![gel](https://static.igem.wiki/teams/5932/logo/paida-01.webp)

**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)
Follow the same protocol as Thursday, 26 June. This time we do 60°C and 1 minute.

**Results:** Colonies 1, 5 and 6 have a 1kb, colonies 2 and 4 have the same result as negative control.
So Colonies 1, 5 and 6 have tyr1 in pAIDA.

**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.

### **11 July 2025**

**Miniprep of pAIDA Tyr1 1,5 and 6**

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.

Nanodrop results: pAIDA Tyr1 1 : 24,75 ng/µL; OD= 1,60
pAIDA Tyr1 5 : 18,75 ng/µL; OD= 1,50
pAIDA Tyr1 6 : 37,45 ng/µL; OD= 1,78

Samples 1 and 5 do not meet the required purity and concentration criteria for sequencing. A new culture and miniprep will be necessary to obtain higher quality DNA.

### **15 July 2025**

**Pre-culture preparation of pAIDA Tyr1 6**

Inoculate colony 6 of pAIDA Tyr1 into a 5 mL Erlenmeyer flask of LB with Chloramphenicol. Shake overnight at 37°C.

**Miniprep of pAIDA Tyr1 1,5 and 6**

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.

Nanodrop results: pAIDA Tyr1 1 : 33,64 ng/µL; OD= 2,00
pAIDA Tyr1 5 : 34,6 ng/µL; OD= 1,44
pAIDA Tyr1 6 : 36,047 ng/µL; OD= 1,65

We placed the tubes from colony 1 (today) and colony 6 (last week) in the speedvac because their OD values are good, and we are going to send them for sequencing tomorrow.

**Motility Test of W3110 Δ amp T 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 = (0.3x20mL)/1.5 = 4mL of LB agar

Vf - VLB agar = VLB liquid = 20mL - 4mL = 16mL LB liquid

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 and we do the same for a W3110 Δ amp T colony.

Incubate at 37°C (carefully, since de LB agar is not completely solid for this test)

### **17 July 2025**

**Result of motility test :**

![]()

**Test for verification of melanin expression : transformation of pAIDA-Tyr1 into w3110ΔompT and w3110**

**Preparation of supercompetent cells W3110ΔompT and W3110**  Follow the same procedure as described on Tuesday, 03/06.

**Transformation of pAIDA-Tyr1 and pAIDA (for the control) into w3110ΔompT and w3110** Follow the same procedure as described on Tuesday, 03/06. And use chloramphenicol petri dishes (CM) + Tyrosine for the transformation with pAIDA-tyr1 and chloramphenicol petri dishes for the transformation with pAIDA.

### **21 July 2025**

**Results of the transformation**

![plates](https://static.igem.wiki/teams/5932/logo/paida-02.webp)

We have colonies on all the plates, so the transformation worked.

**Sequencing of pAIDA Tyr1 with oLS4.2** With a reverse primer, we do a sequencing of pAIDA-tyr1.

The results show us that we find Tyr1 in pAIDA with the colonie 1 and we have not a signal in colonie 6.

**Sequencing of pAIDA Tyr1 with oLS4.1** With a forward primer, we do a sequencing of pAIDA-tyr1.

The results with the two primers show us that Tyr1 is in pAIDA.

### **22 July 2025**

**Preculture Dilution**Collect the precultures of pAIDA-tyr1 into W3110 ΔompT, W3110 and and the contrôle tag His of the Laetitia team

* 1. Add 1 mL of preculture to a 500 mL flask containing100 mL of LB for each samples
* 2. Incubate for 3h to observe the stationary phase and perform the western Blot
* 3.At OD = 0,5, induction with 200 uL of IPTG

**OD results for e.coli W3110 delta ompT and W3110**

|  | t=1h | t=1h30 | t=2h | t=2h30 | t=2h40 | t=3h |
| --- | --- | --- | --- | --- | --- | --- |
| W3110 delta ompT | 0,244 | 0,502 | 0,933 | 1,17 | 1,2 | 1,3 |
| W3110 | 0,202 | 0,533 | 0,905 | 1,16 | 1,17 | 1,3 |
|  | t=1h | t=1h30 | t=1h40 | t=1h50 | t=2h20 | t=2h30 | t=2h40 | t=3h |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Control | 0,109 | 0,293 | 0,429 | 0,577 | 0,908 | 0,98 | 1,01 | 1,086 |

**western Blot :**Follow the same procedure as described on Friday 04/07

The total length of the protein is actually 1008pb , which corresponds to 37KDa.

![western-blot](https://static.igem.wiki/teams/5932/logo/paida-03.webp)

The results show us we have expression only of the contrôle and no in w3110 ΔompT and w3110. So we will make preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team

### **23 July 2025**

**Test for verification of melanin expression : western Blot**

Preparation of LB+tyr+IPTG+Cm plates :

* 200ml of LB
* 260uL of Cm
* 100uL of IPTG
* 100mg of Tyr for 1g/L de concentration
* Streaking on plates of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT and w3110 .

**Preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team.** Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C. For the control tag His of the Laetitia team, inoculated 1 colony into a 5 mL LB + Km Erlenmeyer flask. Shake overnight at 37°C.

### **24 July 2025**

**Preculture Dilution of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team.**

Under sterile conditions (Bunsen Burner flame)

* 1. Collect the precultures of pAIDA-tyr1 into pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team.
* 2. Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
* 3. incubate for 3h to observe the stationary phase and perform the western Blot .
* We perform 2 dilutions in two different Erlenmeyer flasks: one that will be induced with 100uL of IPTG, and the other without IPTG .

**OD Mesurements 600nm**

|  | t=1h30 | t=3h |
| --- | --- | --- |
| pAIDA-Tyr1 W3110 delta ompT | 0,7 | 1,24 |
| pAIDA-Tyr1 W3110 | 0,75 | 1,2 |
| pAIDA w3110 | 0,7 | 1,3 |
| pAIDA w3110 delta ompT | 0,76 | 1,2 |
| W3110 delta ompT | 0,7 | 1,4 |
| W3110 | 0,7 | 1,2 |
| Control tag His of the Laetitia team | 0,5 | 1,05 |

**Western Blot**Follow the same procedure as described on Friday 04/07

western Blot Results without IPTG : The total length of the protein is actually 1008pb , which corresponds to 37KDa.

We found the same result like the first western blot.
Conclusion: our colony 1 doesn’t exprime the tyrosinase.
western Blot Results with IPTG

![western-blot3](https://static.igem.wiki/teams/5932/logo/paida-04.webp)

Here we have expression only of pAIDA in w3110 ΔompT and w3110 no in pAIDA-tyr1w3110 ΔompT and pAIDA-tyr1w3110. Conclusion: our colony 1 doesn’t exprime the tyrosinase.
So we must make another PCR Colony with the colony 5,6,7,8,9,10,13 and 14.
The samples that successfully contain tyr1 in pAIDA will be used to perform another western blot, and our control will be pAIDA in w3110 ΔompT or pAIDA in w3110.

### **25 July 2025**

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

We do this for 8 colonies of pAIDA Tyr1 and 1 colony of pAIDA (negative control)
Streaking on LB+Cm plates over the weekend for colonies 5,6,7,8,9,10,13,14.

### **28 July 2025**

**pAIDA-I stock**Colony PCR Protocol with oLS 4.1, oLS 4.2 and pAIDA Tyr 1 cells

Follow the same protocol as Thursday, 26/06.
This time we do 60°C and 1 minute.

![colony-pcr2](https://static.igem.wiki/teams/5932/logo/paida-05.webp)

Colonies 5, 6 and 7 have a 1kb like tyr1, colonies 8, 9, 10, 13 and 14 have the same result as negative control.
So Colonies 5, 6 and 7 have tyr1 in pAIDA.
So we will make pre-culture of colony Colonies 5, 6, 7 and pAIDAw3110ΔompT.

**Test for verification of melanin expression : Preculture of colonies 5,6,7 and pAIDA w3110**

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C

### **29 July 2025**

**Test for verification of melanin expression** Preculture Dilution of colonies 5,6,7 and pAIDA w3110

Under sterile conditions (Bunsen Burner flame)

* Collect the precultures of colonies 5,6,7 and pAIDAw3110 (the control).
* Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
* Incubate for 3h to observe the stationary phase and perform the western Blot .
* At OD=0,5 induction with 100uL of IPTG.

|  | t=1h | t=1h30 | t=2h | t=2h20 | t=3h |
| --- | --- | --- | --- | --- | --- |
| pAIDA W3110 control | 0,228 | 0,534 | - | - | 1,3 |
| pAIDA-Tyr1 W3110 delta ompT colonie 5 | 0,102 | 0,185 | 0,382 | 0,527 | 1,3 |
| pAIDA-Tyr1 W3110 delta ompT colonie 6 | 0,104 | 0,199 | 0,386 | 0,527 | 1,25 |
| pAIDA-Tyr1 W3110 delta ompT colonie 7 | 0,109 | 0,203 | 0,393 | 0,538 | 1,3 |

**Western Blot**Follow the same procedure as described on Friday 04/07

We do not have expression of tyrosinase in colonies 5,6 and 7 with the anti-His tag antibody. The anti-His tag antibody did not detect any protein bands in these samples.

![western-blot4](https://static.igem.wiki/teams/5932/logo/paida-06.webp)

### **30 July 2025**

**Test for verification of melanin expression : Preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110 and pAIDAw3110 (the control)**

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

### **31 July 2025**

**Test for verification of melanin expression : Preculture Dilution of colonies pAIDA W3110, pAIDA-tyr1 w3110 and pAIDA-tyr1 3110ΔompT**

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

Under sterile conditions (Bunsen Burner flame)

* 1. Collect the precultures of pAIDA-tyr1 inw3110 and pAIDA-tyr1 inw3110ΔompT and pAIDAw3110 (the control).
* 2. Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
* 3. Incubate for 3h to observe the stationary phase and perform the western Blot .
* 4. At OD=0,5 induction with 100uL of IPTG 200uM.

**OD Mesurements 600nm**

|  | t=1h | t=1h30 | t=2h45 | t=3h | t=4h |
| --- | --- | --- | --- | --- | --- |
| pAIDA W3110 Δ ompT | 0,035 | 0,094 | 0,3 | 0,604 | 1,33 |
| pAIDA W3110 | 0,2 | 0,625 | - | 1,25 | - |
| pAIDA Tyr1 w3110 | 0,193 | 0,624 | - | 1,4 | - |

**Western Blot**Follow the same procedure as described on Friday 04/07

![western-blot5](https://static.igem.wiki/teams/5932/logo/paida-07.webp)

In this western Blot, we used :
pAIDA W3110 as control, which has His tag at the N-terminal and a Myc tag at the C-terminal. And the mouse anti Myc antibody.
We can see the expression of la tyrosinase in w3110 Δ ompT even if it is weak . This means that the sequencing results are correct : the tyrosinase gene is present in pAIDA W3110Δ ompT.
So, we will do another western Blot with colony PCR samples 5,6 and 7 transformed in W3110Δ ompt ;to check if these colonies also express tyrosinase

**Test for verification of melanin expression**Preculture of colonies PCR 5,6 and 7 transformed in W3110Δ ompt
Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C

### **1 August 2025**

**Test for verification of melanin expression : Preculture dilution of colonies PCR 5,6 and 7 transformed in W3110Δ ompt**

**OD Mesurements :**

|  | t=1h | t=3h |
| --- | --- | --- |
| pAIDA W3110 delta ompT | 0,4 | 1,3 |
| pAIDA-Tyr1 W3110 delta ompT colonie 5 | 0,02 | 0,04 |
| pAIDA-Tyr1 W3110 delta ompT colonie 6 | 0,4 | 1,3 |
| pAIDA-Tyr1 W3110 delta ompT colonie 7 | 0,4 | 1,3 |

**Western Blot**Follow the same procedure as described on Friday 04/07

We did the western Blot only with the control and colonies 5 and 7 because we got the sequencing results of colony 6 and the signal was not good .
In colonies 1 et 7 we see the tyrosinase experession. But we schooled have one band, not two.Maybe the tyrosinase was cut.
So, we will do another western Blot with and without IPTG, both in dans pAIDA-Tyr1 w3110ΔompT and pAIDA-Tyr1 w3110, for the colonies 5 and 7 to show clearly that tryrosinase expression is in strain w3110ΔompT.

Preculture of colonies PCR 1and 5 transformed in W3110Δ ompt , colonies PCR 1 and 5 transformed in W3110, pAIDAW3110Δ ompt ( control) and pAIDAW3110 ( control)

### **04 August 2025**

**Test for verification of melanin expression**

Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt colonies PCR 1 and 5 transformed in W3110, pAIDAW3110Δ ompt ( control) and pAIDAW3110 ( control)

**OD Mesurements :**

|  | t=1h | t=1h10 | t=1h20 | t=1h30 | t=3h | t=3h20 |
| --- | --- | --- | --- | --- | --- | --- |
| pAIDA W3110 delta ompT | 0,5 | - | - | - | 1 | 1,2 |
| pAIDA-Tyr1 W3110 delta ompT colonie 1 | 0,47 | 0,38 | 0,5 | - | 1 | 1,2 |
| pAIDA-Tyr1 W3110 delta ompT colonie 5 | 0,41 | 0,44 | 0,5 | - | 1 | 1,2 |
| pAIDA W3110 | 0,34 | 0,32 | - | 0,5 | 1 | 1,3 |
| pAIDA-tyr1 W3110 colonie 1 | 0,26 | 0,33 | - | 0,5 | 1 | 1,3 |
| pAIDA-tyr1 W3110 colonie 5 | 0,3 | 0,36 | - | 0,5 | 1,2 | 1,4 |

**Western Blot**Follow the same procedure as described on Friday 04/07

![western-blot7](https://static.igem.wiki/teams/5932/logo/paida-08.webp)
![western-blot7](https://static.igem.wiki/teams/5932/logo/paida-08-2.webp)

In this western blot, we see expression of colonies 1 and 5 only in the Δ+ strain, which is w3110Δompt induced with 1 mM IPTG. In w311, we only have the control expressed. This strain does not have the same physiological properties as w3110Δompt, so tyrosinase is not expressed.
This confirms that tyrosinase is well expressed in w3110Δompt when induced.
Therefore, we will do more western blots with colonies 1 and 5 at different IPTG concentrations to know at which concentration tyrosinase is expressed in a good amount.

Preculture of colonies 1 and 5 transformed in W3110Δompt.

### **5 August 2025**

**Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt**

OD measurement

For colony 1

|  | t=1h30 | t=2h5 | t=3h |
| --- | --- | --- | --- |
| 0uM IPTG | 0,2 | 0,56~0,6 | 1,2 |
| 50uM IPTG | 0,2 | 0,6 | - |
| 200uM IPTG | 0,2 | 0,6 | - |
| 500 uM IPTG | 0,2 | 0,6 | - |
| 1 mM | 0,2 | 0,6 | 1,4 |

For colony 5

|  | t=1h30 | t=2h5 | t=3h |
| --- | --- | --- | --- |
| 0uM IPTG | 0,2 | 0,6 | 1,4 |
| 50uM IPTG | 0,2 | 0,6 | - |
| 200uM IPTG | 0,2 | 0,6 | - |
| 500 uM IPTG | 0,2 | 0,6 | - |
| 1 mM | 0,2 | 0,6 | 1,2 |

**Western Blot**Follow the same procedure as described on Friday 04/07

![western-blot8](https://static.igem.wiki/teams/5932/logo/paida-09.webp)

From these results, we see that tyrosinase is well expressed in colony 1 at 200 um and 500 um [IPTG], and in colony 5 at 1 mM.
Therefore, we will do other western blots of these colonies at three different temperatures and times: 30°C, 37°C and 16°C overnight, to see if the amount of tyrosinase expression changes or stays the same.

### **06 August 2025**

**Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt.**

OD Mesurements

For colony 1 at 37°C

|  | t=1h | t=1h30 | t=2h | t=2h30 | t=3h | t=16°C over night |
| --- | --- | --- | --- | --- | --- | --- |
| 200uM IPTG | 0,1 | 0,2 | 0,4 | 0,7 | 1,7 | 1,9 |
| 500uM IPTG | 0,09 | 0,2 | 0,4 | 0,7 | 1,7 | 1,9 |

For colony 5 at 30°C

|  | t=1h | t=2h | t=3h | t=16°C over night |
| --- | --- | --- | --- | --- |
| 200uM IPTG | 0,1 | 0,2 | 0,4 | 0,7 | 1,7 | 1,9 |
| 500uM IPTG | 0,09 | 0,2 | 0,4 | 0,7 | 1,7 | 1,9 |

For colony 5 at 30°C

|  | t=1h | t=2h | t=3h | t=16°C over night |
| --- | --- | --- | --- | --- |
| 1mM IPTG | 0,1 | 1,1 | 0,3 | 0,2 | 0,6 | 1,4 |

**Western Blot**

![western-blot9](https://static.igem.wiki/teams/5932/logo/paida-10.webp)

With the different concentrations and temperatures, we can see that tyrosinase is expressed, even if it is cut at different sizes.

### **18 August 2025**

The PCR Colony doesn’t necessary because the researchers from Sweden optimized them plasmid pAIDA-tyr1 and we see in them article the expression of tyrosinase and melanin expression . So we will make western blot to verify if we have the same thing like our plasmid pAIDA-Tyr from addgene.

Preculture of pAID-Tyr1 w3110, pAIDA w3110ΔompT, colony 1´, 3´ and 4´ pAID-Tyr1 w3110ΔompT

Inoculate 1 colony in to 5 ml LB + cm Erlenmeyer flask. Shake overnight at 37C.

These preculture will be used both to perform western blot and to test the enzymatic activity of tyrosinase.

### **19 August 2025**

**Results of transformation of the pAIDA-Tyr1 With Supercompetent Cells W3110 and W3110 Δ ompt**

**Colony PCR Protocol** with oLS 4.1 and 4.2 and pAIDA Tyr1 cells Follow the same protocol as Thursday, 26/06. This time we do 60°C and 1 minute.

![colony-pcr3](https://static.igem.wiki/teams/5932/logo/paida-11.webp)

The PCR Colony doesn’t necessary because the researchers from Sweden optimized thème plasmid pAIDA-tyr1 and we see in them article the expression of tyrosinase and melanin expression . So we will make western blot to verify if we have the same thing like our plasmid pAIDA-Tyr from addgene.

### **20 August 2025**

**Test for verification of melanin expression : visual test**

Preparation plates :

For our plasmide pAIDA FROM addgene( induced by IPTG) :

* 10 mL LB
* 20uL of 10mM Cu2+
* 20uL IPTG (200uM)
* 13uL Cm

For the plasmide of David Hornstrom team( induced by rhamnose) :

* 10 mL LB
* 20uL Cu2+ to weigh 10 mg for 4 mL H2O
* 100 uL Rhamnos ( 1g/L)
* 13uL Cm

Take 1 colony for each plasmid and steak on each plate, then incubate at 37C over night.

![colony-pcr3](https://static.igem.wiki/teams/5932/logo/paida-12.webp)

We have the same results with the plasmide of Sweden team or ours, whatever antibody used.

### **21 August 2025**

**Test for verification of melanin expression : visual test results**

We see the melanin production in the plamsmid induced by rhamnose and no in the plasmid induced by IPTG.

So we will make another test of plasmid induced by IPTG.

Take 1 colony and steak on the plate, then incubate at 37C over night.

**Preparation plates :**

plasmide induced by IPTG :

* 10 LB
* 20uL of 10mM Cu2+
* 20uL IPTG (200uM)
* 13uL Cm

Enzymatic activity assay

![](https://static.igem.wiki/teams/5932/logo/paida-13.webp)
![](https://static.igem.wiki/teams/5932/logo/paida-13-2.webp)

### **22 August 2025**

**Test for verification of melanin expression : visual test results**

![](https://static.igem.wiki/teams/5932/logo/paida-14.webp)

We can clearly see the appearance of some black spots, which indicate that melanin is being produced.

### **26 August 2025**

**Activity test for pAIDA-Tyr1 Sweden**

Preculture

### **27 August 2025**

**Preparation of M9 media**

For 1L :

* 100 mL M9 10x
* 887 mL water sterile
* 10 mL Glucose 40%
* 2 mL MgSO4 1M
* 1 mL CaCl2 0,1M

**Preparation of M9 media agarose 1,5 %**

* 150 mL M9 media
* Add 1,5 g of agarose
* Boil to dissolve the agarose and sterilize in the microwave

**Activity test with pAIDA Tyr-1 Sweden**

We adjust the DO at 0,05 for a final volume of 25ml.

Delta ompt pAIDATry-1: 4,6mL of preculture (DO: 0,27)W3110 pAIDATry1: 4,3mL of preculture (DO: 0,29)

Add in each erlenmeyer 25mL of Lb and 200µL of Rhamnose with the right volume of periculture

Incubate at 37°C and 180rpm during 3hours

Centrifugate at 3270g during 10minutes

Resuspend the pellet in 10mL of tampon

Tampon préparation :

* 40µL of cooper
* 5mL of Tris-HCL pH 7.2
* 100mg of tyrosine
* Add water of a finale volume of 100mL
* Put the solution in the microwave until it’s clear

Incubate at 30°C, 220rpm during 15hours

### **28 August 2025**

**Activity test with pAIDA Tyr-1 Sweden**

**Measurement of Melanin in the supernatant**Centrifugate at 13 000 rpm during 5min, Measure the supernatant at 400nm, the blank is the tampon made

## Shikimate pathway amplification: Mutation AroF

▼

This section contains the construction of the AroF mutation.

### 17 June 2025

**Shikimate pathway amplification: Mutation AroF**

Preparation of E. coli CC118 and E. coli CC118 pKNG101 Pre-cultures for Supercompetent Cell Preparation

Inoculate 1 colony of E. coli CC118 into a 5 mL LB flask.

Inoculate 1 colony of E. coli CC118 pKNG101 into a 5 mL LB flask.

Incubate overnight at 37°C with shaking.

### 18 June 2025

**Preculture Dilution**

Follow the same procedure as described on Tuesday, 03/06 but this time with E.coli CC118

**OD Measurement by Spectrophotometer**

Measure the OD of the cells in 1 mL cuvettes

CC118 : 0,77

CC118 pKNG 101 : 0,32

**Centrifugation of CC118 cells**

Follow the same procedure as described on Friday 13/06 (with DH5 alpha) this time with 50 mL of CC118, so only one falcon.

**Preparation of Supercompetent Cells CC118**

Follow the same procedure as described on Tuesday, 03/06.

**Plasmid Miniprep Kit for E. coli CC118 pKNG 101**

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

**Buffer Preparation Guidance (NEB #T1110)**

* Add supplied Monarch RNase A to Monarch Buffer B1 for a final concentration of 100 μg/ml. Mix and store at 4°C.
* For T1110S (50-prep) kit, add 60 μl of Monarch RNase A to Monarch Buffer B1.
* Add isopropanol to the Monarch Buffer BZ prior to use (0.43 volumes of isopropanol per volume of Monarch Buffer BZ).
* For T1110S (50-prep) kit, add 3.6 ml of isopropanol to Monarch Buffer BZ.
* Add ethanol to the Monarch Buffer WZ prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Buffer WZ).
* For T1110S (50-prep) kit, add 20 ml of ethanol to Monarch Buffer WZ.
* Always keep all buffer bottles tightly closed when not in use.

**Plasmid Miniprep Protocol using Centrifugation (NEB #T1110)**

Plasmid Miniprep Protocol

* 1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard the supernatant. For a standard miniprep to prepare plasmid for restriction digestion or PCR, we recommend 1.5 ml of culture, which is sufficient for most applications. Ensure cultures are not overgrown; 12–16 hours is usually ideal for optimal growth.
* 2. Resuspend the pellet in 200 μl of Monarch Buffer B1 (pink •). Vortex or pipet mix to ensure cells are completely resuspended. There should be no visible clumps.
* 3. Add 200 μl of Monarch Buffer B2 (blue •), gently invert the tube 5-6 times, and incubate at room temperature for 1 minute. Do not vortex. The color should change to dark pink, and the solution should be transparent and viscous. Handle the sample gently to reduce the risk of shearing chromosomal DNA, which can be co-purified as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.
* 4. Add 400 μl of Monarch Buffer B3 (yellow •), and gently invert the tube until neutralized. Do not vortex. The color should be uniformly yellow and a precipitate will form. Incubate for 2 minutes. Gentle but uniform mixing will ensure complete neutralization without shearing chromosomal DNA.
* 5. Centrifuge the lysate for 2–5 minutes. The pellet should be compact; spin longer if needed. Spin time should not be less than 2 minutes. For culture volumes > 1 ml, we recommend a longer spin (~5 minutes) to ensure efficient RNA removal by RNase A and a more compact pellet, which will lower the risk of clogging the column.
* 6. Carefully transfer the supernatant to the Monarch Spin Column S2D and centrifuge for 1 minute. Discard the flow-through.
* 7. Re-insert the Monarch Spin Column S2D in the Monarch Spin Collection Tube and add 200 μl of Monarch Buffer BZ (wash 1). Centrifuge for 1 minute and discard the flow-through. This is a high-salt wash step that helps remove any residual RNA, protein, and other contaminants. Incubate for 5 minutes after adding Monarch Buffer BZ and before centrifugation if the plasmid will be used for transfection.
* 8. Wash by adding 400 μl of Monarch Buffer WZ (wash 2) and centrifuge for 1 minute and discard the flow-through. Centrifuge again for 1 minute and discard the flow-through.
* 9. Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not touch the flow-through. If in doubt, re-spin for 1 minute.
* 10. Add ≥ 30 μl of Monarch Buffer EY (preference if it’s with H2O but this time I did it with the Buffer EY) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. Nuclease-free water (pH 7– 8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of Monarch Buffer EY is used, but the DNA will be less concentrated. For larger size plasmids (≥ 15 kb), incubate the column with an elution buffer at room temperature for 5 minutes to maximize the yield. Alternatively, the elution buffer can be heated to 50°C before use.
* 11. Use a Nanodrop Spectrophotometer to measure pKNG DNA.
* 12. Store the DNA at -20°C.
* 13. Nanodrop result : pKNG 101 : 52 ng/µL

### **20 June 2025**

**Test : Transformation with Supercompetent Cells with CC118 and pKNG 101**

Follow the same procedure as described on Tuesday, 03/06.

For the last step, spread 6µl of bacteria onto a LB + Streptomycine plate for each plasmid (CC118 (control) and CC118 pKNG 101) (using a spreader/pasteur pipette)

**Contamination Test**

Spread 6 µL of E. coli CC118 on half of 3 plates and 6 µL of E. coli CC118 pKNG 101 on the other half of each plate (on LB agar + ampicillin, LB agar + kanamycin and LB agar + chloramphenicol)

### **24 June 2025**

**PCR and Electrophoresis of genomic DNA of W3110 with oLS 2.1 + 2.2 and oLS 2.3 + 2.4 using Q5® High-Fidelity 2X Master Mix following NEB Protocol**

Follow the same procedure as described on Thursday 19/06. This time we do 2 PCR tubes.

For W3110, 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 2.1, oLS 2.2 ; Template DNA = W3110

F.2 : primers = oLS 2.3, oLS 2.4 ; Template DNA = W3110

For the 25-35 cycles, we put the 2nd step at 72°C.

F.1 and F.2 = 0.5 kB

![](https://static.igem.wiki/teams/5932/logo/arof-notebook-01.webp)

### **Wednesday 25/06**

**Digestion of pKNG101 with BAMH1:**

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

Incubate for 3 hours at 37°C

**PCR and DNA Cleanup of F.1 and F.2, following NEB Protocol (#T1130)**

**Buffer Preparation**

* Add isopropanol to the Monarch Buffer BZ prior to use (0.43 volume of isopropanol per volume of Buffer BZ).
* For T1130S (50-prep) kit, add 18 ml of isopropanol to Monarch Buffer BZ.
* Add ethanol to the Monarch Buffer WZ prior to use (4 volumes of ≥ 95% ethanol per volume of Buffer WZ).
* For T1130S (50-prep) kit, add 20 ml of ethanol to Buffer WZ.
* Always keep all buffer bottles tightly closed when not actively in use.

**Standard Cleanup Protocol using Centrifugation**

1. Add 5 volumes (e.g., 250 μl) of Monarch Buffer BZ to 1 volume (e.g., 50 μl) of sample. Mix well by pipetting up and down or flicking the tube. Do not vortex. Using a sample volume of 20-100 μl is recommended. For samples less than 20 μl, adjust the volume with TE or nuclease-free water to 20-100 μl. For diluted samples larger than 800 μl, load 800 μl first, proceed with step 2, and repeat as needed.

1. 2. Insert the Monarch Spin Column S1A into the Monarch Spin Collection Tube and load the sample onto the column. Spin for 1 minute, then discard the flow-through.
2. Re-insert the column into the collection tube. Wash by adding 200 μl of Monarch Buffer WZ and spin for 1 minute. Discarding flow-through is optional.
3. Repeat wash (step 3).
4. Centrifuge again for 1 minute and discard the flow-through.
5. Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not touch the flow-through. If in doubt, re-spin for 1 minute.
6. Add 5-20 μl of H2O to the center of the matrix to elute DNA. Wait for 1 minute, and spin for 1 minute.

**SLIC**

Two different T4 polymerases are used (from two different kits, including one from the former iGEM 2024 team). Test in two different ratios: 1-1(1µL vector and 1µL of each primer) or 1-2 for each T4 polymerase. To be performed on ice

**Preparation of tubes:**

In tube ratio 1-1:

* 5,6 µL H20
* 1µL Buffer
* 1 µL PKNG vector
* 1 µL PCR fragment F1
* 1 µL PCR fragment F2
* 0,4 µL T4 polymerase

In tube ratio 1-2:

* 3,6 µL H20
* 1 µL Buffer
* 1 µL PKNG vector
* 2 µL PCR fragment F1
* 2 µL PCR fragment F2
* 0,4 µL T4 polymerase

Lastly, T4 polymerase is added to the tubes on ice.

Incubate for 2min45 at room temperature

Return to ice

CC118 cell transformation: Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (CC118 ratio 1-1 T4 pol 2024 and CC118 ratio 1-2 T4 pol 2024, and CC118 ratio 1-1 T4 pol 2025 and CC118 ratio 1-2 T4 pol 2025) (using glass beads)

Incubate at 37°C overnight

### **26 juin**

**Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2024 ratio 1-2 cells**

We do this for 24 colony.

1. Pick a well-isolated colony with a toothpick of CC118 pKNG 101 AroF
2. Transfer the colony into a Sm + LB plate, than into a PCR tube
3. 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
4. For 24 PCR tubes, we prepare 260 µL of PCR mix so everything x26 + 104 µL of H2O
5. To finish, vortex the PCR mix
6. Add 10 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

**Thermocycling Conditions for a Routine PCR:**

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 94°C | 5 minutes |
| 30 Cycles | 94°C | 30 seconds |
| \*50–72°C | we did 60°C | 30 seconds |
| 72°C | 1 min/kb | we did 1 min 30 |
| Final Extension | 72°C | 5 minutes |
| Hold | 16°C |  |

**Electrophoresis:**

* We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel.
* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB. (I forgot)

Every vector is empty. We have to try again.

**Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2025 ratio 1-2 cells**

We do this for 8 colony. Follow the same protocol as today. The electrophoresis will be done tomorrow.

![Electrophoresis results](https://static.igem.wiki/teams/5932/logo/arof-notebook-02.webp)

### **27 June**

### **Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2025 ratio 1-2 cells**

We finished yesterday's protocol with the electrophoresis.

Every vector is empty. We have to try again.

![](https://static.igem.wiki/teams/5932/logo/arof-notebook-03.webp)

### **01 July**

**PCR and Electrophoresis of genomic DNA of W3110 with oLS 2.1 + 2.2 and oLS 2.3 + 2.4 using Q5® High-Fidelity 2X Master Mix following NEB Protocol**

* Follow the same procedure as described on Thursday 19/06. This time we do 2 PCR tubes.
* For W3110, 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 2.1, oLS 2.2 ; Template DNA = W3110
* F.2 : primers = oLS 2.3, oLS 2.4 ; Template DNA = W3110
* For the 25-35 cycles, we put the 2nd step at 72°C.

![PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-04.webp)

X = PCR 1 (we did it a second time)

**Plasmid Miniprep Kit for E. coli CC118 pKNG 101, pAIDA-I**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
* Result Nanodrop :
  + pKNG 101 : 19,78 ng/µL
  + pAIDA-I : 18 ng/µL

### **03 July**

**Digestion of pKNG101 with BAMH1:**

* Follow the same protocol as described on Wednesday 25/06

**DNA Cleanup of pKNG 101, following NEB Protocol (#T1130)**

* Follow the same protocol as Wednesday, 25/06.
* Result Nanodrop : pKNG 101 : 52.23 ng/µL

**SLIC of pKNG 101 with fragments 1 and 2.**

* We followed a new SLIC protocol.
* We use CC118 cells for the transformation

**Preparation of Cloning reaction:**

| Component | Volume | Final Concentration |
| --- | --- | --- |
| Linearized vector (pKNG) | 0.5 µL (10ng/µL) | 100ng |
| Insert (fragment 1 and 2) | 1.5 µL (a 1/10 dilution) |  |
| 10X BSA | 1 µL |  |
| 10X NEB Buffer 2 | 1 µL |  |
| H2O | Up to 10 µL |  |

Calculate the amount of the insert needed as follows:

ng of insert needed = [100 ng (ng of vector used) × size of the insert (kb)] / size of vector (kb) × molar ratio of insert:vector

Here we have:

* Vector : 6.982 kb, 100 ng
* Insert : 0.43 kb
* Molar ratio of the vector:insert = 1:3

Insert needed = 100 ng vector × 0.43 kb insert / 6.982 kb vector × 3/1 = 18.5 ng

We calculate how much of F.1 and F.2 we need to have 18.5 ng.

* F.1 : Insert needed/Concentration of the fragment = 18.5/37.76 = 0.4 µL
* F.2 : 18.5/101.91 = 0.2 µL

[Since we put H2O to have our fragment, we add 0.2 µL of Tris pH 8 for our 10mM fragment.]

**Attention:** As you need to treat the reaction mixture with T4 DNA polymerase for a short duration you should keep the vial of reaction mixture at room temperature rather than on ice. Be sure to mix the vector and insert completely before adding the T4 DNA polymerase

1. Add 0.5 µL of T4 DNA Polymerase (3U/µL, NEB) to the mixture, mix immediately, and incubate at room temperature (~25°C) for 1 min or 50°C for 30 sec.
2. To stop the reaction, place the reaction mixture on ice immediately and incubate on ice for 10 min.
3. (You may use 10 µL (final volume) of the reaction mixture in 100µL of competent cells.)
4. Keep chemically competent E.coli cells on ice for 10 minutes to thaw.

**CC118 cell transformation:** Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (CC118 ratio 1-1 T4 pol 2024 and CC118 ratio 1-2 T4 pol 2024, and CC118 ratio 1-1 T4 pol 2025 and CC118 ratio 1-2 T4 pol 2025) (using glass beads)

Incubate at 37°C overnight

### **Friday 04/07**

**Result of the SLIC:**

![SLIC results](https://static.igem.wiki/teams/5932/logo/arof-notebook-05.webp)

It didn't work, we have to do it again and change the ratio.

**SLIC of pKNG 101 with fragments 1 and 2.**

* Follow the same procedure as described on Thursday 03/07
* This time we do it with a ratio 1:5 and we do 4 tubes (2 tubes with a ratio 1:3 like yesterday and 2 tubes with ratio 1:5)

**CC118 cell transformation:** Follow the same procedure as described on Tuesday, 03/06. with 1 tube 1:5 and 1 tube 1:3

For the 2 other tubes:

1. Incubate the cells on ice for 20 min
2. Heat shock the cells at 42°C for 45 sec
3. Incubate the cells on ice for 2 min
4. Add 200 µL LB broth to the cells
5. Incubate the cells at 37°C for 1 hr
6. Spread the cells onto agar plates containing appropriate antibiotics
7. Incubate the plates at 37°C overnight

For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube

### **07 July**

**Respread of contaminated SLIC colonies**

Results of Transformation with SLIC from last week : Our petri dishes were contaminated, we repoured the colonies we thought could correspond to our on new petri dishes and incubated them at 37°C

![](https://static.igem.wiki/teams/5932/logo/arof-notebook-06.webp)

### **08 July**

**Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F 2024 ratio 1:3 et 1:5 cells**

* Follow the same protocol as Thursday, 26/06

**Results:**

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-07.webp)

we did not find our SLIC desired product

### **09 July**

**Electrophoresis of Digested Pkng101 overnight**

* Follow the same protocol as Thursday 19/06 for the electrophoresis

![Electrophoresis results](https://static.igem.wiki/teams/5932/logo/arof-notebook-08.webp)

The plasmid wasn't digested

**Digestion of pKNG101 with BAMH1, following Promega Protocol (Usage information)**

In a 1.5 mL Eppendorf tubes add (to get 1ng for digested plasmid):

* 16,6µL pkng101
* 2µL E Buffer
* 0,2 µL BSA
* 0,5 µL BAMHI

Overnight at 37°C no shaking

### **10 July**

**PKNG**

**Electrophoresis of digested Pkng101 overnight**

* Follow the same protocol as Thursday 19/06

![Electrophoresis results](https://static.igem.wiki/teams/5932/logo/arof-notebook-09.webp)

The plasmid was digested, it was at 6KB

**SLIC aroF**

Test in two different ratios: 1-2(1µL vector and 2µL of each primer) or 1-3 with the T4 polymerase of our kit. To be performed on ice

**Preparation of tubes:**

In tube ratio 1-3:

* 1,6 µL H20
* 1µL Buffer (R.1)
* 1 µL PKNG vector
* 3 µL PCR fragment F1
* 3 µL PCR fragment F2
* 0,4 µL T4 polymerase

In tube ratio 1-2:

* 3,6 µL H20
* 1 µL Buffer (R 2.1)
* 1 µL PKNG vector
* 2 µL PCR fragment F1
* 2 µL PCR fragment F2
* 0,4 µL T4 polymerase

Lastly, T4 polymerase is added to the tubes on ice.

Incubate for 2min45 at room temperature

Return to ice

**CC118 cell transformation:** We used 3µL of our mix and 50µL CC118. Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (using glass beads)

Incubate at 37°C overnight

### **11 July**

**Colony PCR Protocol with oLS 2.5, oLS 3.6 and CC118 pKNG 101 Aro F 2024 ratio 1-2 cells**

* Follow the same protocol as Thursday, 26/06. We do this for 5 colony

The plasmid is empty we have to try again

**result**

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-10.webp)

**Slic**

* Follow the same protocol as Wednesday, 25/06. This time we did the ratio 2/2 (2µL of pkng for 2µL fragment)

### **15 July**

**Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F 2024 ratio 2/2**

* Follow the same protocol as Thursday, 26/06.

### **17 July**

**Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F**

Follow the same procedure as described on Thursday 26/06

![SLIC results](https://static.igem.wiki/teams/5932/logo/arof-notebook-11.webp)

### **18 July**

**result slic**

![SLIC results](https://static.igem.wiki/teams/5932/logo/arof-notebook-12.webp)

### **21 July**

**Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F**

* Follow the same procedure as described on Thursday 26/06

The vector contains AroF !!!!

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-13.webp)

**Preculture DH5alpha pKng101-AroF\* colony 6, 8, 11 for Miniprep**

**Test for verification of melanin expression : western Blot**

**Pre-culture of pAIDA-tyr1 into W3110 ΔompT, W3110 and the contrôle tag His of the Laetitia team (CHI)**

**Control tag His of the Laetitia team**

* Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

For the contrôle tag His of the Laetitia team, inoculated 1 colony into a 5 mL LB + Km Erlenmeyer flask. Shake overnight at 37°C.

### **22 July**

**Miniprep pkng 101 AroF\*, colony 6, 8, 11**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.

### **23 July**

**Result of transformation pkng101-AroF\* in W3110 ΔompT:**

![Transformation results](https://static.igem.wiki/teams/5932/logo/arof-notebook-14.webp)

W3110 is resistant to streptomycin so we need to change pkng101, and do the Slic again.

### **24 July**

**Transformation of pKng101 AroF\* colony 6 in W3110**

* Follow the same procedure as described on Tuesday, 17/06.

**Preculture of CC118 pkng AroF\* colony 11, 8, and 4**

* Inoculate colony 3 into a 5 mL Erlenmeyer flask of LB with Streptomycin

![Transformation results](https://static.igem.wiki/teams/5932/logo/arof-notebook-15.webp)

### **25 July**

Result of the transformation of pKng101 AroF\* colony 6 in W3110

![](https://static.igem.wiki/teams/5932/logo/arof-notebook-16.webp)

### **28 July**

**Slic of AroF**

* Follow the same protocol as Wednesday 25/06. This time we did the ratio 2/2 (2µL of Plasmid for 2 µL of DNA fragments)

**Result of Slic AroF**

**Preculture of DH5 alpha pko3**

* Add 5mL of LB and 6,5µL of Cm incubate overnight at 30°C

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

* Follow the same procedure as described on Thursday 19/06. For the 72°C step we did 2:50

### **29 July**

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

* Follow the same procedure as described on Thursday 19/06.

**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**

* Follow the same procedure as described on Monday 23/06.

**Slic of AroF**

* Follow the same protocol as Wednesday 25/06. This time we did the ratio 2/2 (2µL of Plasmid for 2 µL of DNA fragments)

### **31 July**

**slic**

We did it again

* Follow the same protocol as Wednesday 30/07/2025

![SLIC results](https://static.igem.wiki/teams/5932/logo/arof-notebook-17.webp)

**PCR for linearized PKO3 using Pfu Turbo with oLS 4.3 and oLS 4.4**

Add for 50 µL final:

* 2,5 µL oLS 4.3
* 2,5 µL oLS 4.4
* 2µL pKO3
* 5µL dNTP
* 5µL Buffer
* 1 µL Pfu Turbo

For the second step we did 56°C and the elongation time was 6min.

For the Slic Follow the same protocol as Wednesday 30/07/2025

For the Transformation incubate at 30°C with shaking during 1H20

### **Monday 04/08/2025**

**Results of Slic PCR for linearized PKO3 using Pfu Turbo and PKO3 linearized with Q5 master Mix**

![PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-18.webp)

### **05 August**

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

* Follow the same procedure as described on Thursday 19/06. For the 72 step we did 3min30 and put the Tm at 56°C. We did that with 4 tubes using our primer and PI's primers. He did the manipulation in parallel

![PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-19.webp)

**Slic**

* Follow the same protocol as Wednesday 25/06. We did the transformation in DH5alpha

### **06 August**

**Slic**

* Follow the same protocol as Wednesday 25/06

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

* Follow the same protocol as Tuesday 24/06

![PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-20.webp)

### **07 August**

**Colony PCR with olS 4.7 and 4.8**

There was an issue with olS 4.8. It didn't hybridize completely on the plasmid.

So we did 3 difference Colony PCR with 3 couple of oligos différents to try to amplify the fragment of AroF

* Tube a: olS 4.5 and olS 2246 (it hybridizes on PKO3, gave by our PI)
* Tube b: olS 4.8 and olS 4.5
* Tube c: olS 4.7 and olS 4.6

Follow the same protocol as Thursday 26/06

**Results**

![Results](https://static.igem.wiki/teams/5932/logo/arof-notebook-21.webp)

### **08 August**

**Result of electrophoresis of the 3 Colony PCR:**

* Tube a: olS 4.5 and olS 2246 (it hybridizes on PKO3, gave by our PI)
* Tube b: olS 4.8 and olS 4.5
* Tube c: olS 4.7 and olS 4.6

The oligos of the tubes A are going to be used for the next Colony PCR.

Only the colony 3, 4, 6 on the Pfu petri dish contain the fragment AroF.

**Colony PCR Protocol with oLS 4.7/4.8 and oLS 4.5 and 2246(iGEM Slic)**

* Tube a: olS 4.5 and olS 2246 (gave by our PI it hybridizes on PKO3)
* Tube d: olS 4.7 and olS 4.8

For the PI's Slic i lost the products. So I only did 2 Colony PCR. None of this colony have the fragment.

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-22-1.webp)
![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-22-2.webp)
![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-22-3.webp)

### **12 August**

**Shikimate pathway amplification: Mutation AroF**

**Plasmid Miniprep Kit for PKO3 AroF of colony 3,6,4**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.
* 3, 4,6: 15 ng/µL

**Transformation of PKO3 AroF colony 3,6,4 into W3110 Δ OmpT**

* Follow the same procedure as described on Tuesday, 03/06.
* Transform 3µL plasmid into 50µL of Cell
* Incubate with shaking at 30°C during 1H20
* Spread 50µL on CM and incubate overnight at 42°C

### **13 August**

**Shikimate pathway amplification: Mutation AroF**

**Result Transformation pko3 AroF\* into W3110 ΔompT (first recombination event)**

**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.

![Preparation results](https://static.igem.wiki/teams/5932/logo/arof-notebook-25.webp)

### **14 August**

**Transformation pko3 AroF\* into W3110 ΔompT**

**Streak into plate:**

* Streak positives clones () into LB agar + Sucrose 6% (1 plate for 1 colony)
* Incubated for 72 hours at 12°C

### **18 August**

**Results of spreading W3110 Δ OmpT pko3 AroF**

We put this plates at 37°C

![Results](https://static.igem.wiki/teams/5932/logo/arof-notebook-26.webp)

### **19 August**

**Shikimate pathway amplification: Mutation AroF**

**Results of spreading W3110 Δ OmpT pko3 AroF after we put the plate at 37°C**

![](https://static.igem.wiki/teams/5932/logo/arof-notebook-27.webp)

### **20 August**

**Shikimate pathway amplification: Mutation AroF**

The sequencing results are horrible. I may have extracted another plasmid.

![Sequencing results](https://static.igem.wiki/teams/5932/logo/arof-notebook-28-1.webp)
![Sequencing results](https://static.igem.wiki/teams/5932/logo/arof-notebook-28-2.webp)

### **21 August**

**Shikimate pathway amplification: Mutation AroF**

**Colony PCR DH5 alpha PKO3 Arof with oLS 2246 and oLS 4.5**

* Follow the same protocol as Thursday, 26/06.

I have one colony right (the 9)

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/arof-notebook-30.webp)

### **Friday 22/08/2025**

**Shikimate pathway amplification: Mutation AroF**

**Miniprep of the colony 9**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.

**Slic**

* Follow the same protocol as Wednesday, 25/06. We did the ratio 2/2.

### **Monday 25/08/2025**

**Shikimate pathway amplification: Mutation AroF**

**Colony PCR W3110 Delta OmpT PKO3 Arof with oLS 2246 and oLS 4.5**

* Follow the same protocol as Thursday, 26/06.

### **27 August**

**Shikimate pathway amplification: Mutation AroF**

**Colony PCR DH5 alpha PKO3 Arof with oLS 2246 and oLS 4.5**

* Follow the same protocol as Thursday, 26/06.

## Construction of isogenic mutant murI of W3110 Δ ompT

▼

This section contains all the steps involved in constructing the isogenic mutant murI of W3110 Δ ompT for auxotrophy

### 17 June 2025

Preparation of 10% glycerol solution for W3110 :

* Vi = 10% (Cf) \* 200 mL (Vf) / 80% (Ci) = 25 mL glycérol 80%
* Added H2O to final volume of 200 mL

**Preparation of Electrocompetent Cells W3110 cells**

* 1. Pre-chilled on ice for 15min
* 2. Two washes with 35mL of 10% glycerol (keep on ice)
* 3. Transferred 35 mL from the Erlenmeyer into a 40mL Falcon tube
* 4. Centrifuged at 5000 rpm 10 min 4°C
* 5. Discarded supernatant and resuspended in 35mL 10% glycerol (using two sticks)
* 6. Repeated centrifugation 5000 rpm 10 min 4°C
* 7. Discarded supernatant
* 8. Resuspended in 35mL 10% glycerol (using two sticks)
* 9. Final Centrifugation 5000 rpm 10 min 4°C
* 10. Discarded supernatant
* 11. Resuspended in 250 uL 10% glycerol (using two sticks)

**Electroporation of pKOBEG Vector into W3110**

* Prepared two 1,5mL Eppendorf tubes:
  + CONTROL : 60 µL W3110 in 10% glycerol
  + pKOBEG : 60 uL W3110 in 10% glycérol + 1 uL vecteur pKOBEG
* 1. Incubated on ice for 15min
* 2. Transferred les 60 µL into electroporation cuvettes
* 3. Performed electroporation using the appropriate machine
* 4. Add 1mL of LB to each cuvette (control and vector-containing sample) and resuspend.
* 5. Transfer into two 1.5 mL Eppendorf tubes
* 6. Incubate in a shaker at 37°C, 750 rpm for 1 hour
* 7. Centrifuge at 5000 rpm for 5 minutes
* 8. Remove 900 µL of supernatant
* 9. Resuspend the pellet in the remaining 100 µL
* 10. Plate serial dilutions on LB agar + Chloramphenicol for selection
* 11. Store the remaining WT and pKOBEG bacteria at 4°C

### **01 July**

**Pre-culture Dilution**

Under sterile conditions (Bunsen Burner flame)

1. Collect the precultures of E. coli W3110 ΔompT
2. Add 250 µL of preculture to a 125 mL erlenmeyer flask containing 25 mL of LB for W3110 ΔompT
3. Incubate with with chalking for 2 hours at 37°C

**OD Measurement via spectrophotomètre**

* Measure the OD of cells in 1 mL cuvettes, targeting OD600 = 0.5.
* W3110 ΔompT:

**Preparation of Electrocompetent Cells W3110 ΔompT Cells:**

* Follow the same protocol as Friday 13/06.

### **09 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG :**

**Preculture Dilution of W3110 Δ ompT pKOBEG :**

Under sterile conditions (Bunsen burner flame)

1. Retrieve the W3110 Δ ompT pKOBEG preculture
2. Add 1 mL of preculture to a 500 mL Erlenmeyer flask containing 100 mL of LB
3. Incubating at 30°C with shaking at 180 rpm until A600 reaches 0,2 - 0,4

**DO measure of W3110 Δ ompT pKOBEG :**

* After 3 hours : DO 0,35

**Induction of W3110 Δ ompT pKOBEG :**

* When the DO reaches 0,2-0,4 : Add 0,05% of L-arabinose in the W3110 Δ ompT pKOBEG culture.
* Incubate at 30°C until an A600 of 0,6-0,8.

**DO measure of W3110 Δ ompT pKOBEG :**

* Result DO 0,8

**Optional : (we did it)**

* Incubate 15 min at 42°C

**Prepare electrocompetent cells :**

* Follow the same protocol as Friday 13/06

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol :**

* Here we use 1 µL of Template DNA (pKD4)
* Reaction Setup: Follow the same protocol as Thursday 19/06 for 25µL

Transfer PCR tubes to a PCR machine and begin thermocycling.

**Thermocycling Conditions for a Routine PCR :**

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 98°C | 3 minutes |
| 25–35 Cycles | 98°C | 40 seconds |
| \*57°C | 57°C | 40 seconds |
| 72°C | 72°C | 40 seconds |
| Final Extension | 72°C | 2 minutes |

**Electrophoresis :**

* We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

**2 : PCR pKD4 (oLS 1.3 and oLS 1.4)**

* The band of the kanamycin is very light, so we did it again with a different temperature of hybridization.

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol :**

* Reaction Setup: Follow the same protocol as Thursday 19/06 for 25µL
* Thermocycling Conditions for a Routine PCR : Follow the same protocol as Wednesday 09/07 but for the hybridization : 62 °C

**Electrophoresis :**

* We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

![](https://static.igem.wiki/teams/5932/logo/delta-muri-01.webp)

**Prepare electrocompetent cells :**

* Follow the same protocol as Friday 13/06.
* And freeze aliquots (100 µL) in liquid nitrogen, then store at -80°C.

### **10 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG :**

**PCR clean up of pKD4 :**

* Follow the same protocol as Wednesday, 25/06
* We mix the 2 tubes of PCR that we did Wednesday 09/07
* Nanodrop result : pKD4 : 64,85 ng/µL

**Electroporation 600ng of PCR pKD4 :**

* Follow the same protocol as Friday 13/06
* We had 10 µL of pKD4 into 50 µL of cells competent two times to test on different concentrations of DL glutamic acid on plate LB.
* Select on Kan LB 10 mM DLGlu plates and Kan LB 2 mM DLGlu.
* Incubate on at 37°C

**Preparation of DL-glutamic acid monohydrate (DL-glu) and LB agar, Kan plates :**

**Preparation of DL glu stock :**

* Molecular Weight of DL-glu : 165,145 g/mol
* Preparation of the stock solution : DL-Glu is miscible in water : solubility 8,83 mg/ml
* Weigh 441,5 mg of DL-glu
* Add 50 mL of distilled water to dissolve
* Filter to sterilize
* Store at 4°C
* The concentration : C = m / (V × M) = 0,4415 g / (0,05 L × 165,145) = 0,053 mol/L so it's 53 mM

**Plate of 20 µL LB, Kanamycin and 10 mM DL Glu :**

* Volume of DL glu stock to preleve : 20 mL × (10 mM / 53 mM) = 3,8 mL

**Plate of 20 µL LB, Kanamycin and 2 mM DL Glu :**

* Volume of DL glu stock to preleve : 20 mL × (2 mM / 53 mM) = 0,8 mL

**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.

### **11 July**

**Result of Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 :**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 :**

**Colony PCR Protocol with oLS 1.1 and 5'KAN, oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4 :**

* We do this for 8 colony and in two tubes of different primers :
* oLS 1.1 and 5'KAN
* oLS 1.2 and 3'KAN

1. Pick a well-isolated colony with a toothpick
2. Transfer the colony into a Kan + DL Glu LB agar plate (and incubate 37°C), than into a PCR tube
3. Prepare a PCR mix in an Eppendorf tube with oLS 1.1 and 5'KAN
   * 90 µL of EconoTaq (2X)
   * 3,6 µL of Forward Primer
   * 3,6 µL of Reverse Primer
   * 83 µL H2O to complete until final volume
4. Prepare a PCR mix in an Eppendorf tube with oLS 1.2 and 3'KAN
   * 90 µL of EconoTaq (2X)
   * 3,6 µL of Forward Primer
   * 3,6 µL of Reverse Primer
   * 83 µL H2O to complete until final volume
5. To finish, vortex the PCR mix
6. Add 20 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

**Thermocycling Conditions for a Routine PCR:**

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 94°C | 5 minutes |
| 30 Cycles | 94°C | 30 seconds |
| 56°C | 56°C | 30 seconds |
| 72°C | 72°C | 1 min 30 |
| Final Extension | 72°C | 5 minutes |

**Electrophoresis :**

* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

![](https://static.igem.wiki/teams/5932/logo/delta-muri-02.webp)

Colonies 6 and 7 have positive results!

The band is about 2kb == strange. Normally it gives 1.2kb.

### **15 July**

**DO preculture of E. coli W3110 Δ ompT pKOBEG pKD4**

* It's the same as the zero LB.

**The result of the save of the PCR colony:**

![]()

We think that maybe the cells die on the plate because there is so little colony :/

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Colony PCR Protocol with oLS 1.1 and 5'KAN, oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:**

* We do this for 8 colony and in two tubes of different primers (clone 9 to 16):
* oLS 1.1 and 5'KAN
* oLS 1.2 and 3'KAN

1. Pick a well-isolated colony with a toothpick
2. Transfer the colony into a Kan + DL Glu LB agar plate (and incubate 37°C), than into a PCR tube
3. Prepare a PCR mix in an Eppendorf tube with oLS 1.1 and 5'KAN:
   * 90 µL of EconoTaq (2X)
   * 3,6 µL of Forward Primer
   * 3,6 µL of Reverse Primer
   * 83 µL H2O to complete until final volume
4. Prepare a PCR mix in an Eppendorf tube with oLS 1.2 and 3'KAN:
   * 90 µL of EconoTaq (2X)
   * 3,6 µL of Forward Primer
   * 3,6 µL of Reverse Primer
   * 83 µL H2O to complete until final volume
5. To finish, vortex the PCR mix
6. Add 20 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

**Thermocycling Conditions for a Routine PCR:**

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 94°C | 5 minutes |
| 30 Cycles | 94°C | 30 seconds |
| 56°C | 56°C | 30 seconds |
| 72°C | 72°C | 1 min 30 |
| Final Extension | 72°C | 5 minutes |

**Electrophoresis:**

* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

### **17 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* Here we use 2 µL of Template DNA (pKD4)
* Reaction Setup: Follow the same protocol as Thursday 19/06 for 50µL
* Transfer PCR tubes to a PCR machine and begin thermocycling.
* Thermocycling Conditions for a Routine PCR as Wednesday 09/07

**Electrophoresis:**

* We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

![](https://static.igem.wiki/teams/5932/logo/delta-muri-04.webp)

**PCR clean up of pKD4:**

* Follow the same protocol as Wednesday, 25/06
* Nanodrop result: pKD4 : 31 ng/µL

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent
* Select on Kan LB 20 mM DLGlu plates
* Incubate overnight at 37°C

### **18 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Colony PCR Protocol with oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:**

* Follow the same procedure as described on Tuesday 15/07

![](https://static.igem.wiki/teams/5932/logo/delta-muri-05.webp)

**The result of W3110 Δ ompT pKOBEG pKD4**

The colonies 4, 5 and 10 are positive (~1200 pb) !

### **21 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Preparation of pre-cultures for E. coli W3110 Δ ompT pKOBEG pkd4**

* Inoculated 1 colony into a 5 mL LB KAN 20 mM DL Glu Erlenmeyer flask for the colonies 4, 5 and 10.
* Incubated overnight at 37°C with shaking

**Streak into plate:**

* Streak positives clones (4, 5 and 10) into LB agar + chloramphenicol plates to verify the sensitivity of cells
* Streak positives clones (4, 5 and 10) into LB agar + KAN + 20 mM DL Glu plate
* Streak positives clones (4, 5 and 10) into LB agar + KAN plate
* Incubated overnight at 37°C

### **22 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Result of positive clone on Kan DL Glu or only KAN:**

The colony 10 didnt grow without the DL Glu !!!!

**Result of the test for the cm sensitivity**

The plasmide pKOBEG is cmR so it's still in the cells…

![](https://static.igem.wiki/teams/5932/logo/delta-muri-06.webp)

**Verification of W3110 Δ ompT if they are AMP, CM, SM sensible:**

* Streak a colony of W3110 Δ ompT into LB agar plate and different antibiotic
* Streak a colony of BL21 (our control) into LB agar plate and different antibiotic

**Preparation of Electrocompetent Cells W3110 Δ ompT pKOBEG pkd4:**

* Follow the same protocol as Friday 13/06, the centrifugation is made with the program: 5000 rpm for 8 min.
* And at the end of the preparation of electrocompetent cells, resuspend in 100 µL of glycerol 10%.
* Stockage at -80°C

### **23 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Results of W3110 Δ ompT on LB and antibiotic plates:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-07.webp)

W3110 Δ ompT grow on Streptomycin plate :/

### **24 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Streak the colony 10 of W3110 Δ ompT pKOBEG pKD4 Δ murI into LB agar KAN 20 mM GLU plate and incubate at 42°C (to eliminate pKOBEG)**

### **25 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Streak multiple colonies of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB agar KAN 20 mMGLU did the 24 july into a LB agar Kan plate and into a LB agar Cm plate to compare which colonies grow on plate CM.**

**Incubate on paillasse over weekend**

### **28 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**results: W3110 Δ ompT pKOBEG pKD4 Δ murI**

After the incubation on the Δ murI at 42°C they are no longer Cm resistant!

**Preparation of pre-cultures for E. coli W3110 Δ ompT pKOBEG pkd4 Δ murI**

* Inoculated 1 colony into a 5 mL LB KAN 20 mM DL Glu Erlenmeyer flask.
* Incubated overnight at 37°C with shaking

![](https://static.igem.wiki/teams/5932/logo/delta-muri-08.webp)

### **29 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Preparation of Electrocompetent Cells W3110 Δ ompT pKOBEG pKD4 Δ murI:**

* The dilution of the preculture is made with 2µL of the preculture (DO: A600 nm: 1) in 20 mL LB KAN 20mM DL GLU.
* Follow the same protocol as Friday 13/06, the centrifugation is made with the program: 5000 rpm for 8 min.
* And at the end of the preparation of electrocompetent cells, resuspend in 100 µL of glycerol 10%.

**Electroporation of pCP20:**

* Electroporation with 1 µL of pCP20 and follow the same protocol as Friday 13/06 but for the incubation it's 1h20 at 30°C
* Streak into LB agar CM, AMP and 20mM DL Glu plate
* Incubate two night at 30°C

### **31 July**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**result of the electroporation of pCP20 into W3110 Δ ompT Δ murI pKD4:**

Left: control W3110 Δ ompT Δ murI pKD4

And right: W3110 Δ ompT Δ murI pKD4 pCP20

![](https://static.igem.wiki/teams/5932/logo/delta-muri-09.webp)

**W3110 Δ ompT pkd4 pCP20 Δ murI:**

* Grow W3110 Δ ompT, pKD4, pCP20 Δ murI colonies in 1 mL of LB 20 mM DL Glutamate (without antibiotic) at 30°C to an A600 of 0,5.
* DO at 11 am: 0,2
* DO at 4 pm: 0,09
* DO at 6 pm: 0,1
* Incubation overnight at 30°C with shaking

### **01 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**W3110 Δ ompT pkd4 pCP20 Δ murI:**

* Grow W3110 Δ ompT, pKD4, pCP20 Δ murI colonies in 1 mL of LB DL Glutamate (without antibiotic) at 30°C to an A600 of 0,5:
* DO after 14h incubation: 5
* Dilution of the culture: 10 µL culture in 1 mL LB 20 mM DL Glu
* Incubation at 30°C with shaking
* DO after 2h30: 0,3
* DO after 3h10: 0,3

Streak on LB 20 mM DL Glu plate and LB plate and incubate over weekend on bench

### **04 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-10.webp)

It is not supposed to grow without D-L-glutamate, which means that our strain is no longer auxotrophic to D-L-glutamate. We have to find a strain which is auxotrophic.

* Streak multiple colonies of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB agar Kan did Friday 25/07/2025 into a LB agar Kan plate and into a LB agar Kan plate +20 mM D-L glutamate to see if they are still auxotrophic to D-L glutamate.
* Incubate 30°C overnight.
* Streak again the 22 colonies of the plate from Thursday 17/07 into a LB agar Kan plate and into a LB agar Kan plate + 20 mM D-L glutamate to see if they are still auxotrophic to D-L glutamate.
* Incubate 30°C overnight.

### **05 August**

**Result of clone on Kan DL Glu or only Kan:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-11.webp)

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Verification of colony 10 W3110 Δ ompT pKD4 to see if we can use it:**

* Streak colony 10 of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB Kan 20mM D-L glutamate did Monday 21/07 into a LB agar Kan plate to see if they are still auxotrophic to D-L glutamate.
* Incubate 30°C overnight.

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
* With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
* Select on Kan LB 20 mM DLGlu plates
* Incubate overnight at 37°C

### **06 August**

**Result of clone on Kan DL Glu and colony 10 on Kan:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-12.webp)

It did not work, we need to do the electroporation again

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
* With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 900 µl of 2YT)
* Select on Kan LB 20 mM DLGlu plates
* Incubate overnight at 37°C

### **07 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* We don't have PKD4 anymore. Here we use 2 µL of Template DNA (pKD4)
* Follow the same protocol as Thursday 17/07

**Thermocycling Conditions:**

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 98°C | 3 minutes |
| 25 Cycles | 98°C | 10 seconds |
| 57°C | 57°C | 30 seconds |
| 72°C | 72°C | 45 seconds |
| Final Extension | 72°C | 2 minutes |
| Hold | 16°C |  |

**Result of clone on Kan DL Glu and PCR**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-13.webp)

Nothing has grow

**PCR clean up of pKD4:**

* Follow the same protocol as Wednesday, 25/06
* Nanodrop result: pKD4: 15,7 ng/µL

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
* With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
* Select on Kan LB 20 mM DLGlu plates
* Incubate overnight at 37°C

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Pre-culture of W3110 ΔompT pKOBEG**

* Inoculate 1 colony of W3110 ΔompT pKOBEG into 5 mL LB + Cm in a sterile 25 mL flask.
* Shake overnight at 30°C

### **08 August**

**Result of electroporation of pKD4**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-14.webp)

It didn't work, we need to retry by creating new W3110 ΔompT pKOBEG electrocompetent

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Preculture Dilution of W3110 Δ ompT pKOBEG**

* Follow the same protocol as Wednesday 09/07

**Prepare electrocompetent cells:**

* Follow the same protocol as Friday 13/06

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
* With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
* Select on Kan LB 20 mM DLGlu plates
* Incubate overnight at 37°C

### **11 August**

**Result of electroporation of pKD4 in W3110 Δ ΔompT pKOBEG**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-15.webp)

No results, we need to try again and create new electrocompetent cells

**Pre-culture of W3110 ΔompT pKOBEG and W3110 ΔompT**

* Inoculate 1 colony of W3110 ΔompT pKOBEG into 5 mL LB + Cm in a sterile 25 mL flask. Shake overnight at 30°C
* Inoculate 1 colony of W3110 ΔompT into 5 mL LB in a sterile 25 mL flask. Shake overnight at 30°C

### **12 August**

**Construction of isogenic mutant murI of W3110 Δ ompT:**

**Preculture Dilution of W3110 Δ ompT until a DO 0,6:**

* in 20 mL LB, dilution 1/100

**Preparation of Electrocompetent Cells W3110 ΔompT Cells:**

* Follow the same protocol as Friday 13/06.

**Electroporation of PCR pKOBEG in W3110 ΔompT Cells:**

* Follow the same protocol as Friday 13/06
* Streak on LB Cm plate
* Incubate at 30°C

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Preculture Dilution of W3110 Δ ompT pKOBEG:**

* Same protocol as Wednesday 09/07 but in 20 mL LB, dilution 1/100

**Induction of W3110 Δ ompT pKOBEG with arabinose when DO 0,2 - 0,4:**

* Same protocol as Wednesday 09/07

**Prepare electrocompetent W3110 Δ ompT pKOBEG cells:**

* Follow the same protocol as Friday 13/06

**Electroporation 600ng of PCR pKD4 in W3110 Δ ompT pKOBEG:**

* Follow the same protocol as Friday 13/06
* We had 10 µL of pKD4 into 50 µL of cells competent
* Select on Kan LB 20 mM DLGlu plates
* Incubate on at 37°C

### **13 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Colony PCR Protocol with oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:**

* Follow the same procedure as described on Tuesday 15/07

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Colony PCR oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:**

* Follow the same procedure as described on Tuesday 15/07

**The result of W3110 Δ ompT pKOBEG pKD4**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-16.webp)

The colony 1 is positive (~1200 pb)!

### **14 August**

**Construction of isogenic mutant murI of W3110 Δ ompT:**

**Preculture Dilution of W3110 Δ ompT until a DO 0,6:**

* in 20 mL LB, dilution 1/100

**Preparation of Electrocompetent Cells W3110 ΔompT Cells:**

* Follow the same protocol as Friday 13/06.

**Electroporation of PCR pKOBEG in W3110 ΔompT Cells:**

* Follow the same protocol as Friday 13/06
* Streak on LB Cm plate
* Incubate at 30°C

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**Preculture Dilution of W3110 Δ ompT pKOBEG: (30°C)**

* Same protocol as Wednesday 09/07 but in 20 mL LB, dilution 1/100

**Induction of W3110 Δ ompT pKOBEG with arabinose when DO 0,2 - 0,4:**

* Same protocol as Wednesday 09/07

**When DO 0,8:**

* Incubate 15 min at 42°C

**Prepare electrocompetent W3110 Δ ompT pKOBEG cells:**

* Follow the same protocol as Friday 13/06

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* Follow the same protocol as Wednesday 09/07

The electrophoresis reveal no band …

### **18 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-17.webp)

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* Follow the same protocol as Wednesday 09/07

**Result: no band**

There is a problem with our plasmide, maybe there is a DNAase in it … So we tried another plasmid pKD4 from someone else.

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* Follow the same protocol as Wednesday 09/07

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 colony 10:**

* The isolated colonies from colony 10 are analyzed by PCR on colony (primer oLS 1,2 and 3'KAN).
* Incubate the backup to 37°C.

**Result: There was a problem with the tube caps, which were not suitable, and the water evaporated in the PCR machine. So there is no band …**

### **19 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 colony 10:**

* The isolated colonies from colony 10 are analyzed by PCR on colony (primer oLS 1,2 and 3'KAN).
* Incubate the backup to 37°C

**Result: no band at 1200 pb**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:**

**PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:**

* Follow the same protocol as Wednesday 09/07
* We did it with 5 differents Q5 master mix to verify if it's the problem

**Result:**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-18.webp)

**PCR clean up of pKD4:**

* We did the clean up on the mix 1, 2 and 4 of pKD4
* Follow the same protocol as Wednesday, 25/06
* Nanodrop result:
  + pKD4 tube 1: 22 ng/µL
  + pKD4 tube 2: 45 ng/µL
  + pKD4 tube 4: 66 ng/µL

**Electroporation 600ng of PCR pKD4:**

* Follow the same protocol as Friday 13/06
* We had 15 µL of pKD4 into 50 µL of cells competent two times to test the addition of DL GLU during the 1 hour incubation at 37°C with shacking.
* Select on Kan LB 20 mM DLGlu plates.
* Incubate on at 37°C

### **21 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**PCR Colony with W3110 Delta ompT pKOBEG pKD4 (primer oLS 1,2 and 3'KAN):**

* Streak the backup on Kan 20 mM DL GLU and on Kan to verify if the clones grow without the Glutamate and if the auxotrophy are working.
* Incubate the backup to 37°C

Control: W3110 Delta ampT pKOBEG

Red dot: clones of the plates when we add DL GLU in the 1 hour incubation after the electroporation

1 à 6: clones of the plates when we do not add DL GLU in the 1 hour incubation after the electroporation

All clones are positive (1200 pb)

But there are also capable to grow without DL GLU …

It's possible that there is a mutation or maybe a duplication of the gene murI …

**Results PCR colony and growth**

![](https://static.igem.wiki/teams/5932/logo/delta-muri-19.webp)

### **25 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Pre-culture preparation**

* Inoculate 1 colony of W3110 delta ampT delta murI pKOBEG pKD4 (the colony red dot) into a 5 mL Erlenmeyer flask of LB Kan 20 mM DL GLU and same with the W3110 delta ampT.
* Shake overnight at 37°C.

### **26 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

**Preculture Dilution**

* Follow the same procedure as described on Tuesday, 03/06

**Growth curve:**

* Incubate the culture at 37°C with shaking and every 15 min take the DO 600 nm and report it
* For the blank: use LB for the WT and for W3110 delta ampT murI pKOBEG pKD4 whitout Glu
* and use LB 20mM DL GLU for W3110 delta ampT murI pKOBEG pKD4 which grow on LB 20mM GLU

| Time (min) | 20 mM DL GLU add | W3110 delta ampT murI pKOBEG pKD4 | W3110 delta ampT |
| --- | --- | --- | --- |
| t=0 | 0,061 | 0,046 | 0,052 |
| 15 | 0,047 | 0,059 | 0,09 |
| 25 | 0,038 | 0,064 | 0,125 |
| 40 | 0,053 | 0,126 | 0,243 |
| 55 | 0,054 | 0,214 | 0,37 |
| 70 | 0,065 | 0,355 | 0,575 |
| 85 | 0,072 | 0,555 | 0,755 |
| 95 | 0,083 | 0,67 | 0,95 |
| 105 | 0,105 | 0,84 | 1,124 |

The problem is that we dilute the LB when we add the 20mM GLU so there is no more the same concentration of composites of LB. This is not possible to compare the result.

But we can say that the bacteria are not auxotrophic for glutamate since they grow as the WT

### **27 August**

**Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:**

* Streak the colony red dot on M9 media agarose 1,5% KAN 20mM GLU
* and on M9 media agarose 1,5% KAN
* to see if they can grow without glutamate
* But nothing has grow on it

## Construction of complementation plasmid of Δ murI : pMurI

▼

This section contains all the manipulations to constructs the complementation plasmid pMurI for the Δ murI mutant.

### **17 June**

**Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid**

1. Puncture the desired DNA brick tube with a clean pipette tip (specify which DNA blocks being used)
2. Add 10 µL of sterile H2O and mix by pipetting up and down, then transfer to a 1.5mL Eppendorf tube
3. Aliquot 1 µL into a new Eppendorf tube (placed horizontally to visualize droplet) keep this aliquot on ice for transformation. Store remaining 9 µL at -20°C
4. Add 50 µL of DH5 alpha competent cells to the 1 µL DNA aliquot
5. Incubate on ice for 30 minutes
6. Perform heat shock at 42°C for 45 seconds
7. Immediately return to ice for 2 minutes
8. Add 700 µL of sterile LB broth
9. Incubate with shaking at 37°C for 1 hour
10. Centrifuge at 5000 rpm for 5 minutes
11. Plate cells on appropriate selective agar plates
    * A1, I19, C1, O10: Cm
    * O9, G7: Amp

### **18 June**

**Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid**

* Follow the same procedure as described on Tuesday, 17/06.

### **23 June**

**Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid**

* Follow the same procedure as described on Tuesday, 17/06, this time with A12 and C1.
* For the last step, plate cells on appropriate selective agar plates (C1 on Chloramphenicol and A12 on Kanamycin)

### **27 June**

**Golden gate assembly pMurI**

In a 0.5 µL PCR tube add:

* 75 ng plasmide A12: volume to pipette from undiluted tube: 2,36 µL
* 2 ng promoteur A1: volume to pipette from tube 1/20: 1,89 µL
* 1,2 ng RBS I19: volume to pipette from tube 1/100: 1,7 µL
* 6,8 ng terminator C1: volume to pipette from tube 1/20: 1,9 µL
* 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 1,75 µL
* 2 µL T4 DNA ligase buffer (10x)
* 1 µL NEB Golden Gate mix
* 7.4 µL H2O (for a final volume of 20 µL)

**Thermocycler program:**

| STEP | TEMP | TIME | CYCLES |
| --- | --- | --- | --- |
| Cycle | 37°C | 1 minute | 30 cycles |
| Cycle | 16°C | 1 minute |
| Final Extension | 65°C | 5 minutes |  |

**Transformation with Supercompetent Cells**

* Follow the same procedure as described on Tuesday, 03/06.
* For step 1 and 2 we used 2 µL of PCR product (GG assembly = pMurI-HA) and 50 µL of DH5alpha supercompetent cells.
* Before step 8, we spread 100 µL of each tube on the LB + Kanamycine + IPTG 0.1M + xGal plate.
* Incubate overnight at an ambient temperature protected from light.

**Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB**

1. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

* At step 2, we add 2 µL of GG assembly (pMurI-HA)
* At the end, we spread 100 µL of each tube on the LB + Kanamycine + IPTG 0.1M + xGal plate. Incubate overnight at an ambient temperature protected from light.

### **30 June**

**Result**

![Transformation results](https://static.igem.wiki/teams/5932/logo/pmuri-notebook-1-boite.webp)

**Colony PCR Protocol with OG35, OG36 and pMurI-HA cells**

* We do this for 23 colony, 22 white colonies and 1 blue colony (negative control).
* Follow the same protocol as Thursday, 26/06.
* This time we do 56°C and 45 seconds.

![Colony PCR results](https://static.igem.wiki/teams/5932/logo/pmuri-notebook-1-gel.webp)

It didn't work, we have to try again.

**Western Blot of pMurI-HA**

The total length of the protein is 258pb, which corresponds to 28,6KDa.

We will prepare 4 gels at 15%.

**Preparation of the lower gel (separating gel):**

Add in the following order:

* 7 mL of H2O
* 10 mL of 30% Acrylamide
* 2,6 mL of 3M Tris, pH 8,8
* 200 uL of 10% SDS
* Add 20 uL of TEMED under a fume hood
* Add 150 uL of 10% APS, (weigh 10g for 10 mL)

**Preparation of the upper gel (stacking gel)**

Add in the following order:

* 5,6 mL of H2O
* 1,32 mL of 30% Acrylamide
* 1 mL of 1M Tris, pH 6,8
* 200 uL of 10% SDS
* Add 12 uL of TEMED under a fume hood
* Add 80 uL of 10% APS, (weigh 10g for 10 mL)

**Pouring the gels:**

* Pour the separating gel into the casting frame, add water on top to prevent polymerization at the surface, blot with absorbent paper once set, add the stacking gel.

### **01 July**

**Plasmid Miniprep Kit for pMurI 8, 12, 17 and 19**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
* Result Nanodrop:
  + pMurI 8: 37 ng/µL
  + pMurI 12: 40 ng/µL
  + pMurI 17: 36 ng/µL
  + pMurI 19: 64 ng/µL

**Colony PCR Protocol with OG35, OG36 and pMurI**

* We do this for 4 different pMurI (8, 12, 17, 19) and A12 (negative control, empty vector).
* Follow the same protocol as Thursday, 26/06.
* For 5 PCR tubes, we prepare 60 µL of PCR mix so everything x6 + 18 µL of H2O
* Instead of picking a colony, we need 10ng of DNA (the µL depends on the DNA concentration) so we dilute DNA in H2O to have 3 µL final volume and take 1µL of it.

| DNA | DNA (µL) | µL of H2O |
| --- | --- | --- |
| pMurI 8 | 0.8 | 2.2 |
| pMurI 12 | 0.75 | 2.25 |
| pMurI 17 | 0.8 | 2.2 |
| pMurI 19 | 0.5 | 2.5 |

This time we do 56°C and 1 minute 30 seconds for the PCR.

Abnormal results

**Colony PCR Protocol with oLS 3.2 oLS 3.3 and pMurI**

* We do the same with primers oLS 3.2 and oLS 3.3 for 4 different pMurI (8, 12, 17, 19) and A12 (negative control, empty vector)

Abnormal results

**Digestion and Electrophoresis of pMurI with Ecor1/Xma1 and Ecor1/Spe1**

**Reaction Mix Setup:**

* 2 µL of rCutSmart Tampon
* 0.2 µL of Enzyme 1
* 0.2 µL of Enzyme 2
* H2O QSP 10 µL
* The DNA must be 300ng

| DNA | DNA (µL) | µL of H2O |
| --- | --- | --- |
| pMurI 8 | 8.1 | 1.9 |
| pMurI 12 | 7.5 | 2.5 |
| pMurI 17 | 8.3 | 1.7 |
| pMurI 19 | 4.7 | 5.3 |
| A12 | 9.4 | 0.6 |

* Add 10 µL of DNA in a 1.5 Eppendorf Tube, than add 10 µL of Mix.
* Vortex and heat at 37°C for 30 minutes.
* This time, we do this for pMurI 8, 12, 17, 19 and A12 (empty vector) with Ecor1/Xma1 and Ecor1/Spe1 enzymes. So we need 60 µL of Reaction Mix:
  + 12 µL of rCutSmart Tampon
  + 1 µL of Enzyme 1
  + 1 µL of Enzyme 2
  + H2O 46 µL

**Electrophoresis:**

* We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel.
* We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

![Electrophoresis results](https://static.igem.wiki/teams/5932/logo/pmuri-notebook-3.webp)

We added too much Ladder.

For Ecor1/Xma1 we wanted 2931pb and 220pb for A12, and a linearized band for the rest.

For Ecor1/Spe1 we wanted ~2980pb and ~460pb and for A12, and 2931pb and 1134pb for the rest

**Starter**

* Follow the same protocol as described on Monday, 30/06 for the clone pMurI 17, 8, 12, 19.

### **02 July**

**Sequencing**

* We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer (OG35) and 2.5 µL of H2O
* JFB470 = pMurI 19
* JFB469 = pMurI 12

**Pre-culture preparation of pAIDA-I and iGEM Brick A12 to make a stock**

* Inoculate 1 colony of A12 into a 5 mL Erlenmeyer flask of LB and with Kanamycin. Same for pAIDA-I with Chloramphenicol.
* Shake overnight at 37°C.

### **03 July**

**Pre-culture preparation of pMurI 12, pMurI 19 and pok-HA for Western Blot**

* Inoculate 1 colony of pok-HA into a 5 mL Erlenmeyer flask of LB and with Kanamycin. Same for pMurI 12 and 19.
* Shake overnight at 37°C.

### **04 July**

**OD measurement using a spectrophotometer for pMurI (colonie 12 and 19) and pok-HA**

* Measured cell OD in 1 mL cuvettes to target OD₆₀₀ ≈ 0.8.
* pMurI 12: 2.9
* pMurI 19: 3.6
* pok-HA: 2.9

**Western Blot of pMurI-HA**

The total length of the protein is actually 858pb, which corresponds to 32KDa.\*

The pok-HA is our positive control to ensure that our anti-HA antibodies are working. We are testing two different anti-HA antibodies (the iGEM anti-HA antibodies and one from the team of Laeticia).

**Gel preparation (Monday 30/06)**

* Centrifuge 1 mL of pre-culture at 9000 rpm for 4 min.
* Discard the supernatant and resuspend the pellet in loading buffer as follows:
  + 72.5 µL of TPC 2X Buffer for pok-HA,
  + 62.5 µL for pMurI 12,
  + 90 µL for pMurI 19 to have a 0.4 OD/10µL

The calculation used helps determine the volume of loading buffer to add based on the culture's OD. We aim for an OD of 0,4 in 10 µL for each electrophoresis well. Use the formula Vf × Cf = Ci × Vi, therefore Vf = (Ci × Vi) / Cf

1. Incubate at 95°C for 10 min.
2. Assemble the gel system and fill the space between the plates with a 10X ZY running buffer to the top.
3. Load the gel: 5 µL of protein ladder and 10 µL of each sample (2 times to test the two different antibodies)
4. Run the gel 200 V, 50 min for a 10% gel and 55 min for a 15% gel
5. Transfer to nitrocellulose: Prepare the transfer sandwich as follows, soaking all components in transfer buffer beforehand:
   * 3 whatman papers
   * The gel
   * 1 nitrocellose membrane
   * 3 whatman papers
6. Note: Proteins migrate downward, so the gel must be placed directly on top of the nitrocellulose membrane.
7. Transfer using a semi-dry transfer system: 7 min at 25 V, 1,3 A
8. Blocking buffer preparation:
   * For a 3% BSA: weigh 300 mg BSA and add 10 mL of 1X PBS
   * For a 5% milk: weigh 500 mg powdered milk and add 10 mL of 1X PBS
9. Place the nitrocellulose membrane into trays on a shaker and discard the other papers and gel
10. Add 10 mL of 3% BSA for the membrane which would be incubated with iGEM anti-HA antibody and add 10 mL of 5% milk for the membrane which would be incubated with anti-HA antibody from the Laeticia teams.
11. Incubate 1 h
12. Add 10µL of the corresponding anti HA antibodies in each tray to obtain a 1:1000 dilution.
13. Incubate 1h
14. Wash 3 times with 1X PBS 5 min each
15. Add 10 mL of dilution buffer containing the alkaline phosphatase conjugated secondary antibody.
16. Wash 2 times with 1X PBS 5 min each
17. Wash once with 1X PA buffer 5 min
18. Revelation buffer preparation:
    * 10 mL of 1X PA buffer
    * 10 µL MgCl2
    * 70 µL NBT
    * 30 µL BCIP
19. Add the revelation buffer and monitor the color development

### **07 July**

**Result of Western Blot:**

![Western Blot results](https://static.igem.wiki/teams/5932/logo/pmuri-westernblot-07-july.webp)

We don't have positive result: lack of tag HA of our pMurI

**Golden gate assembly pMurI**

* Follow the same procedure as described on Friday 27/06
* In a 0.5 µL PCR tube add:
  + 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  + 2 ng promoteur A1: volume to pipette from tube 1/20: 1 µL
  + 1,2 ng RBS I19: volume to pipette from tube 1/100: 1 µL
  + 6,8 ng terminator C1: volume to pipette from tube 1/20: 1 µL
  + 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 0.7 µL
  + 2 µL T4 DNA ligase buffer (10x)
  + 1 µL NEB Golden Gate mix
  + 7.4 µL H2O (for a final volume of 20 µL)

**Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB**

* Follow the same procedure as described on Friday 27/06

### **08 July**

**Rehydration of murI oligos from IDT**

| Primer Name | µL H2O for Rehydration |
| --- | --- |
| oLS 4.1 tyr1UP | 265 |
| oLS 4.2 tyr1DO | 252 |

→ Stock concentration: 100µM

**Dilution to 10µM (Final volume: 200µL):**

1. Vortex the stock oligo solution
2. Incubate on ice for 1 hour
3. Vortex again
4. In a 1.5 mL Eppendorf tube, add 20µL of oLS and 180µL of H2O

**Colony PCR Protocol with OG35, OG36 and pMurI-HA cells**

* We do this for 7 colonies, 6 white colonies and 1 blue colony (negative control).
* Follow the same protocol as Thursday, 26/06.
* This time we do 56°C and 1 minute 30 seconds.

**Colony PCR Protocol with OG35, oLS 3.3 and pMurI-HA cells**

* We do the same with primers OG35 and oLS 3.3 for the same 7 colonies, 6 white colonies and 1 blue colony (negative control).

**Result of Transformation with pMurI and PCR colony**

![Transformation results](https://static.igem.wiki/teams/5932/logo/pmuri-notebook-4.webp)

**Pre-culture preparation of pMurI 4, 5 and 6**

* Inoculate 1 colony of pMurI 4, 5 and 6 into a 5 mL Erlenmeyer flask of LB with Kanamycin.
* Shake overnight at 37°C

### **09 July**

**Plasmid Miniprep Kit for pMurI 4, 5 and 6.**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.
* pMurI 4 = 29.5 ng/µL
* pMurI 5 = 64.7 ng/µL
* pMurI 6 = 76.05 ng/µL

### **11 July**

**Golden gate assembly pMurI**

* Follow the same procedure as described on Friday 27/06
* In a 0.5 µL PCR tube add:
  + 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  + 2 ng promoteur A1: volume to pipette from tube 1/20: 1 µL
  + 1,2 ng RBS I19: volume to pipette from tube 1/100: 1 µL
  + 6,8 ng terminator C1: volume to pipette from tube 1/20: 1 µL
  + 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 0.7 µL
  + 2 µL T4 DNA ligase buffer (10x)
  + 1 µL NEB Golden Gate mix
  + 7.4 µL H2O (for a final volume of 20 µL)
* In another 0.5 µL PCR tube add:
  + 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  + 75 ng promoteur A1: volume to pipette from tube 1/20: 1.1 µL
  + 75 ng RBS I19: volume to pipette from tube 1/100: 0.8 µL
  + 75 ng terminator C1: volume to pipette from tube 1/20: 0.6 µL
  + 45 ng PCR murI purified (clean up): volume to pipette from tube 1/10: 1.5 µL
  + 2 µL T4 DNA ligase buffer (10x)
  + 1 µL NEB Golden Gate mix
  + 12 µL H2O (for a final volume of 20 µL)
* For the program, we do 60°C during 5 minutes instead of 65°C.

**Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB**

* Follow the same procedure as described on Friday 27/06

### **15 July**

**Colony PCR Protocol with OG35, OG36 and pMurI-HA cells**

* We do this for 8 colonies, 7 white colonies and 1 blue colony (negative control).
* Follow the same protocol as Thursday, 26/06.
* This time we do 56°C and 1 minute 30 seconds.

**Colony PCR Protocol with OG35, oLS 3.3 and pMurI-HA cells**

* We do the same with primers OG35 and oLS 3.3 for the same 7 white colonies.

**Result of Transformation with Golden Gate and PCR colony:**

![Transformation results](https://static.igem.wiki/teams/5932/logo/pmuri-notebook-5.webp)

### **16 July**

**Pre-culture preparation of pMurI 3, 4 and 5 for a Miniprep and POK-HA**

* Inoculate colony 4 of pMurI into a 10 mL Erlenmeyer flask of LB with Kanamycin.
* Same for pMurI 3, 5 and pok-HA with Kanamycin,
* Shake overnight at 37°C

### **17 July**

**Miniprep of pMurI 3, 4 and 5**

* Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
* Result Nanodrop:
  + pMurI 3: 51 ng/µL
  + pMurI 4: 41 ng/µL
  + pMurI 5: 39 ng/µL
* We sent pMurI 3, 4 and 5 for sequencing.

**Pre-culture preparation of pMurI 3, 4, 5 and Pok-HA for WesternBlot**

* Inoculate colony 3 of pMurI into a 5 mL Erlenmeyer flask of LB with Kanamycin.
* Same for pMurI 4, 5 and pok-HA with Kanamycin,
* Shake overnight at 37°C

### **18 July**

**Western Blot of pMurI-HA**

The total length of the protein is actually 858pb, which corresponds to 32KDa.\*

The pok-HA is our positive control. We use our antibodies.

* Follow the same procedure as described on Friday 04/07

![Western Blot results](https://static.igem.wiki/teams/5932/logo/pmuri-westernblot-18-july.webp)

It is difficult to read, but we think 3 and 5 have HA and not 4.

### **21 July**

**Sequencing of pMurI 3, 4 and 5 with oLS 3.3**

* With a Reverse primer (murDO) we do a sequencing of pMurI 3, 4 and 5
* KCH602 = Colonie 3
* KCH605 = Colonie 4
* KCH608 = Colonie 5

3 has a mutation

4 is unreadable

5 is good but has some empty space, we need to do another sequencing to be sure everything is alright and redo a Western blot only with 5.

**Pre-culture preparation of pMurI 5 and Pok-HA for WesternBlot**

* Inoculate colony 3 of pMurI into a 5 mL Erlenmeyer flask of LB with Kanamycin.
* Same for pok-HA with Kanamycin,
* Shake overnight at 37°C

### **22 July**

**Western Blot of pMurI-HA**

The total length of the protein is actually 858pb, which corresponds to 32KDa.\*

The pok-HA is our positive control. We use our antibodies.

* For step 3, in 450 µL of loading buffer, add 50µL of β-mercaptoéthanol 25% to obtain a loading buffer + β-mercaptoéthanol 2.5%.
* Discard the supernatant and resuspend the pellet in loading buffer, to aim for an OD of 0,2 in 10 µL, as follows:
  + 220 µL of TPC 2X Buffer + β-mercaptoéthanol 2.5% for pok-HA,
  + 110 µL for pMurI 12.
* Follow the same procedure as described on Friday 04/07

![Western Blot results](https://static.igem.wiki/teams/5932/logo/pmuri-westernblot22-july.webp)

We have murI with HA at the top and maybe some remains at the bottom of it.

### **28 July**

**Sequencing of pMurI colony 5**

* We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer and 2.5 µL of H2O
* KCH594: pMurI 5 with primer oLS 3.5
* KCH595: pMurI 5 with primer oLS 3.4

### **07 August**

**Pre-culture of pMurI 5 for Miniprep and W3110 ΔompT pKOBEG**

* Inoculate 1 colony of pMurI 5 into 5 mL LB + Kan in a sterile 25 mL flask.
* Shake overnight at 37°C

### **08 August**

**Sequencing of pMurI colony 5**

* The sequencing tubes have been lost, we have to send it again.
* We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer and 2.5 µL of H2O
* KCH603: pMurI 5 with primer oLS 3.5
* KCH604: pMurI 5 with primer oLS 3.4

[Download Laboratory Notebook (PDF)](https://static.igem.wiki/teams/5932/footer/notebook-living-scaffolds-igem-2025-amu.pdf)

{% endblock %}

### Engineering success

* [Introduction](#section1)
* [Cycle 1](#section2)
  + [Design](#subsection1-1)
  + [Build](#subsection1-2)
  + [Tests](#subsection1-3)
  + [Learn](#subsection1-4)
* [Cycle 2](#section3)
  + [Design](#subsection1-1)
  + [Build](#subsection1-2)
  + [Tests](#subsection1-3)
  + [Learn](#subsection1-4)
* [Cycle 3](#section4)

### Table of contents

* [Introduction](#section1)
* [Cycle 1 : Design and testing of the AIDA-Tyr1 system in a wild-type strain](#section2)
* [Cycle 2 : Host Engineering](#section3)
* [Perspective Cycle 3 : Amplification of metabolic flux](#section4)

## Overcoming the barrier of proteolytic degradation of extracellular tyrosinase Tyr1

The development of our biotherapy for osteoporosis hinges on the ability of an engineered Escherichia coli strain to produce melanin. By anchoring tyrosinase to the outer membrane via the AIDA autotransporter system, we achieve localized production and controlled release of melanin within the hydrogel. This strategy is crucial for our application, as it allows melanin to diffuse through the hydrogel network to the bone lesion site, where it neutralizes reactive oxygen species and creates a microenvironment favorable for regeneration.

To build this system, we followed the Design-Build-Test-Learn (DBTL) cycle. This framework for biological engineering relies on four iterative stages: Design, where a hypothesis is formulated and the genetic construct is planned; Build, where molecular tools are assembled and introduced into the host; Test, where performance is evaluated through experiments; and Learn, where results are analyzed to identify limitations and improve the design.

![](https://static.igem.wiki/teams/5932/footer/boucle-inge.webp)

Figure 1: The iterative cycle of biological engineering.

Implementing our concept required several iterations of this cycle. A major challenge emerged early on: the degradation of our fusion protein by host proteases.

## Cycle 1: Design and testing of the AIDA-Tyr1 system in a wild-type strain

### Design

Our initial design involved a plasmid fusing the tyrosinase gene from Bacillus megaterium (Tyr1) with the AIDA (Adhesin Involved in Diffuse Adherence) system. This system was chosen for its ability to anchor protein domains to the outer membrane and expose the linked Tyr1 protein extracellularly. The host strain for this first expression attempt was the standard laboratory strain, E. coli K12 W3110.

![](https://static.igem.wiki/teams/5932/footer/figure-experience-paida-tyr1.webp)

Figure 2: Plasmid design for pAIDA-Tyr1.

Reference : Hörnström D, Larsson G, van Maris AJA, et al. (2019) Molecular optimization of autotransporter-based tyrosinase surface display. Biochimica et Biophysica Acta (BBA) - Biomembranes 1861: 486–494.

### Build

The build phase was successful. The pAIDA-Tyr1 plasmid was assembled via megaprimer PCR and transformed into wild-type E. coli W3110. Positive clones were selected and sequenced to confirm the absence of mutations.

### Tests

Clones were cultured in liquid and solid media induced with IPTG and supplemented with tyrosine and copper. A blackening of the medium, indicating melanin production, was expected.

Progress was monitored by visual observation, spectrophotometry at 405 nm, and Western Blot. The results were minimal: coloration was faint, absorbance was very low (figure 4), and Western Blots showed a protein of a smaller size than expected (Figure 5).

Enzymatic Assay Activity:

![](https://static.igem.wiki/teams/5932/footer/activity-tyrosine-engeniering.webp)

Figure 3 : tyrosinase activity measurements

In vivo tyrosinase activity measurements showing the strong increase in absorption at 400 nm after incubation of bacteria with IPTG, tyrosine and Cu2+ in W3110 bacteria containing our pAIDA1-Tyr plasmid after about 10 hours, and in the Delta OmpT strain (which grew less well) after about 30 hours.

### Learn

The results suggested instability of the fusion protein. Literature indicates that the protease OmpT, present in the outer membrane of E. coli K12, is often responsible for such cleavage. OmpT is an endoprotease known to specifically cleave linker domain sequences in surface-displayed proteins, making the AIDA-Tyr1 construct a prime target. This led us to our second cycle. Instead of a laborious and potentially detrimental redesign of our construct to remove cleavage sites, we opted for a more efficient host engineering strategy.

## Cycle 2: Host Engineering

### Design

To overcome the barrier of proteolytic degradation, we designed a second cycle using the same W3110 strain, but genetically optimized by deleting the ompT gene. This directly removes the primary source of Tyr1-AIDA degradation.

### Build

The build phase for this cycle was identical to the first: the pAIDA-Tyr1 plasmid was transformed into the new E. coli W3110 ΔompT host.

### Tests

Tests were repeated under the same conditions as before. This time, the results were clear: the medium turned intensely black, indicating strong melanin production. Absorbance measured by spectrophotometry was significantly higher. Most importantly, the Western Blot showed a single, intact protein band at the expected size, with no evidence of cleavage.

![](https://static.igem.wiki/teams/5932/footer/engeniering-figure-3.webp)

Figure 4: Results with the optimized strain ΔompT vs with the WT

![](https://static.igem.wiki/teams/5932/footer/engeniering-figure-4.webp)

Figure 5: Western Blot results confirming intact protein.

(Lane M: Protein marker. Lane T-: Negative control (pAIDA W3110 ΔompT). Lanes 1', 3', 4': Colonies of pAIDA-Tyr1 W3110 ΔompT showing the full-length protein at ~97 kDa. The expected size of the AIDA-Tyr1 fusion protein is 97 kDa; the control and marker confirm the specificity of the result.)

![](https://static.igem.wiki/teams/5932/footer/engeniering-figure-5.webp)

Figure 6 : schematic representation of melanin production results between the WT and deltaompT strains

### Learn

We learned that the performance of a genetic system is inextricably linked to the physiological context of its host. For the export, secretion, or surface display of proteins, it is critical to select mutant strains lacking proteases that can interfere with production. Our host engineering approach successfully resolved the primary bottleneck, validating a core principle of synthetic biology.

## Cycle 3: Amplification of metabolic flux

Looking forward, a third cycle was designed to further enhance melanin production. The idea was to amplify the metabolic flux towards tyrosine, the limiting substrate for the reaction. The design involves introducing a point mutation (S34A) into the aroF gene, which codes for DAHP synthase (DAH7PS). This mutation relieves the allosteric inhibition by tyrosine, thereby significantly increasing precursor availability.

The suicide plasmid necessary for implementing this chromosomal modification was successfully constructed. While this optimization could not be experimentally validated due to time constraints, the design is complete and paves the way for even more efficient melanin production in future work.

{% block scripts %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block header\_image %}
![Bannière de la page](https://static.igem.wiki/teams/5932/footer/wet-lab-experiments-1-nadia.webp)
{% endblock %}
{% block page\_content %}

![Chat qui dort](https://static.igem.wiki/teams/5932/photos/image-1-1.webp)

### Experiments

* [Introduction](#section1)
* [Construction of the Melanin Production System pAIDA-tyr](#section2)
* [Enhancement of Tyrosine Biosynthesis (aroF)](#section3)
* [The Biocontainment Strategy](#section4)
  + [Construction of W3110 Δ ompT ΔmurI for auxotrophy](#subsection4-1)
  + [Construction of complementation plasmid of Δ murI : pMurI](#subsection4-2)
* [Hydrogel Encapsulation and Functionality](#section5)
* [Conclusion](#section6)

### Table of contents

* [Introduction](#section1)
* [Construction of the Melanin Production System pAIDA-tyr](#section2)
* [Enhancement of Tyrosine Biosynthesis (aroF)](#section3)
* [The Biocontainment Strategy](#section4)
* [Hydrogel Encapsulation and Functionality](#section5)
* [Conclusion](#section6)

## 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](protocol).

## 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](protocol).

* 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

OD Measurements at 600nm

| Strain | t=1h30 | t=3h |
| --- | --- | --- |
| pAIDA-tyr1 W3110 ΔompT | 0.7 | 1.24 |
| pAIDA-tyr1 W3110 | 0.75 | 1.2 |
| pAIDA W3110 ΔompT | 0.7 | 1.3 |
| pAIDA W3110 | 0.76 | 1.2 |
| W3110 ΔompT | 0.7 | 1.4 |
| W3110 | 0.69-0.7 | 1.2 |
| Control | 0.5 | 1.05 |

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

OD Measurements for pAIDA W3110 Δ ompT

| Time | t=1h | t=1h30 | t=2h45 | t=3h | t=4h |
| --- | --- | --- | --- | --- | --- |
| OD600 | 0.035 | 0.094 | 0.3 | 0.604 | 1.33 |

OD Measurements for pAIDA W3110 and pAIDA Tyr1 W3110

| Strain | t=1h | t=1h45 | t=3h |
| --- | --- | --- | --- |
| pAIDA W3110 | 0.2 | 0.625 | 1.25 |
| pAIDA Tyr1 W3110 | 0.193 | 0.624 | 1.4 |

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

OD Measurements for Colony 1 at Different IPTG Concentrations

| IPTG Concentration | 1h30 | 2h5 | 3h |
| --- | --- | --- | --- |
| 0 μM IPTG | 0.2 | 0.56-0.6 | 1.2 |
| 50 μM IPTG | 0.2 | 0.6 | 1.3 |
| 200 μM IPTG | 0.2 | 0.6 | 1.3 |
| 500 μM IPTG | 0.2 | 0.6 | 1.3 |
| 1 mM IPTG | 0.2 | 0.6 | 1.4 |

For detailed protocols of western blot, please visit our [Protocols page](protocol).

#### 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](protocol).

#### 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

![](https://static.igem.wiki/teams/5932/footer/figure-experience-delta-muri.webp)

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).

### 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

### Construction of complementation plasmid of Δ murI : pMurI

#### Transformation of iGEM Distribution Kit Parts into DH5-ɑ

To obtain a pmurI plasmid complementary to a DL-glutamate auxotrophic strain, we must first transform the parts we will use from the IGEM distribution kit into E. coli DH5-ɑ cells.

To do this, we follow the supercompetent transformation protocol with a few modifications:

* Puncture the desired DNA brick tube with a clean pipette tip (BBa\_J23119, BBa\_J428038, BBa\_J428092, BBa\_J428330)
* Add 10 µL of sterile H2O and mix by pipetting up and down, then transfer to a 1.5mL Eppendorf tube
* Aliquot 1 µL into a new Eppendorf tube (placed horizontally to visualize droplets) keeps this aliquot on ice for transformation. Store remaining 9 µL at -20°C
* Instead of 20µL, add 50 µL of DH5-ɑ competent cells to the 1 µL DNA aliquot
* Incubate on ice for 30 minutes
* Follow the protocol for step 4 to 7
* Centrifuge at 5000 rpm for 5 minutes
* Remove ~900 µL of supernatant and resuspend the remaining 100 µL by pipetting
* Spread the cells onto their respective agarose plates for each DNA brick (using a spreader/Pasteur pipette)
* Chloramphenicol: BBa\_J23119, BBa\_J428038, BBa\_J428092
* Kanamycin: BBa\_J428330

#### Miniprep of cells containing the biobricks

* Following the instructions in the NEB Plasmid Miniprep Kit protocol, DNA was extracted from cells containing the bricks for use in Golden Gates
* The products are then diluted in order to have a large quantity of stock to use
* Dilution of minipreps at 1/100 : 1 µL of biobricks + 99 µL H2O
* Dilution of minipreps at 1/20 : 1 µL of biobricks + 19 µL H2O

For detailed protocols of miniprep, please visit our [Protocols page](protocol).

#### Preparation of XGal, IPTG, Kanamycine Petri dishes

To ensure that the cells have successfully incorporated the plasmid, the blue-white screening technique is used to identify the recombinant bacteria.

This technique is based on the activity of the β-galactosidase enzyme present in E. coli and encoded by the lacZ gene. To screen for clones containing our golden gate, we add X-gal, a substrate which, in the presence of β-galactosidase production, dimerizes into a pigment that turns the bacteria blue. IPTG is used to induce expression of the lacZ gene, along with dimethylformamide (DMF) to solubilize the X-gal and an antibiotic, in this case kanamycin, to select our clones. When our plasmid inserts itself into the vector plasmid, it disrupts the lacZ gene, which prevents β-galactosidase from being produced. The colonies therefore remain white

Material:

* X-Gal
* Dimethylformamide (DMF)
* Isopropyl β-D-1-thiogalactopyranoside, IPTG
* Kanamycin
* LB agar
* Plates

* Actual weight : 7,8 mg X Gal in a Eppendorf tube
* Add 390 µL of DMF under the fume hood
* In 200 mL of LB :
  + Add 1 mL of Kanamycine 10 µg/mL
  + 200 µL of X Gal
  + 400 µL of IPTG 0,1 M
* Pour the plates and cover them with aluminum foil
* Store in the fridge, protected from light (X-Gal is light-sensitive)

#### Golden Gates Assembly

* Using the NEB Golden Gates Mix, we assemble all our DNA fragments with the murI gene accompanied by an HA tag allowing its expression to be verified
* This gene was synthesized by IDT to create our complementation plasmid pMurI

For detailed protocols of golden gate procedures, please visit our [Protocols page](protocol).

#### Blue-white screening

* Our product is transformed into supercompetent E. coli DH5α cells transformed by us, as well as E. coli DH5α cells from NEB in order to obtain potentially more colonies with the NEB cells
* Compared to the initial protocol, we use 2 µL of PCR product, in this case our golden gates assembly, which we transform into 50 µL of supercompetent cells
* Since a blue-white screening is performed to verify that the plasmid created by Golden Gates has been successfully integrated into our cells, before step 8, we spread 100 µL of each tube on the LB + Kanamycine + IPTG 0.1M + xGal plate and it is incubated overnight at an ambient temperature protected from light

For detailed protocols of transformation, please visit our [Protocols page](protocol).

#### Colony PCR

* The white colonies obtained during blue-white screening are used in a colony PCR technique to verify the presence of the murI gene

For detailed protocols of PCR and electrophoresis, please visit our [Protocols page](protocol).

#### Enzymatic digestion

* The white colonies obtained during blue-white screening are used in a colony PCR technique to verify the size of our plasmid and whether it corresponds to the expected size

For detailed protocols of the enzyme-digestion, please visit our [Protocols page](protocol).

#### Western Blot HA

* We performed a Western blot to verify the expression of the murI protein
* Since it is linked to an HA tag, an anti-HA antibody was used along with a positive control to ensure that the antibody was functioning properly
* To obtain a readable Western blot, a step involving β-mercaptoethanol is added to reduce the disulfide bridges that cause this band stacking in the Western blot results :
* For step 3, in 450 µL of loading buffer, add 50µL of β-mercaptoéthanol 25% to obtain a loading buffer + β-mercaptoéthanol 2.5%
* Discard the supernatant and resuspend the pellet in loading buffer, to aim for an OD of 0,2 in 10 µL, as follows :
  + 220 µL of TPC 2X Buffer + β-mercaptoéthanol 2.5% for pok-HA (positive control)
  + 110 µL for pMurI

For detailed protocols of westernblot, please visit our [Protocols page](protocol).

## Hydrogel Encapsulation and Functionality

For detailed protocols of the encapsulation, survival test and escape test, please visit our [Protocols page](protocol).

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 - VLB agar = VLB 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:

![](https://static.igem.wiki/teams/5932/photos/esxperiennces-hydrogel-tableau1-1.webp)

**References:**
1, 2, 3 corresponds to the triplicate of each cell sample
XM corresponds to W3110 non motile + pBAD33
Δ corresponds to W3110 ΔompT + pBAD33
\* corresponds to the tubes in which the marbles have been crushed
t = time

### 4) Hydrogel Bacterial Survival Test

#### Tube Preparation:

Prepare 36 sterile Eppendorf tubes and name them as follows:

| Time Point | Tube Names |
| --- | --- |
| t=0 | mX1 t=0, mX2 t=0, mX3 t=0, Δ1 t=0, Δ2 t=0, Δ3 t=0 |
| t=30' | mX1 t=30', mX2 t=30', mX3 t=30', Δ1 t=30', Δ2 t=30', Δ3 t=30' |
| t=1h | mX1 t=1h, mX2 t=1h, mX3 t=1h, Δ1 t=1h, Δ2 t=1h, Δ3 t=1h |
| t=3h | mX1 t=3h, mX2 t=3h, mX3 t=3h, Δ1 t=3h, Δ2 t=3h, Δ3 t=3h |
| t=6h | mX1 t=6h, mX2 t=6h, mX3 t=6h, Δ1 t=6h, Δ2 t=6h, Δ3 t=6h |
| t=24h | mX1 t=24h, mX2 t=24h, mX3 t=24h, Δ1 t=24h, Δ2 t=24h, Δ3 t=24h |

**References:**
1, 2, 3 corresponds to the triplicate of each cell sample
mX corresponds to W3110 non motile + pBAD33
Δ corresponds to W3110 ΔompT + pBAD33
t = time of measurement

#### 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

![ELISA Plate Layout Diagram 1](https://static.igem.wiki/teams/5932/footer/experience-hydrogel-fig-1.webp)

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

![ELISA Plate Layout Diagram 2](https://static.igem.wiki/teams/5932/footer/experience-hydrogel-fig-2.webp)

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

![ELISA Plate Layout Diagram 3](https://static.igem.wiki/teams/5932/footer/experience-hydrogel-fig-3.webp)

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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block header\_image %}
![Bannière de la page](https://static.igem.wiki/teams/5932/footer/wet-lab-results-nadia.webp)
{% endblock %}
{% block page\_content %}

![Chat qui dort](https://static.igem.wiki/teams/5932/photos/image-1-1.webp)

### Results

* [Introduction](#section1)
* [Construction of the Melanin Production System pAIDA-tyr](#section2)
  + [Plamid Design](#subsection2-1)
  + [Cloning Strategy : clonage PCR of tyr 1 into pAIDA](#subsection2-2)
  + [Validation of construction](#subsection2-3)
  + [Visual tests](#subsection2-4)
  + [Enzyme activity tests](#subsection2-5)
* [Enhancement of Tyrosine Biosynthesis](#section3)
  + [Use of pKng101, suicide plasmid](#subsection3-1)
  + [Use of a new vector thermosensitive plasmid Pko33](#subsection3-2)
  + [Final result](#subsection3-3)
* [The Biocontainment Strategy](#section4)
  + [Construction of W3110 Δ ompT ΔmurI for auxotrophy](#subsection4-1)
    - [Conception and validation of the murI gene deletion](#subsection4-1-1)
    - [Failure of phenotypic validation: absence of D-glutamate auxotrophy](#subsection4-1-2)
    - [Further investigation and consequences](#subsection4-1-3)
  + [Construction of complementation plasmid of Δ murI : pMurI](#subsection4-2)
    - [Blue-white screening of E. coli DH5ɑ with pMurI](#subsection4-2-1)
    - [Colony PCR, Enzymatic Digestion Electrophoresis](#subsection4-2-2)
    - [Western Blot of pMurI with an anti-HA antibody and sequencing of pMurI](#subsection4-2-3)
    - [Further investigation and consequences](#subsection4-2-4)
* [Hydrogel Encapsulation and Functionality](#section5)
* [Futurs Works](#section6)
  + [Experiment to test the mutation on AroF](subsection6-1)
  + [Plan for phenotypic characterization of the ΔmurI strain](subsection6-2)
  + [Poly-DL-γ-glutamate (PGA) hydrogel for biocontainment](subsection6-3)
  + [Genetic Modifications to Enhance Containment](subsection6-4)
  + [Biocompatibility and Biodegradability Testing](subsection6-5)
  + [Injectability of the hydrogel](subsection6-6)
  + [Melanin Diffusion Control](subsection6-7)
  + [Microstructure Optimization](subsection6-8)
  + [In Vivo Testing](subsection6-9)
* [Conclusion](#section7)

### Table of contents

* [Introduction](#section1)
* [Construction of the Melanin Production System pAIDA-tyr](#section2)
* [Enhancement of Tyrosine Biosynthesis](#section3)
* [The Biocontainment Strategy](#section4)
* [Hydrogel Encapsulation and Functionality](#section5)
* [Futurs Works](#section6)
* [Conclusion](#section7)

## Introduction

This section presents the experimental results of our strategies to engineer E. coli for controlled melanin production and biocontainment. We detail the successful construction and functional validation of the pAIDA-tyr1 plasmid for extracellular tyrosinase activity, leading to visible melanin synthesis. Furthermore, we report on the challenges faced in optimizing the tyrosine biosynthetic pathway through aroF mutagenesis and in establishing a functional ΔmurI auxotrophic system for biocontainment, alongside the successful creation of a complementary pMurI plasmid. Finally, we demonstrate the efficacy of alginate hydrogel encapsulation as a physical barrier, confirming its ability to confine cells while maintaining their viability and metabolic activity. These results collectively outline the construction and validation of a foundational platform for a safe and functional microbial system.

## 1/ Construction of the Melanin Production System pAIDA-tyr

### Plasmid Design

Melanin is a natural pigment known for its antioxidant and radioprotective properties. Our project is to use melanin for its ability to scavenge free radicals and protect cells from oxidative stress.

To construct our melanin production system, we designed a plasmid based on the AIDA autotransporter system, which mediates the export of heterologous proteins to the bacterial surface or extracellular space.

In this construct, the tyr1 gene encoding tyrosinase was genetically fused to the aida gene sequence using a flexible linker to preserve the correct folding and activity of both protein domains.

To facilitate detection and purification, a MYC tag was added at the C-terminus and a His tag at the N-terminus of the tyrosinase domain.

This design is expected to enable the efficient extracellular export of active tyrosinase, thereby allowing melanin biosynthesis to occur outside the bacterial cell.

![Plasmid Design](https://static.igem.wiki/teams/5932/footer/figure-experience-paida-tyr1.webp)

**Figure 1.1:** Plasmid Design - AIDA autotransporter system fusion with tyrosinase gene (tyr1) via a linker with addition of MYC tag (C-terminal) and His Tag (N-terminal) to facilitate detection and purification. Goal: Efficient extracellular export of tyrosinase → melanin production outside the cell.

![Melanin Production System](https://static.igem.wiki/teams/5932/footer/figure-description-zoom-hydrogel-paida-tyr.webp)

**Figure 1.2:** Melanin production system based on tyrosinase exposure at the bacterial cellular surface through the AIDA autotransporter.

### Cloning Strategy: Introduction of the tyr1 sequence into the pAIDA plasmid by insertional PCR cloning

To construct the pAIDA-tyr1 plasmid, we first amplified the tyrosinase gene (tyr1) by PCR. The PCR amplification produced the expected tyr1 fragment of ~1000 bp, as shown in the agarose gel figures 1.3.a and 1.3.b.

Several colonies were screened by colony PCR (Figure 1.3.a and 1.3.b). Colonies 1, 5, and 6 were positive for the presence of the pAIDA-tyr1 construct.

![Colony PCR Results](https://static.igem.wiki/teams/5932/footer/result-paida-fig3.webp)

**Figure 1.3:** Colony PCR showing amplification of the tyr1 gene, attesting successful cloning in the pAIDA plasmid. PCR signal from colonies 2 and 4 similar to the control (T- = pAIDA), indicating that cloning failed in the corresponding plasmids. However, PCR on colonies 1, 5 and 6 show 1kb amplification product, as expected for successful insertion of tyr1 in pAIDA.

### Validation of Construction

After sequencing verification, only colonies 1 and 5 were confirmed to contain the correct tyr1 sequence within the pAIDA plasmid. The plasmids from colony 1 and colony 5 were selected for further experiments on tyrosinase expression and melanin production.

#### Validation of Protein Production

Then, the recombinant pTyr1-AIDA plasmid was transformed into E. coli W3110 and W3110 ΔompT super competent cells. Western blot analysis with anti-His and anti-Myc antibodies were used to confirm production of the pAIDA-tyr1 construct.

In the E. coli W3110 ΔompT strain, a clear band at the expected molecular weight (~97 kDa) was observed, corresponding to the full-length Tyr1-AIDA fusion protein using anti-Myc antibodies (Figure 1.4.a). In contrast, the anti-His antibody revealed a protein with an apparent molecular weight of 64 kDa corresponding to the full-length AIDA was observed.

These results allowed us to conclude that there is partial cleavage of construct at the C-terminus of the Tyrosinase domain, before the C-Myc tag. Very interestingly, AIDA-production was stronger and more stable in the ΔompT genetic background compared to the wild-type W3110 strain.

#### Expression Conditions

Induction by IPTG (200 µM and 500 µM) at different temperatures (16°C, 30°C, and 37°C) showed robust expression of tyrosinase, with optimal detection at 16°C and 37°C for colony 1. Therefore, the Tyr1-AIDA construct was successfully produced.

![Western Blot Anti-His](https://static.igem.wiki/teams/5932/footer/result-paida-fig4a.webp)

**Figure 1.4.a:** Western blot revealed with anti-His tag antibody showing production of His-tagged tyrosinase AIDA. T- = 64 KDA = control of Laetitia's team with His Tag. pAIDA-tyr1 = 97 KDA. Gel 12%. C-Myc (BSA).

![Western Blot Anti-Myc](https://static.igem.wiki/teams/5932/footer/result-paida-fig4b.webp)

**Figure 1.4.b:** Western blot result with anti-Myc antibody.

![Western Blot Optimization](https://static.igem.wiki/teams/5932/footer/result-paida-fig4.webp)

**Figure 1.4.c:** Western blot result at different concentrations and different temperatures with antibody anti-Myc.

To verify partial cleavage of construct at the C-terminus of the Tyrosinase domain, before the C-Myc tag, we collaborated with David Hörnström's team in Sweden to compare their tyrosinase expression with ours.

![Collaboration Results](https://static.igem.wiki/teams/5932/footer/result-paida-fig5.webp)

**Figure 1.5:** Western blot results comparison with David Hörnström's team. Conclusion: we have same results.

### Visual Tests

At the same time, enzyme activity tests confirmed the functionality of tyrosinase: cultures induced by IPTG and supplemented with L-tyrosine and Cu²⁺ showed visible black pigmentation, characteristic of melanin production. This phenomenon was absent in the non-induced controls.

![Melanin Production Visual](https://static.igem.wiki/teams/5932/footer/engeniering-figure-3.webp)

**Figure 1.6:** Melanin production of bacteria containing our Tyr1-AIDA construction on plates supplemented with IPTG, tyrosine and Cu2+. Results: clear melanin production was observed in the bacterial strains.

### Enzyme Activity Tests

In vivo tyrosinase activity measurements showing the strong increase in absorption at 400 nm after incubation of bacteria with IPTG, tyrosine and Cu2+ in W3110 bacteria containing our pAIDA1-Tyr plasmid after about 10 hours, and in the Delta OmpT strain (which grew less well) after about 30 hours.

![Enzyme Activity Results](https://static.igem.wiki/teams/5932/footer/activity-tyrosine-engeniering.webp)

**Figure 1.7:** Enzyme activity tests showing tyrosinase functionality through melanin production measurement.

### Conclusion

In conclusion, this first part enabled the design, construction, and functional validation of our melanin production system. We successfully designed a plasmid, pAIDA-tyr1, exploiting the AIDA autotransporter system for the extracellular export of tyrosinase. The cloning strategy was validated by PCR and sequencing, identifying clones 1 and 5 as carriers of the correct genetic construct.

The expression of the Tyr1-AIDA fusion protein was confirmed by Western blot, revealing the production of the protein at the expected molecular weight (~97 kDa). A key observation was the discovery of partial cleavage of the construct, releasing the AIDA domain (~64 kDa) from tyrosinase. In addition, we demonstrated that the E. coli W3110 ΔompT strain provided a much more stable and efficient genetic background for the expression of our construct.

## 2/ Enhancement of Tyrosine Biosynthesis

We wanted to introduce the mutation S181A into AroF gene which codes for the DAH7PS, the first enzyme of the shikimate pathway. According to Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-D arabino-heptulosonate-7-phosphate synthase from Escherichia coli by Di Cui this mutation reduces the interaction between DAH7PS and tyrosine as a result the feedback self-regulation decreases. Thus we should have an increase in melanin production. To accomplish that we used the SLIC cloning method

### Use of pKng101, suicide plasmid

First, we attempted to introduce our insert into the suicide vector pKng101 which bears streptomycin resistance and is only replicative in the strain CC118. We encountered three issues:

1. One of our flanking sequences for our insert was incorrect, it only led to empty vectors during colony PCR

![](https://static.igem.wiki/teams/5932/footer/result-arof-01.webp)

**Figure 2.1:** Example of an empty vector

2. With the aid of our instructors we eventually managed to introduce the the insert into pKng101

![](https://static.igem.wiki/teams/5932/footer/result-arof-02.webp)

**Figure 2.2:** Result of the SLIC realized by our PI: The insert in pkng101

3. Unfortunately the opmT- strain we were using carried streptomycin resistance so could not be used with the pKng101 plasmid

Therefore, we designed new oligonucleotides.

### Use of a new vector thermosensitive plasmid Pko33

We decided to use Pko3, a thermosensitive plasmid only replicative at 30°C. This slowed our progress as our strain grew more slowly. Once again, there were two problems:

1. One of the oligonucleotides for the verification (3’VerifpKO3) was designed on the wrong version of Pko3 which led to insufficient amplification. At that time it was impossible to order new oligos

![](https://static.igem.wiki/teams/5932/footer/result-arof-03.webp)

**Figure 2.3:** Electrophoresis showing that the amplified fragment was too small

C: Control, it’s the miniprep of Pko3. 1 and 2: Number of colonies

2. After testing three different types of primers pairs a, b, c (Cf:Notebook). Only the couple with olS 4.5/2246 seemed to give the correct amplification. We pursued the second recombination event only to find that all the vectors were empty according to the sequencing. This primer pair does not seem 100% reliable

![](https://static.igem.wiki/teams/5932/footer/result-arof-04.webp)

**Figure 2.4:** PCR with the 3 different primer pairs shows that the first pairs of oligos seems rights. Even though of the sequencing all the vectors were empty

**Tube a**: oligo olS 4.5 and olS 2246 (it hybridizes on PKO3, gave by our PI). **Tube b:**  oligo olS 4.8 and olS 4.5 **Tube c:** oligo olS 4.7 and olS 4.6

After further PCR on colonies, we found a vector that contained our insert but there were other unwanted mutations.

### Final result

To reduce the number of mutations in our insert we decided to connect the two fragments of our insert by PCR using the Master Mix. The sequencing results were of higher quality, but there was a base deletion that was also found in our other sequencing results. This indicates that our template for making these fragments was mutated. Our time was limited, and we couldn’t obtain the mutation S181A.

Comparison of the insert sequencing made with oLS 4.7;

![](https://static.igem.wiki/teams/5932/footer/result-arof-05.webp)

**Figure 2.5:** Direct assembling

![](https://static.igem.wiki/teams/5932/footer/result-arof-06.webp)

**Figure 2.6:** PCR to connect the two fragments of the insert

Reference : Cui D, Deng A, Bai H, et al. (2019) Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Journal of Structural Biology 206: 322–334

## 3/ The Biocontainment Strategy

## Construction of W3110 Δ ompT ΔmurI for auxotrophy

### Conception and validation of the murI gene deletion

In order to create a robust metabolic containment system, we targeted the chromosomal murI gene, which codes for glutamate racemase, an enzyme essential for peptidoglycan biosynthesis. This modification was intended to establish the first layer of our biocontainment system. Inactivation of this gene was designed to render the bacterium auxotrophic for D-glutamate, an amino acid not available in human tissues, thereby preventing proliferation of our bacterium outside of our control.

When designing the murI gene deletion, we deliberately designed a deletion that does not affect the contiguous vitamin B12 transporter gene. This strategic choice was made to create auxotrophy while maintaining the integrity of essential metabolic pathways unrelated to our containment goal.

Agarose gel electrophoresis results after murI deletion consistently revealed the presence of a single amplification product of approximately 1.2 kbp (Figure 3.1) for the majority of clones tested. We used a primer that hybridizes to the kanamycin resistance gene KanR and a primer that hybridizes to the conserved region of murI. This size corresponds precisely to that expected for the ΔmurI deletion, confirming that homologous recombination occurred and that the coding sequence of the murI gene was excised.

![](https://static.igem.wiki/teams/5932/logo/resultats-muri-figure-1.webp)

**Figure 3.1:** Genotypic validation of the murI deletion by colony PCR

**Lane M:** Molecular weight marker. **Lane C:** W3110 Δ ompT strain showing no PCR product because it lacks the KanR resistance cassette. **Lane with the dot :** clone that grew when DL glutamate was added during culture one hour after electroporation. **Lanes 1 to 6:** Different independent candidate clones showing a single PCR band at ~1.2 kbp, the molecular signature of the deletion.

### Failure of phenotypic validation: absence of D-glutamate auxotrophy

Based on the literature, an E. coli strain lacking functional glutamate racemase should have a strict nutritional requirement for DL-glutamate and be unable to grow on standard media (Dougherty et al., 1993). To test this prediction, clones genotypically confirmed as ΔmurI were inoculated onto LB agar medium without any D-glutamate supplement (figure 3.2).

This prototrophic phenotype was consistently reproduced in several biological replicates, ruling out the possibility of accidental contamination

![](https://static.igem.wiki/teams/5932/logo/resultats-muri-figure-2.webp)

**Figure 3.2 :**D-glutamate dependency test on medium LB agar.

**A/**Clones growing on LB Kanamycin agar. **B/** Clones growing on LB Kanamycin 20 mM DL-Glutamate agar

**The dot:** clone that grew when DL glutamate was added during culture one hour after electroporation. **Clone 1 to 6:**Different independent candidate clones, no DL glutamate added during the one hour culture after electroporation.

The absence of any visible difference in growth conclusively demonstrates that ΔmurI clones do not exhibit the expected auxotrophic phenotype.

The growth profiles of the mutant strains W3110 ΔompT and ΔmurI were evaluated by measuring optical density (DO600) in LB liquid medium without DL glutamate supplementation. No significant differences were observed, with both strains exhibiting superimposable growth profiles with no detectable delay. These results confirm that the murI deletion does not induce DL glutamate auxotrophy or even slow cell growth under these conditions.

### Further investigation and consequences

The phenotype we observed shows that the deletion of murI is not sufficient to induce DL glutamate auxotrophy. We were therefore unable to establish the Δ murI base strain as functionally auxotrophic. As a result, the basis of our containment system could not be achieved.

However, faced with this contradiction between genotype and phenotype, we would have liked to have pursued the investigation further to identify the problem, but due to lack of time we were unable to do so. We would have liked to sequence the 1.2 kbp PCR product. Analysis of the sequence could confirm the absence of the murI coding sequence, which would validate the integrity of the deletion.

The planned steps of complementation with the pMurI plasmid, which carries a functional copy of murI, could not be carried out. Neither could the steps of mutation of the aroF pathway in our final strain, nor the melanin production test and encapsidation in the hydrogel. Thus, the first and main layer of our biological containment system could not be validated experimentally. This failure to reproduce the expected phenotype raises important questions about metabolic redundancy or the adaptation of E. coli strains.

## Construction of complementation plasmid of Δ murI : pMurI

### Blue-white screening of E. coli DH5ɑ with pMurI

In order to provide a functional copy of the murI gene to a genetically modified bacterial strain that is auxotrophic for D-glutamate, we attempted to construct a complementary plasmid pMurI using the Golden Gates assembly technique, then transformed the newly created plasmid into DH5α cells and verified its integration using white-blue screening.

![](https://static.igem.wiki/teams/5932/logo/resultats-figure-pmuri-1.webp)

**Figure 4.1:**Example of results for our DH5α E. Coli cells and NEB DH5α (C29871) E. Coli cells transformed with pMurI on an XGal+IPTG+Kanamycin plate as part of a blue-white screening.

After cell transformation and screening visualization, we first observe a higher number of white cells with NEB cells compared to our transformed cells, which produce more blue cells, i.e., cells that have not incorporated the plasmid. This means that NEB cells are more competent than the ones we created and that they have successfully integrated the plasmid.

### Colony PCR, Enzymatic Digestion Electrophoresis

![](https://static.igem.wiki/teams/5932/logo/resultats-figure-pmuri-2.webp)

**Figure 4.2:**Example of PCR results and enzymatic digestion with EcoRI/XmaI and EcoRI/SpeI of pMurI after its transformation in DH5α E. Coli

We tested digesting our plasmid with two different pairs of restriction enzymes. In the case of EcoRI/XmaI, these enzymes cut within LacZ; if the vector is empty, we obtain two bands (2931 bp and 220 bp), otherwise we obtain a linearized band. In the case of EcoRI/SpeI, the opposite is true. That is, if the vector is empty, we obtain the size of lacZ, which is 460 bp; otherwise, we obtain two bands (2931 bp and 1134 bp).

Unfortunately, after electrophoresis, this is not very visible in terms of markers. We can only conclude that for EcoRI/XmaI there are no empty plasmids except for the control. As for the results of the second digestion, the results cannot be used without the marker. Since subsequent experiments showed the presence of our plasmid, we did not attempt another enzymatic digestion.

A PCR colony assay was performed to observe whether the DNA in our cells contained an empty plasmid (460 bp), lacZ, or our plasmid constructed by Golden Gates (1100 bp). We can see that among the colonies tested, colonies 3, 4, and 5 are the expected size in the case of the presence of the plasmid we integrated which means that the transformation worked well. (fig4.2A)

### Western Blot of pMurI with an anti-HA antibody and sequencing of pMurI

![](https://static.igem.wiki/teams/5932/logo/resultats-figure-pmuri-3.webp)

**Figure 4.3:**Example of Western blot results for our pMurI plasmid with HA tag

For our initial results, we observed expression in two of the three plasmids tested (fig4.3.A), but visibility was poor. Assuming that the cause was the presence of disulfide bridges, we performed another Western blot, this time adding β-mercaptoethanol to the loading buffer. This time, the results are much clearer, as is the expression in the complementary plasmid tested.

Since the murI protein normally has a molecular weight of 32 kDa, we can clearly see a band between 28 and 38, which corresponds to the expected size. This experiment shows that our complementary plasmid produces the murI gene, enabling the complementation of a strain in which this same gene has been inactivated. It is assumed that the following bands correspond to remains.

Finally, our plasmid is sequenced and then aligned with the expected sequence.

![](https://static.igem.wiki/teams/5932/logo/resultats-figure-pmuri-4.webp)

**Figure 4.4:**Example of alignment of the expected cloning results with the sequenced plasmid after its transformation DH5α E. Coli

By aligning the sequenced fragment with the expected sequence, we observe a very good overall alignment. After manually checking the multiple sequences that were produced, we note that there are no gaps or mutations and that those shown in Figure 4.4 are in fact sequencing errors. Ultimately, the complementary plasmid we created is as expected and successfully produces the murI protein.

### Further investigation and consequences

As we were unable to finalize the DL-glutamate auxotrophic E. coli W3110 strain, it was not possible to test its complementation with our complementary pMurI plasmid. Nevertheless, a new composite part remains that can be tested by future teams and will enable future iGEM teams to complement a DL-glutamate auxotrophic E. coli strain.

## 4/ Hydrogel Encapsulation and Functionality

After setting up our protocols for hydrogel synthesis and characterization, we tested the system through a series of experiments. As a first step, we confirmed the difference between our motile and non-motile E. coli strains with a motility assay. Surprisingly, our W3110 ΔompT strain (the final strain used in our project) turned out to be motile (Figure 5.1). We had initially assumed it would be non-motile, since we believed that the ompT deletion might have been generated using the λ-Red recombination system, which is known to occasionally introduce secondary mutations in nearby loci. Such mutations can, in some cases, disrupt genes involved in flagellar assembly and result in a loss of motility.

![](https://static.igem.wiki/teams/5932/footer/measurement-fig-1-1.webp)

**Figure 5.1:** Motility test results for E.coli W3110 ΔompT and E.coli W3110

The motility test successfully validated the difference between the motile ΔompT and the non-motile strains, allowing us to use them as a reliable model to evaluate containment.

Next, we transformed the pBAD33 plasmid (arabinose-inducible) into both strains, enabling us to track the cells during experiments using the Magic Box system and to monitor GFP fluorescence as a reporter of cellular activity (Figure 5.2).

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-figb-1.webp)

**Figure 5.2:** Transformation results of pBAD33 transformation intoE.coli W3110, E.coli W3110 ΔompT induced by arabinose and E.coli W3110 non-motile.

We proceeded by developing our own alginate hydrogel synthesis protocol and evaluated both the ability of the cells to escape from the matrix and their survival rate over time. To demonstrate cell confinement, we designed a bacterial escape assay, which we repeated several times until the protocol was optimized (Figure 5.3)

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-figc.webp)

**Figure 5.3**Our experiments confirmed the effectiveness of the alginate hydrogel in physically containing E. coli. We demonstrated that the matrix significantly limited bacterial escape, whether the hydrogel was carefully preserved or mechanically disrupted to simulate escape (in which case OD measurements increased slightly faster than under the intact condition). Escape curves revealed a clear difference between the two strains, with motile cells showing a higher propensity to move through the hydrogel compared to the non-motile strain. Nevertheless, the majority of encapsulated cells remained confined within the hydrogel throughout the tested time frames

These results are promising and suggest that introducing a motility mutation could be considered in future engineering cycles to further reduce bacterial escape for potential applications

A bacterial survival assay was also performed to assess the viability of cells within the hydrogel matrix over time (Figure 5.4).

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-figd.webp)

**Figure 5.4:** Survival assays showed that encapsulation did not compromise cell viability. The survival histograms indicate that a substantial proportion of cells remained alive inside the matrix, demonstrating that the hydrogel provides a protective environment compatible with bacterial growth, even after 72 hours.

To better understand the structure of the hydrogel and the position of the cells inside it, we also performed SEM imaging of our hydrogel marbles (Figure 5.5.a, 5.5.b and 5.5.c)

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-fige1.webp)

**Figure 5.5.a:** SEM images of 1.5% alginate marbles. 10µL marbles containing E.coli W3110 non motile cells

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-fige2.webp)

**Figure 5.5.b:** SEM images of 1.5% alginate marbles. 10 µL marbles without cells

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-fige3.webp)

**Figure 5.5.c:** SEM images of 1.5% alginate marbles. 10 µL marble containing E.coli W3110 Δ ompT cells

SEM imaging further confirmed the structural integrity of the hydrogel and showed the embedded cells distributed throughout the matrix in agglomerates.

Finally, we used fluorescence microscopy (Figure 5.6) to confirm that our encapsulated engineered strains were still functional and able to express GFP inside the hydrogel.

![](https://static.igem.wiki/teams/5932/footer/results-hydrogel-figf.webp)

**Figure 5.6:** Fluorescence microscopy provided additional confirmation that our engineered strains were successfully encapsulated and remained functional, as indicated by GFP expression inside the hydrogel.

Together, these results demonstrate that our alginate hydrogel is an efficient and biocompatible system for containing genetically modified E. coli, while preserving their viability and capacity for molecular production. However, its performance could be further improved by modifying certain aspects, as discussed in the Future Work section.

## 5/ Futurs Works

### Experiment to test the mutation on AroF

In order to test our mutation on AroF which is supposed to increase the production of tyrosine by E.Coli, we planned 2 experiences:

The first one is to analyze the production of tyrosine by HPLC ( High Performance Liquid Chromatography). This kind of chromatography allows us to measure the concentration of tyrosine. Here is the protocol we planned to use:

* 1.Take 500 µl of bacterial culture
* 2.Dilute the sample in 1 N HCl
* 3.Incubate at 55°C for 30 minutes, shaking occasionally with a vortex mixer
* 4.Centrifuge the sample and collect the supernatant
* 5.Dillute the supernatant with the appropriate amount of distilled water
* 6.Inject the prepared sample into an Agilent 1200 Series HPLC system equipped with a photodiode array detector (PDA) set at 210, 254, and 280 nm
* 7.Perform seperation on a reverse phase C18 column(Inertsil; 2.1 x 250 mm, 3.5µm; GL Sciences) at a flow rate of 0.15mL/min
* 8.Use a mobile phase of water (A)/ methanol (B) with the following gradient:
  + 0-8 min: 5 % B
  + 8-13min: 40 %B
  + 13-16min: maintain at 40% B
  + 16-21 min: 5% B
  + 21-31min: equilibrate at 5%
* 9.Quantify L-tyrosine using a 5 points calibration curve (14 to 448 mg/L)
* 10.Check the quality of calibration (R² = 0.99)

After the HPCL we’re going to do a measurement activity test to see if we obtain more melanin

Reference : Juminaga D, Baidoo EEK, Redding-Johanson AM, et al. (2012) Modular Engineering of l-Tyrosine Production in Escherichia coli. Appl Environ Microbiol 78: 89–98.

### Plan for phenotypic characterization of the ΔmurI strain

If we had succeeded in obtaining the ΔmurI auxotrophic strain, we would have undertaken a phenotypic characterization.

We would first have performed a kinetic analysis of growth in LB medium. This study would have compared the growth profiles of four strains: the wild-type W3110 ΔompT strain as a control, the ΔmurI auxotrophic strain, the ΔmurI strain complemented with the pMurI plasmid, and the wild-type strain complemented with pmurI. We would have measured the growth curves in LB medium with and without 20 mM DL-glutamate supplement. And we would have performed three biological replicates for each condition. This analysis would have allowed us to determine essential parameters such as the growth delay or absence of growth without glutamate supplement and the final cell yield.

At the same time, we would have evaluated the survival profile of the strains under deficiency conditions. The protocol would have consisted of resuspending stationary cultures in nutrient-free PBS, then monitoring cell viability by counting CFU/mL for 48 hours. Analysis of the survival curves would have provided information on the respective mortality rates of the different strains in the absence of DL-glutamate.

A study of the response to different concentrations of D-glutamate was also conducted. We tested a full range of concentrations (0, 5, 10, 20, and 50 mM) to establish the minimum concentration required for normal growth. For each concentration, we produced complete growth curves and measured viability after 24 hours of deprivation.

To complete this characterization, we would have quantified the expression of the murI gene by real-time qPCR. This analysis would have compared the level of expression of the murI gene in the wild-type strain and in the strain complemented with pMurI. We would have measured this expression under different conditions in the presence and absence of D-glutamate and at different growth stages.

This characterization would have provided us with essential data for evaluating the potential of the metabolic containment system. Although the failure to obtain the auxotrophic strain prevented us from carrying out this work, this detailed protocol remains valid for future attempts to optimize the system.

Reference : Cabral, M. P. et al. Design of live attenuated bacterial vaccines based on D-glutamate auxotrophy. Nat Commun 8, 15480 (2017).

### 3/ Poly-DL-γ-glutamate (PGA) hydrogel for biocontainment

Building on our current results with alginate hydrogels, future work could explore the use of a poly-DL-γ-glutamate (PGA) hydrogel, as described in the scientific paper Li, J., Huang, Y., Wang, Y. & Han, Q. A Poly-γ-Glutamic Acid/ε-Polylysine Hydrogel: Synthesis, Characterization, and Its Role in Accelerated Wound Healing. Gels 11, 226 (2025). Since our engineered E. coli strains are auxotrophic for D-glutamate, a PGA-based matrix could provide an additional layer of biocontainment: cells would only survive and remain active within the hydrogel, while any escaped cells would be unable to grow in the absence of D-glutamate. This strategy could be combined with motility-reducing mutations to further minimize bacterial escape.

Moreover, PGA hydrogels could be optimized to maintain cell viability and molecular production, while allowing controlled diffusion of molecules such as the melanin produced by our strain. Structural characterization of the PGA matrix, including porosity and degradation over time, would provide insights into cell confinement and metabolite exchange. Finally, future applications could include scaling up the hydrogel for safe bioproduction, designing hybrid matrices combining alginate and PGA, and integrating sensors to monitor metabolites in real time within the hydrogel.

### 4/ Genetic Modifications to Enhance Containment

To further improve the biocontainment of our engineered E. coli within the hydrogel, targeted genetic modifications could be considered. For example, deleting the fliC gene, which encodes the flagellin protein essential for flagellar assembly, would abolish motility and reduce the ability of cells to escape the hydrogel matrix (similar to our ΔompT strain).

### 5/ Biocompatibility and Biodegradability Testing

To further optimize hydrogel-based scaffolds, future work could focus on evaluating their biocompatibility and biodegradability. Biocompatibility can be assessed in vitro by measuring cell viability, proliferation, and adhesion using assays such as Live/Dead staining. For osteogenic applications, differentiation markers such as alkaline phosphatase (ALP) activity or mineralization can be monitored to confirm that the hydrogel provides a supportive environment for osteoblast function. Additionally, inflammatory responses could be evaluated by exposing immune cells to the hydrogel and measuring cytokine production.

Biodegradability could be tested by monitoring hydrogel mass loss or structural changes over time under physiological conditions (for example in PBS at 37°C) or in the presence of relevant enzymes such as lysozymes or proteases. Mechanical properties, porosity, and release of embedded molecules could provide complementary insights into scaffold degradation. Combined approaches, such as co-culturing cells on the hydrogel while tracking degradation, or using microscopy (SEM or confocal) to visualize structural changes and cell distribution, would provide a comprehensive understanding of both cell compatibility and controlled scaffold resorption.

### 6/ Injectability of the hydrogel

For potential applications, it is important that hydrogels exhibit injectability. This means that the gel should be in a liquid or semi-liquid state under specific conditions, such as temperature or pH, allowing for easy injection into the desired site. After injection, the hydrogel should rapidly solidify to form a stable gel, maintaining its structural integrity and providing a supportive environment for encapsulated cells.

Future work could focus on optimizing the physicochemical properties of the hydrogel to achieve this transition efficiently, while preserving cell viability and molecular production. Techniques such as rheological measurements could be used to assess the gel’s flow properties and gelation kinetics under different conditions.

### 7/ Melanin Diffusion Control

To ensure effective ROS reduction, future work could focus on controlling melanin release from the hydrogel. Simple diffusion assays could help us track melanin concentration in surrounding media over time, while ROS scavenging tests would help us verify antioxidant activity. Mathematical modeling could help predict release kinetics and optimize hydrogel properties. These tests together would guide the design of our hydrogel that maintains local ROS reduction while keeping cells contained and viable.

**Protocol Overview:**

* **Prepare hydrogel:** Embed melanin-producing E. coli in alginate or PGA hydrogel. Place hydrogel in PBS or culture medium
* **Diffusion assay:** Collect small aliquots of the surrounding medium at regular time points (e.g., 0, 2, 4, 8, 24, 48 h). Measure melanin concentration using UV-Vis or fluorimetry
* **ROS scavenging assay:**Test supernatants on ROS-sensitive dye or cells to verify antioxidant activity over time.
* **Data analysis:** Plot melanin release curves and ROS reduction activity. Compare hydrogel formulations to identify optimal conditions for controlled diffusion.

### 8/ Microstructure Optimization

To improve the functionality of our hydrogel in tissue engineering applications, advanced techniques such as microfluidics and 3D printing can be explored. These approaches allow the design of specific porosity patterns, optimizing both the controlled diffusion of therapeutic molecules, such as melanin, and the confinement of cells within the scaffold. For example, Wang et al. (2011) demonstrated the fabrication of highly organized alginate scaffolds using microfluidic technology, resulting in well-defined porous structures that promote cell proliferation and cartilage formation in tissue engineering (Wang, C.-C., Yang, K.-C., Lin, K.-H., Liu, H.-C. & Lin, F.-H. A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology. Biomaterials 32, 7118–7126 (2011).). Additionally, 3D printing enables the creation of scaffolds with complex and controlled geometries, facilitating the customization of scaffold architecture according to the specific needs of the target tissue. Combining these technologies we could significantly enhance hydrogel performance, providing an optimal environment for cell adhesion, proliferation, and differentiation.

### 9/ In Vivo Testing

For future development, in vivo studies could be conducted to evaluate the safety, biocompatibility, and efficacy of the hydrogel system in animal models. These tests would provide critical information on how the scaffold behaves in a complex physiological environment, including degradation rate, immune response, cell viability, and controlled release of molecules such as melanin. In addition, in vivo studies would allow assessment of tissue regeneration, such as bone repair, and confirm that the hydrogel can effectively contain engineered cells while supporting host tissue integration. These experiments are essential before considering potential clinical applications.

### 10/ Implementation of a Toxin-Antitoxin Biocontainment System

To reinforce our multi-layered biocontainment strategy, a genetic "dead-man's switch" based on the CcdB/CcdA toxin-antitoxin system could be implemented. The CcdB toxin poisons DNA gyrase, leading to rapid cell death, while the CcdA antitoxin neutralizes CcdB.

#### Proposed Design:

**Constitutive Toxin Expression:** The ccdB gene would be placed under the control of a constitutive promoter, ensuring constant production of the lethal toxin to prevent any plasmid conjugation.

#### Validation Experiments:

* **Plasmid Construction:** Clone the ccdB and ccdA genes into a bicistronic operon or on compatible plasmids with the specified promoters.
* **Killing Efficiency Test:** Transform the system into our production strain and measure the loss of cell viability (by CFU count) over time after removal of the antitoxin inducer.
* **Containment Efficacy:** Co-encapsulate the engineered strain with the toxin-antitoxin system in our hydrogel and measure the number of viable cells that escape into an inducer-free medium, comparing it to a control strain without the system.

This system would provide a potent, genetically encoded fail-safe mechanism, working synergistically with our physical (hydrogel) and metabolic (ΔmurI auxotrophy) containment layers to ensure maximum environmental safety.

### 11/ Integration of All Modules and System-Level Validation

The ultimate goal is to integrate all developed modules into a single, robust, and safe platform. This involves assembling the melanin production system, the optimized tyrosine pathway, and all biocontainment layers (metabolic, physical, and genetic) into a final chassis strain.

#### Integration Pipeline:

**Strain Consolidation:** Combine the successfully constructed genetic parts—pAIDA-tyr1 (melanin production), the feedback-resistant aroFS181A mutation (tyrosine overproduction), the ΔmurI deletion with pMurI complementation (metabolic containment), and the CcdB/A toxin-antitoxin system (genetic containment)—into the E. coli W3110 ΔompT background.

#### Functional Characterization:

Test the fully integrated strain to ensure that all systems function harmoniously without negative interference. Key assays would include:

* **Melanin Quantification:** Measure the final yield of melanin production under optimized conditions.
* **Containment Verification:** Perform a comprehensive escape assay, combining the hydrogel with all genetic safeguards, to quantify any viable cells in the external environment.
* **Growth and Stability:** Monitor the strain's growth kinetics and genetic stability over multiple generations to ensure robust performance for potential applications.

## Conclusion

In this project, we successfully designed and validated a melanin production system in E.Coli using the AIDA autotransporter to achieve extracellular export of tyrosinase, enabling efficient melanin synthesis.

Cloning, sequencing and expression analyses confirmed the functionality of our constructs, and enzyme activity tests demonstrated robust melanin production. Despite challenges in introducing the S181A mutation in the aroF gene and generating the Δ murI auxotrophic strain, the project established reliable methodologies and genetic tools that can be further optimized in future work.

Encapsulation of engineered bacteria in the alginate hydrogel provided effective physical containment while preserving cell viability and molecular activity, as confirmed by the survival tests, escape tests, SEM imaging and fluorescence microscopy. These results demonstrate the potential of therapies such as biodegradable hydrogel scaffolds for safe and functional deployment on engineered cells.

Future improvements described before will be crucial to validate Living Scaffolds performance and safety for potential therapy application. Overall, our work establishes a solid foundation for a modular, functional system that combines bacterial melanin production and hydrogel based confinement, providing a versatile platform for future synthetic biology and regenerative medicine applications.

{% endblock %}