iGEM 2023 — Team aix-marseille

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Lab notebooks from the Wetlab team.{% endblock %}
{% block page\_content %}

## Truncated mucA

---

It appears you don't have a PDF plugin for this browser.
You can [click here to
download the PDF file.](https://static.igem.wiki/teams/4582/wiki/p-putida-chassis-strain-producing-truncated-muca.pdf)

## Alginate overproduction

---

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You can [click here to
download the PDF file.](https://static.igem.wiki/teams/4582/wiki/overproduction-and-control-of-alginate-production.pdf)

## Growth with a glycerol feedstock

---

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You can [click here to
download the PDF file.](https://static.igem.wiki/teams/4582/wiki/saipol-glycerol-growth.pdf)

## Alginate beads

---

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Overproducing alginate with *P. putida* - Part [BBa\_K4582000](https://parts.igem.org/Part%3ABBa_K4582000) {% endblock %}
{% block page\_content %}

The ultimate goal of our project is to turn alginate into a commodity. To make it inexpensive enough that we can use it at scale in agriculture to fight drought. We’ll need a lot of engineering to achieve this goal and here is our first stone to this building.

# Design

Alginate is traditionally produced from algae. Its production, extraction and purification are expensive and not environmentally friendly.

There is already [research](https://link.springer.com/article/10.1007/s11274-017-2363-x) about producing alginate from bacteria. Actually, it was one of the subjects of one great iGEM project from last year: [Cosmic](https://2022.igem.wiki/duesseldorf/engineering/). They tried (and unfortunately failed) to overproduce alginate in *A. vinelandii* by overexpressing *alg8*.

In our search for an alternative chassis for alginate production, we became interested in Pseudomonas sp. because:

1. certain species naturally produce this polymer
2. in the human pathogen *Pseudomonas putida*, genetic mutations leading to overproduction of alginate have been described in the literature
3. researchers in our host laboratory, the LISM, are specialists of Pseudomonas.

For our work, we selected Pseudomonas putida as the chassis because it is a non-pathogenic bacterium closely related to *P. aeruginosa*.

Alginate production is tightly regulated in the Pseudomonas genus. The genes involved in alginate biosynthesis are organized in operons with their promoter recognized by the alternative sigma factor AlgU, and the major negative regulatory element is the anti-sigma factor MucA.

Even though MucA inhibits alginate production, it is also vital for the bacteria's survival. However, researchers have identified several *mucA* mutations (called *mucA*\*) resulting in a truncated MucA protein. These mutations are not lethal for the bacteria but lead to an increased production of alginate.[1](#footnote-1). As the physiology of *P. putida* is similar to that of *P. aeruginosa*, we decided to introduce the *mucA*\* mutations into the *P. putida* genome in order to obtain an alginate overproducing strain.

To do this, we constructed a mutator plasmid corresponding to the suicide plasmid pKNG101, in which we integrated the mutated *mucA* gene coding for a truncated MucA, along with 50 bp regions homologous to the regions flanking the *mucA* gene on the chromosome of *P. putida* KT2440. In this way, we aimed to replace the wild-type *mucA* gene with the mutated *mucA* gene through homologous recombination after plasmid integration into the strain.

# Build

As our design was based on work carried out on *P. aeruginosa* and not on *P. putida*, we knew that the operation was likely to fail. The risk of failure was all the greater in that we used homologous recombination for the construction of our chassis, a technique that is itself laborious.

Four different *mucA*\* parts [were built](https://2023.igem.wiki/aix-marseille/results#construction-of-muca-mutants) based on the literature and introduced into the suicide plasmid pKNG101. We were tempted to introduce the mutations sequentially in the *P. putida* genome by homologous recombination, testing each time whether it worked, but we thought it would be much quicker to do everything in parallel.

To construct our mutator plasmid, we performed PCR using *P. putida* DNA as a template to obtain fragments corresponding to the truncated mutated gene with the addition of a stop codon and homologous regions. To obtain the mutated gene, we designed primers with a reverse primer hybridizing to the desired stop region of the mutated gene with the addition of a stop codon at the end. Further details are available in the results section. All fragments were integrated and assembled into the pKNG101 plasmid, digested using the NEBuilder Assembly kit, following an unsuccessful attempt with the SLIC method.

![conjugation figure 1](https://static.igem.wiki/teams/4582/wiki/conjugation-figure-1.png)

Figure 1: Diagram of the mutator plasmid and homologous recombination. Black arrows represent possible homologous recombination events. Designed with Biorender. Part [BBa\_K4582000](https://parts.igem.org/Part%3ABBa_K4582000)

After confirming the acquisition of our mutator plasmid for the four constructed forms through sequencing, we integrated each mutator plasmid into a wild-type strain of *P. putida* KT2440 via conjugation. After two homologous recombination events, we aimed to screen the obtained colonies by selecting homologous recombination events that replaced the *mucA* gene with the mutated *mucA* gene by PCR on colonies. This was a challenging step since we initially wanted to differentiate the mutated *mucA* from the wild-type *mucA* on the chromosome by size, but we encountered difficulties in amplifying and distinguishing the different sizes of each mutant. Therefore, we decided to use a different approach not based on the gene size. We thought of using two different primer pairs, one pair hybridizing to the ends of the mutated gene and another pair hybridizing to the ends of the wild-type gene. This way, for a colony that replaced the wild-type *mucA* with the mutated one, only amplification with primers flanking the mutated gene would be detected.

Thus, we succeeded in obtaining a mutant named *mucA*\*34, which replaced the wild-type *mucA* gene with the truncated *mucA* gene of 399 bp in size.

![](https://static.igem.wiki/teams/4582/wiki/electrophoresis-2.png)

Figure 2: **Results of colony PCR analyzed by agarose gel electrophoresis (1%).** Colony PCR was performed under both control conditions for a colony of *P. putida* wild-type (WT), colony 4 of the mucA1 strain (mucA14), and colony 4 of the mucA34 strain obtained after homologous recombination. The marker size (bp) is indicated on the left.

As shown in Figure 2, we succeeded in the construction of one *P. putida* *mucA*\* mutant. Colony PCR indicated that the *P. putida* *mucA*34 clone is successfully mutated as this clone does not contain a WT copy of the *mucA* gene (absence of PCR amplification in the red rectangle).

The *mucA*\* coding sequence used for this construction has been submitted as a part into the registry under reference [BBa\_K4582000](https://parts.igem.org/Part%3ABBa_K4582000)

# Test

To measure the efficiency of *mucA* gene mutation in alginate production, we considered performing an alginate assay with the wild-type *P. putida* strain under conditions of osmotic stress by adding NaCl, which is one of the best-characterized natural conditions for alginate production, under non-stress conditions (LB), and with the mutant strain obtained under non-stress conditions (LB).

![](https://static.igem.wiki/teams/4582/wiki/alg-per-strain.png)

Figure 3: The concentration of Alginate as a function of the strain

The [results](https://2023.igem.wiki/aix-marseille/results#alginate-quantification-assays) showed a 30-fold increase in alginate production in the mutant strain compared to the wild-type strain grown in LB. This result indicates that the truncation of the MucA protein effectively prevents the sequestration of AlgU, leading to higher expression of genes encoding enzymes for alginate biosynthesis, which are naturally expressed under membrane stress conditions, resulting in improved alginate production despite optimal growth conditions. However, it does not produce more alginate than a wild-type strain under high osmotic stress conditions (0.5M NaCl).

# Learn

Throughout this engineering cycle, we learned that the truncation of the MucA protein allows alginate production under non-stress conditions but not in large quantities. This can be explained by the multifactorial nature of alginate production in the wild-type strain, involving factors other than genetic regulation alone.

We observed that modifying genes in cis by homologous recombination is simpler to implement but can be more uncertain, with minimal success probabilities. In the future, other methods described as effective in *P. putida* could be used for genetic modification at the genome level.

We realized the importance of carrying out multiple constructions to ensure obtaining at least one successful result since, of the 4 mutator plasmids constructed, we obtained only one mutant colony. Similarly, having a backup method in a key construction step is essential, as we faced difficulties in the assembly and verification of mutant strain acquisition through colony PCR. This is a practice to keep for future constructions.

# Design 2

This engineering cycle allowed us to highlight the multifactorial aspect of alginate production. To optimize its production, we have considered exploring the metabolic aspect in addition to regulation. Therefore, we aim to add the *alg8* gene in trans to the mutant *P. putida* *mucA*34 strain under the control of an inducible promoter. Alg8 has been shown to overproduce alginate in *P. aeruginosa* and *A. vinelandii*, and it is also a path chosen by the CosMic team. *alg8* is described as a key gene in alginate production because it is involved in polymerization [3](#footnote-3). Integrating the gene into a plasmid allows us to avoid the homologous recombination step but requires the use of antibiotics to prevent plasmid loss. One solution could be to construct a plasmid based on an autotrophy system to make the plasmid essential for the strain's survival.

# Sources

1. *Different mutations in mucA gene of Pseudomonas
   aeruginosa mucoid strains in cystic fibrosis
   patients and their effect on algU gene expression*, Giovanna Pulcrano et al. NEW MICROBIOLOGICA, 2012
2. [The Cosmic 2022 iGEM project](https://2022.igem.wiki/duesseldorf/)
3. *Bacterial alginate production : an overview of its biosynthesis ans potential industrial production*, Viviana Urtuvia et al. World J Microbiol Biotechnol, 2017

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{% block title %}Experiments{% endblock %}
{% block lead %}Molecular biology, alginate production and beads{% endblock %}
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# Molecular biology

## PCR

We performed PCRs to obtain and amplify fragments corresponding to mutated *mucA* from genomic DNA of *P. putida* as the template.

| Materials: |
| --- |
| Template: Genomic DNA from *P. putida* or *P. putida* on LB agar plate |
| Forward and Reverse Primers |
| Q5 High-Fidelity 2X Master Mix (New England Biolabs) |
| MiliQ Water |

### Protocol:

* Dilute a colony of*P. putida* from LB agar plate in 70µL of water (genomic DNA).
* In a PCR tube placed on ice (-4°C), add:

+ 25 μL Q5 master mix (containing buffers and components required for PCR)
+ 2.5 μL Reverse primer
+ 2.5 μL Forward primer
+ 1µL of genomic DNA (=template)
+ Complete with MiliQ water to reach a final volume of 50µL

### PCR program with Q5 polymerase:

| **Cycling Step** | **Temperature** | **Time** |
| --- | --- | --- |
| Cell lysis and initial denaturation | 98°C | 30s |
| 25-30 cycles Denaturation | 98°C | 10s || 25-30 cycles Hybridation | 65°C | **30s/kb** |
| 25-30 cycles Elongation | 72°C | 50s |
| Final elongation | 72°C | 2min |
| Hold | 4°C | ∞ |

![pcr](https://static.igem.wiki/teams/4582/wiki/experiments-pcr.jpg)

## Vector Linearization

We digested our vectors to enable the cloning of mutated *mucA* and *mucA* wt. All vector linearizations were performed by digestion with two restriction enzymes to prevent vector re-circularization.

| Materials: |
| --- |
| Enzyme buffer |
| Restriction Enzyme 1 |
| Restriction Enzyme 2 |
| Vector for digestion |
| MiliQ Water |

### Protocol

* In a 1.5mL tube, add:

+ 5 µL enzyme buffer
+ 1 µL Restriction Enzyme 1
+ 1 µL Restriction Enzyme 2
+ 22 µL of the vector to be linearized.
+ 18 µL of MiliQ water

* Incubate for one hour at 37°C.

## Cloning

We carried out the cloning of mutated *mucA* into the pKNG101 vector and cloning of *mucA* wt into the pJN105 vector using the NEBuilder kit from New England Biolabs.

| Materials: |
| --- |
| Linearized vector |
| Fragment(s) to be assembled |
| NEBuilder MasterMix |
| MiliQ Water |

### Protocol:

* In a 1.5mL tube, add:

+ 2µL of the linearized vector
+ 2µL of the fragment(s)
+ 10µL of NEBuilder MasterMix
+ Complete with MiliQ water to reach a final volume of 20µL

* Incubate for 15 minutes at 50°C.

## Competent cell preparation

We transformed *E. coli* cc118λpir strains with our cloning products to verify the correct insertion of inserts into vectors and amplify cloning products. Prior to transformation, the cells need to be made competent.

| Materials: |
| --- |
| Cells to be made competent |
| LB |
| CaCl2 50mM |
| Glycerol 15% |
| Water |
| Ice |

### Protocol:

* The day before:

+ Inoculate 10mL of LB with 100µL of cc118λpir cells.
+ Incubate overnight at 37°C.

* On the day of the procedure:

+ Prepare the following solutions:

- 50mL of 50mM CaCl2 solution (Buffer 1)
- 50mL of 50mM CaCl2 solution + 15% Glycerol (Buffer 2)

+ Filter the solutions and store them at 4°C. They must remain sterile and be used while kept on ice.
+ Inoculate 100mL of LB with 1mL of the overnight preculture.
+ Incubate at 37°C until the OD600nm reaches between 0.3 to 0.5 (approximately 2 to 3 hours).
+ Once the OD600nm is reached, divide the culture into two 50mL tubes, to be kept on ice (-4°C). All the following steps should be performed on ice.
+ Centrifuge the culture at 4000 rpm at 4°C for 10 minutes.
+ Remove the supernatant and resuspend the pellet in 25mL of Buffer 1.
+ Incubate for 20 minutes on ice.
+ Centrifuge the culture at 4000 rpm at 4°C for 10 minutes.
+ Remove the supernatant and resuspend the pellet in 4mL of Buffer 2.
+ Incubate for 20 minutes on ice.
+ Prepare 350µL aliquots in 1.5mL Eppendorf tubes and store them at -80°C.

## Transformation

We transformed competent *E. coli* cc118ypir cells with our cloning products (mutated *mucA*, *mucA*, *alg8*). The introduction of the recombinant plasmid is achieved through a heat shock.

| Materials: |
| --- |
| Competent cells |
| Cloning product |
| Ice bath |
| LB agar plates + antibiotic |
| Water bath capable of reaching 42°C |
| Shaking incubator at 37°C |

### Protocol:

* Prepare an ice bath and a water bath set to 42°C.
* Thaw a tube stored at -80°C containing competent cells in the ice bath.
* In a 2mL tube, add:

+ 100µL of competent cc118 λpir cells
+ 10µL of the cloning product

* Incubate for 30 minutes in the ice bath (-4°C).
* Incubate for 45 seconds at 42°C.
* Incubate for 10 minutes in the ice bath.
* Add 1mL of LB to the tube.
* Incubate for 1 hour at 37°C under agitation (300rpm).
* During this time, prepare LB agar plates with antibiotic.
* Spread 100µL of the culture onto LB agar plates with antibiotic using a spreader or a bent glass rod.
* Once the plates are dry, incubate the plates overnight at 37°C.

![transformation](https://static.igem.wiki/teams/4582/wiki/experiments-transformation.jpg)

## Colony PCR

We conducted colony PCR to verify the insertion of inserts into vectors after cloning mutated *mucA* and wild type *mucA*. Colonies containing the recombinant plasmid were selected for sequencing after this step. Once the sequencing is confirmed, the cells carrying the recombinant plasmid will be used to amplify the plasmid for extraction. We performed colony PCR using EconoTaq Plus 2X Green Master Mix by Biosearch technologies.

| Materials: |
| --- |
| EconoTaq Master Mix 2X |
| Forward and Reverse Primers |
| emplate = colony on a plate |
| LB agar plates + antibiotic containing the plasmid's resistance marker |

### Protocol:

* Prepare LB agar plates with the appropriate antibiotic containing the plasmid's resistance marker.
* In a PCR tube, add:

+ 12.5µL EconoTaq Master Mix 2X
+ 0.25µL Forward Primer
+ 0.25µL Reverse Primer
+ Complete with water to a final volume of 25µL.

* Pick a colony from the plate with transformed colonies using a toothpick and streak onto a new LB agar plate with the appropriate antibiotic.
* Place the toothpick into the PCR tube and scrape it in the tube to transfer cells (= PCR template).
* Thermocycler Program:

+ Initial denaturation: 95°C for 2 minutes
+ Denaturation: 95°C for 30 seconds
+ Hybridation: 55°C for 30 seconds
+ Elongation: 72°C for 1 minute
+ Final elongation: 72°C for 5 minutes
+ Hold: 4°C

| **Cycling Step** | **Temperature** | **Time** | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 94°C | 2 min | 1 |
| Denaturation | 94°C | 30 sec | 30 |
| Annealing | 65°C | 30 sec | 30 |
| Extension | 72°C | **1kb/min** | 30 |
| Final extension | 72°C | 6min | 1 |
| Hold | 4°C | ∞ | 1 |

## Agarose gel electrophoresis

We conducted agarose gel electrophoresis to verify the linearization of plasmids and the presence of amplicons at the expected size after PCR/colony PCR.

| Materials: |
| --- |
| 1% Agarose powder |
| 1X TBE buffer |
| Water |

### Protocol:

⚠ Caution! TBE buffer is toxic and should be handled with gloves.

* Preparation of Agarose Gel:

+ Prepare 100mL of 1X TBE buffer.
+ Add 1g of 1% agarose.
+ Transfer the solution to a flask and heat it until the agarose melts.
+ Pour the solution into an agarose gel mold.
+ Allow the gel to polymerize.

* Electrophoresis

+ Add the migration buffer (1X TBE) to the electrophoresis.
+ Place the gel, still within the mold, into the electrophoresis chamber.
+ Using a micropipette, dispense between 5 to 10μL of the sample into the well, depending on the well size. Don't forget the size marker.
+ Allow the samples to migrate for 30 minutes at 135 volts.
+ Visualize the gels.

## Conjugation in *Pseudomonas*

We introduced the recombinant pKNG101 plasmids through conjugation to integrate them into the chromosome via homologous recombination. We also used conjugation to introduce pNJ105 carrying *mucA* wt.

| Materials: |
| --- |
| Donor strain (*E. coli* cc118ypir) |
| Recipient strain (*P. putida*) |
| Mobilizing strain (*E. coli* prK2013) |
| LB agar plates |
| LB medium |
| PIA medium |

### Protocol:

* The Day Before the Procedure:
  + Prepare overnight precultures of the recipient, donor, and mobilizing strains in LB medium.
* On the Day of the Procedure:
  + Spot 25µL of the donor and mobilizing strain precultures on LB agar plate.
  + Incubate for 2 hours at 37°C.
  + During this time, incubate the recipient strain preculture at 42°C.
  + Combine with the recipient strain by spotting 25µL of its preculture onto a plate containing the mobilizing and donor strains.
  + Incubate for 6 hours at 37°C.
  + Resuspend the patch in 1mL of LB and incubate for 1 hour at 37°C with agitation.
  + Select clones on PIA with Streptomycin 2000 μg/ml (3 plates with 75μl/150µL/300µL).
  + Incubate overnight at 37°C.

## Recombination in *Pseudomonas*

We used homologous recombination to replace the native *mucA* gene in the genome with a mutated *mucA* gene encoding a truncated MucA. The first homologous recombination event occurs after conjugation. This protocol describes how to proceed with the second homologous recombination event.

![recombination](https://static.igem.wiki/teams/4582/wiki/experiments-pkng101.jpg)

### Protocol:

* The first day:

+ Isolate 6 colonies on 6 plates with LB agar containing 6% sucrose and no NaCl. (It is crucial to perform true isolation to increase the chances of obtaining more isolated clones.)
+ Leave the plates at room temperature (RT) for 2-3 days until isolated clones, approximately 50-100, become SucR (sucrose-resistant).

* The next day:

+ Re-patch the isolated clones on LB plates, LB plates with 2000 μg/ml Sm (Sm2000), and LB plates with 6% sucrose and no NaCl (LB ssNaCl Suc 6%).
+ Incubate at 37°C overnight.
+ Perform colony PCR to screen for the recombination event that occurred.

# Production and testing of alginate beads

## Making alginate beads

In order to optimize the characteristics (component concentration, size, etc.) of our alginate beads to achieve the best water retention possible for the intended application, we created alginate beads using commercially available sodium alginate (ThermoFisher) for various testing purposes.

| Materials: |
| --- |
| Sodium alginate |
| CaCl2 |
| Water |
| Pasteur pipette or bead mold provided by the team |

### Protocol:

Initially, we created the beads as follows:

* Dissolve sodium alginate in water with vigorous stirring to achieve a 1% concentration. Continue stirring until the solution is completely homogeneous.
* Prepare a 0.5 mM CaCl2 solution in a beaker.
* Using a Pasteur pipette, draw up the sodium alginate solution and drop it into the beaker containing the CaCl2 solution. The beads will form immediately.
* Remove the beads using forceps or a small scoop.

Subsequently, we devised a mold to obtain standardized bead sizes, with details provided in the wiki's contribution section. The protocol for using the mold is as follows:

* Prepare solutions of 1% sodium alginate and 1M CaCl2.
* Secure the two parts of the mold using elastic bands or a clamp (preferable).
* Using a micropipette (p1000), draw up 300 µL of 0.5 mM CaCl2 and dispense it into one of the mold's holes.
* Draw up 600 µL of 1% sodium alginate and dispense it into the second hole. Be sure to remove any air bubbles beforehand, as the presence of air may affect bead formation. ⚠ It is essential not to use the same holes for both solutions, as this will prevent bead formation.
* Separate the two mold pieces and retrieve the beads.

## Water retention test by alginate beads

Initially, we pondered on how to demonstrate the water retention capability of alginate beads (alginate 1%; [CaCl2] 1M). Therefore, we devised the following protocol, which we subsequently tested:

* Prepare alginate beads using the dedicated protocol.
* In a Petri dish, add 2 mL of water (condition 1).
* In another Petri dish, add 5 g of soil (condition 2).
* In a third Petri dish, add 5 g of soil + 2 mL of water (condition 3).
* Add 2 g of beads with the same alginate and CaCl2 concentration to each of the Petri dishes.
* Incubate the Petri dishes without lids at 30°C.
* Weigh the Petri dishes every two hours.

Calculation mass of water in the balls:
Allow the beads to dry for several days and record the weight to determine the dry weight of the beads. Subtract the original weight from the dry weight.

## Optimal Concentration Testing of Alginate and CaCl2

We sought to determine the optimal concentrations of alginate and CaCl2 for achieving the best water retention in our beads. To assess water retention, we conducted experiments using beads with varying concentrations of alginate and CaCl2, following this protocol:

* Prepare beads with different concentrations of alginate and CaCl2. We prepared beads at either 0.25% 0.5%, 0.75% or 1% alginate concentration and 0.1M, 0.2M, 0.4M, 0.5M , 0.8M, 1M, 2M, 4M CaCl2 concentration.
* In a Petri dish, add 2 mL of water (condition 1).
* In another Petri dish, add 5 g of soil (condition 2).
* In a third Petri dish, add 5 g of soil + 2 mL of water (condition 3).
* Add 2 g of beads with the same alginate and CaCl2 concentration to each of the Petri dishes.
* Incubate the Petri dishes without lids at 30°C.
* Weigh the Petri dishes every two hours.

The best performing beads will be those that have lost water the slowest.

Calculation mass of water in the balls:
Allow the beads to dry for several days and record the weight to determine the dry weight of the beads. Subtract the original weight from the dry weight.

## Water Retention Tests Based on the Quantity of Beads in Relation to the Amount of Soil

Subsequently, we explored the amount of beads (alginate 1%; [CaCl2] 1M) required in relation to the quantity of soil to achieve optimal water retention. To address this issue, we implemented the following protocol:

* Prepare alginate beads using the dedicated protocol.
* In a Petri dish, add 2 mL of water (condition 1).
* In another Petri dish, add 5 g of soil (condition 2).
* For each of the conditions, conduct tests in multiple Petri dishes with varying amounts of beads. We tested the following quantities: 10%, 20%, 30%, 40%, and 50% (Example: 10% = 5 g of soil + 0.5 g of beads).
* Weigh the Petri dishes every hour for a duration of 4 hours.

From the result in condition 3, substract what you got in condition 1 and 2 to control for the different parameters

# Testing Alginate Production in P. putida:

## Alginate Assay

| Materials: |
| --- |
| LB medium |
| Water |
| Alginate 0.5 mg/mL |
| 96% Ethanol |
| Sulfuric acid |
| Ice |
| *P. putida* cells |

### Sample Preparation for Quantification:

* Start with a bacterial culture in 2 mL of LB (in a Greiner tube) that was inoculated with a single colony and incubated overnight at 30°C.
* Centrifuge the culture at 5,000 rpm for 10 minutes.
* Carefully collect 1 mL of the supernatant and transfer it to a 2 mL Eppendorf tube. Keep this tube on ice.
* Add 1 mL of cold (-20°C) 100% isopropanol to the Eppendorf tube.
* Centrifuge the Eppendorf tube at 14,000 rpm for 30 minutes.
* Discard the supernatant and retain the pellet.
* Resuspend the pellet in 1 mL of distilled water. Use vortexing and sonication if necessary to ensure complete resuspension.

### Preparation of the Reference Range:

* Prepare a stock solution of alginate at 0.5 mg/mL in water. Use vortexing and sonication if necessary to ensure complete dissolution.
* Prepare dilutions as follows:

|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| [Alginate] (µg/mL) | 500 | 400 | 350 | 300 | 250 | 200 | 150 | 100 | 50 |
| Alginate (µg) | 15 | 12 | 10.5 | 9 | 7.5 | 6 | 4.5 | 3 | 1.5 |
| Vi (µL) | 200 | 160.0 | 140.0 | 120.0 | 100.0 | 80.0 | 60.0 | 40.0 | 20.0 |
| VH20 (µL) | 0 | 40.0 | 60.0 | 80.0 | 100.0 | 120.0 | 140.0 | 160.0 | 180.0 |

### Preparation of Solutions:

⚠ To be done in a fume hood:

* Stock Solution:

+ Sulfuric Acid (H2SO4) – Pure
+ Ethanol (96%)
+ Borate Solution: Dissolve 24.74g of H3BO3 in a 4M KOH solution.

* Solutions to Prepare Before Assay:

+ Borate-Sulfuric Acid 0.1M: Dilute the borate solution with pure H2SO4 at a 1/40 ratio (40% for 10 mL, which equals 250 µL).
+ Carbazole 0.1%: Weigh and dissolve the carbazole in ethanol. For 44 wells, prepare 10 mL, which requires 10 mg of carbazole.

### Procedure for Sample Preparation and Measurement:

* Place the 96-well plate on an ice box.
* Add 200 µL of Borate-sulfuric acid 0.1M to each well. Allow it to cool for a few minutes.
* Add 30 µL of the sample to be quantified to each well. Let it cool for a few minutes.
* Add 20 µL of 0.1% carbazole solution to each well. Vortex the carbazole if necessary and perform two pipetting cycles in the wells.
* Place the plate in an incubator at 55°C for 30 minutes. The samples will turn violet during this step.
* After incubation, measure the absorbance of the samples at 530nm using a Tecan plate reader. The reading should be done in two rounds with the Alginate CK program.
* The coloration will remain stable for approximately 2 hours after measurement.

Source: A New Modification of the Carbazole Analysis: Application to Heteropolysaccharides "

## Culture under Water Stress

Since *P. putida* produces alginate as a component of its biofilm during membrane stress, resulting in a mucoid phenotype on agar plates, we aimed to test the appearance of this phenotype under water stress. We cultivated the bacterium in LB medium supplemented with different concentrations of NaCl before quantifying the alginate produced under these conditions.

| Materials: |
| --- |
| LB medium |
| Diluted NaCl solution |
| LB agar medium |
| *P. putida* cells |

### Protocol:

* Inoculate 5 to 6 colonies of *P. putida* in 5 mL of LB.
* Incubate at 30°C with shaking at 180 rpm.
* Measure the OD600nm, at the stationary phase, take 20 µL of the culture and place the drop on an LBa dish plate supplemented or not with NaCl:

+ LB agar
+ LB agar + NaCl 0,1M
+ LB agar + NaCl 0,3M
+ LB agar + NaCl 0,5M

* Streak the liquid drop on the plates and incubate at 30° overnight.
* The next morning, take pictures of the plates.

## Monitoring the growth of P. putida in a glycerol/LB media

| Materials: |
| --- |
| LB medium |
| Glycerol |
| *P. putida* cells |

### Protocol:

* The day before:

+ Make a pre-culture of P. putida: one colony in 10 mL of LB.

* The next day:

+ Inoculate 100 µL of the preculture in 10 mL of the different media: Gly 40%, 20%, 10%, 5% and LB.
+ Place erlens at 30° with agitation.
+ Take the OD every hour until as late as possible in the day. ⚠ Make the blank with the corresponding media
+ Repeat OD the next day and continue measurements if necessary.
+ Make curves with results and calculate generation time.

NB : the medium must be sterile to be sure to detect only the *P. putida*’s growth.

## Building alginate plates

Here is how we built our alginate plates for our collaboration with *Jungle.bio*.

Alginate plate creation protocol:

### Material

* Preferably a box for casting PCR gels (easier to handle), otherwise a rectangular box.
* a 20ml syringe
* 100ml 1% alginate
* 100ml 1M CaCl2

Note: The PCR gel casting box has detachable parts, making it quick and easy to make alginate plates. Prefer this or similar systems.

### Protocol

* Prepare the dish for casting PCR gels.
* Using a syringe, draw up some alginate and place it in a very thin layer on all edges of the dish.
* Then deposit the alginate in the rest of the dish in a regular back-and-forth motion. Don't hesitate to use the syringe (without pouring alginate) to distribute the alginate in any holes.

Note: the layer should be as thin as possible, so don't apply too much alginate. For a 6cm by 13 plate, alginate should be removed 3 times.

Leave to stand for 30 minutes to 1 hour, to allow the alginate to spread into as smooth a surface as possible. During this time, clean the syringe with water. If alginate remains in the syringe, use 96% ethanol.

Draw up CaCl2 and drop into the center of the dish. Once a circle has formed, spread it over the entire dish. Once this is done, add more CaCl2.
The edges of the alginate should shrink and the whole plate will begin to float.

Recover the plate and place in a suitable container with water to prevent drying out. Excess CaCl2 in the dish can be used to make other plates.

P.S: after a week in the open air, the plates start to contain contamination. It's best to store them in a fridge.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Alginate overproduction from bacterias growing on glycerol{% endblock %}
{% block page\_content %}

# Construction of *mucA* mutants

The alginate operon (*algD*) responsible for alginate biosynthesis is transcribed using sigma factor RpoE (σE). When the bacteria finds itself in a favorable environmental condition, it does not need to biosynthesize alginate. Then the anti-sigma factor, MucA, sequesters the RpoE sigma factor at the membrane level. The alginate operon cannot be transcribed. On the other hand, when the bacteria finds itself under stress conditions, such as water stress for example, it will tend to produce more alginate. Thus a regulatory cascade intervenes, allowing the release of RpoE by *mucA*. Thus the sigma factor is released and can allow transcription of the alginate operon.

In the article *Pulcrano, Giovanna et al. The new microbiologica, 2012*, mucoid strains of *P. aeruginosa* (bio synthesizing more alginate than normal) were isolated and sequenced. The researchers noticed that these mucoid strains had one thing in common: the anti-sigma factor was truncated at its C-Terminal domain.

This mutation therefore prevented the sequestration of RpoE while keeping the bacteria alive, because the complete deletion of the *mucA* gene is lethal. Thus, in this condition the bacteria is able to produce alginate constitutively.

## Transformation in *E. coli*

Our idea was therefore to reproduce this mutation in our bacterium *P. putida*. This is an experiment that has never been done but which seemed to us to be the best solution for our bacteria to over-produce alginate. So we created 4 mutants in order to maximize our chances of obtaining one that will produce a high level of alginate. To do this, we attempted to reproduce the same mutants identified in the article by truncating the protein at the same locations identified in the article. We could not create exactly the same ones because the *mucA* gene in *P. aeruginosa* is 585 base pairs while that of *P. putida* is 591 base pairs.

We chose to create the following mutants:

* mucA\*1 → 240 base pairs
* mucA\*2 → 360 base pairs
* mucA\*3 → 399 base pairs
* mucA\*4 → 450 base pairs

Our method was to introduce a stop codon at the place where we wanted the gene to be mutated in order to obtain a truncated protein. For this we used PCR to construct the mutated gene. To allow the wild gene to be replaced by our construction we used homologous recombination. So we did 2 PCRs which introduced a stop codon into the *mucA* gene and which had floating tails homologous to the genome as well as homologous SLIC regions to allow cloning.

We paid close attention to the reading frame, ensuring that the introduction of the stop codon did not shift it. We also took care to keep the RBS of *mucB* so that it could still be translated.

![pcr-diluted-undiluted-dna](https://static.igem.wiki/teams/4582/wiki/pcr-diluted-undiluted-dna.png)

Figure 1: PCR with diluted and undiluted DNA of the different fragments of our construct

![stop inserting strategy](https://static.igem.wiki/teams/4582/wiki/stop-inserting-strategy.png)

Figure 2: Diagram explaining the strategy for inserting the STOP codon into the mucA gene

Once the assembly of our 2 PCR fragments was carried out with the NEBbuilder 2X HiFi DNA assembly Mix Kit, we proceeded to clone the fragment into the suicide plasmid PKNG101.

![cloning slic method](https://static.igem.wiki/teams/4582/wiki/cloning-slic-method.png)

Figure 3: Diagram showing the cloning of our fragment with the SLIC method into the PKNG101 plasmid

Then we transformed competent *E. coli* cc118 λpir strains with this plasmid.

![cloning results](https://static.igem.wiki/teams/4582/wiki/cloning-results-1.png)

![cloning results](https://static.igem.wiki/teams/4582/wiki/cloning-results-2.png)

Figure 4: Cloning results

Then we verified our clones by colony PCR. We tested 6 clones per build because the gel well was too small for the volume used. We repeated 3 clones of each construction on another gel with larger wells to confirm the correct size.
We extracted the plasmids from the 2 clones of each construct that looked good and sent them for sequencing.
Every clones were good except the mucA\*1

![pcr-results-colony](https://static.igem.wiki/teams/4582/wiki/pcr-results-colony.png)

Figure 5: Results of PCR on colonies

## Conjugation in *P. putida*

![conjugation figure 1](https://static.igem.wiki/teams/4582/wiki/conjugation-figure-1.png)

Figure 6: Diagram of the mutator plasmid and homologous recombination. Black arrows represent possible homologous recombination events. Designed with Biorender

Once we had cloned our fragments of interest (the different mutated mucAs) into the suicide plasmid pKNG101 carried by the *E. coli* CC118 strain, we introduced this mutator plasmid into our chassis bacterium *P. putida* by conjugation. Firstly, we brought into contact the donor strain CC118 carrying the mutator plasmid and the mobilizing strain prk2013 then we added the recipient strain *P. putida*. To ensure that we eliminated *E. coli* cells and only selected Pseudomonas strains, we cultured on PIA medium supplemented with Streptomycin. The culture results obtained the next day are presented in the figures below. as negative controls, we cultivated the donor, recipient and mobilizing strains in PIA SM 2000.

![](https://static.igem.wiki/teams/4582/wiki/p-putida-transformants-strains.png)

Figure 7: Selection of the Pseudomonas putida transformants strains in PIA supplemented with Streptomycin.

We consider the clones obtained in the PIA SM 2000 medium as clones having undergone the first recombination event given that they are *P. putida* clones resistant to streptomycin (resistance gene carried by the plasmid pKNG101).

In order to force the 2nd recombination event, we cultivated a few random clones obtained from PIA SM2000 which we isolated on LB medium without NaCl supplemented with 6% sucrose. The colonies obtained were repatched on LB, LB supplemented with Streptomycin, LB without NaCl supplemented with 6% sucrose. The results obtained are presented in the figures below :

![](https://static.igem.wiki/teams/4582/wiki/clone-selection-recombination-1.png)
![](https://static.igem.wiki/teams/4582/wiki/clone-selection-recombination-2.png)

Figure 8: Selection of clones having undergone the 2nd recombination event. The SM sensitive ones are retained.

The clones likely to have undergone the 2nd recombination event are supposed to be sensitive to streptomycin because this would mean that they have excised the mutator plasmid (lethal in the presence of sucrose because of the Levane-sucrase gene carried in the pKNG101 plasmid). Therefore the clones which will have grown on LB and LB ssNaCl 6% sucrose are retained. However, we cannot ensure that the latter have the mucA mutated as we desire, it could be that the 2nd recombination event would have led to the WT phenotype. To screen the mutants, we carried out PCR on colonies using the pairs of primers from table () and the results obtained are presented below :

| The mutant | Forward Primer | Reverse Primer |
| --- | --- | --- |
| *muc*A1 | F1 | R\_SLIC mucB |
| *muc*A2 | F2 | R\_SLIC mucB |
| *muc*A3 | F3 | R\_SLIC mucB |
| *muc*A4 | F4 | R\_SLIC mucB |

![](https://static.igem.wiki/teams/4582/wiki/pcr-colonies-muca-mutants.png)

Figure 9: PCR on colonies of the different mucA mutants to screen the 2nd recombination event

The PCR results on colonies show the amplification of the fragments comprising the truncation point of mucA at different locations (corresponding to mucA1, mucA2, mucA3 and mucA4), but the primers used can just as well hybridize on a wild-type mucA therefore the results obtained do not confirm that our strains have a truncated mucA, for this, it will be necessary to carry out another PCR on colonies using new primers which would hybridize on the regions deleted during the truncation.

## PCR verification

Given that we were unable to confirm the acquisition of mutant strains via PCR solely based on amplicon size, we have devised a novel approach to distinguish strains that have undergone homologous recombination events resulting in the integration of the mutated *mucA* gene from those reverting to a wild-type (wt) phenotype.

To address our issue, since the mutated mucA gene corresponds to a shortened wt *mucA* gene, we contemplated conducting colony PCR under two different conditions based on the choice of primer pairs (Figure 10).

![](https://static.igem.wiki/teams/4582/wiki/schema-genes-primers.jpg)

Figure 10: **Schematic representation of wt and mutant *mucA* genes and primers at their hybridization regions.**The reverse complementary primer (in black) is the same for all matrix under both conditions. The green forward primers correspond to the positive control PCR based on the considered gene, while the red primer is used for the negative control PCR across all matrix. Diagrams were generated using Biorender, with gene schematics from Snapgene.

The so-called positive control PCR corresponds to a colony PCR using primers that anneal to the boundaries of the target mutant gene. The negative control PCR, on the other hand, utilizes primers that anneal to the wt *mucA* gene, where the red forward primer hybridizes to a region unique to the wild-type gene. Thus, confirmation of a mutant strain will be achieved if an amplification of the expected size for the mutant gene is observed in the positive control PCR, and no amplification is detected in the negative control PCR. Both PCR conditions will be conducted on a wild-type strain as a control, and amplification in both conditions is expected, further ruling out the hypothesis that non-amplification in the negative control PCR is due to it not functioning.

![](https://static.igem.wiki/teams/4582/wiki/electrophoresis-2.png)

Figure 11: **Results of colony PCR analyzed by agarose gel electrophoresis (1%).** Colony PCR was performed under both control conditions for a colony of *P. putida* wild-type (WT), colony 4 of the mucA1 strain (mucA14), and colony 4 of the mucA34 strain obtained after homologous recombination. The marker size (bp) is indicated on the left.

Following the colony PCR controls, we only obtained a mutant mucA3 strain of *P. putida* corresponding to colony 4 obtained after homologous recombination. As depicted in Figure 11, amplification is observed in both PCR conditions for the wild-type strain, confirming that the PCR worked. For the mucA34 strain, amplification is only visible in the positive control PCR since those primers anneal to the gene boundaries. Furthermore, the amplicon size matches that of the mutant *mucA4* gene, 450 bp. No amplification is detected in the negative control PCR because the forward primer's hybridization region is only present in the wild-type gene, indicating that the strain has indeed replaced the wild-type *mucA* gene with the mutated *mucA* following homologous recombination.

# Alginate quantification assays

Since we aim for a higher quantity of alginate production by bacteria than the wild-type strains under conditions of membrane stress where alginate is naturally produced (1) and under normal conditions, we have designed *mucA* mutants that affect alginate production with the hope of achieving a substantial quantity.

To address this challenge, we conducted alginate quantification assays and compared the values of the wild-type strains under LB conditions and LB + NaCl hydraulic stress conditions with those of the mutants.

![](https://static.igem.wiki/teams/4582/wiki/alg-per-strain.png)

Figure 12: The concentration of Alginate as a function of the strain

Based on the results presented, it was observed that under LB conditions, there was no detectable alginate production. However, under stress conditions (LB + NaCl), significant alginate production was observed, with values of 17.59 µg/mL, 15.13 µg/mL, and 68.78 µg/mL of alginate for LB + 0.1 M, LB + 0.3 M, and LB + 0.5 M NaCl conditions, respectively. These results suggest a correlation between NaCl concentration and alginate production.

This difference can be explained by the fact that water stress induces a stress response in bacteria, including an increase in alginate production. Bacteria produce alginate as a component of biofilm which allows protection against adverse environmental conditions, aiding in their resistance to stress[[1]](#footnote-1). The mutant mucA34 consistently showed alginate production. The strain produces 37 µg/mL. It appears that this strain likely produces more alginate than the wild-type strain under LB conditions (approximately 30 times more), although its production is lower than that of the wild-type strain under stress conditions.

Indeed, we thought it is normal to observe higher alginate production under stress conditions compared to mutants, as alginate production is a multifactorial process, and the mutants have primarily affected only the regulatory part of this process. The next step will be to integrate *alg8* to influence the metabolic part and optimize polymerization towards alginate production. The goal is to ensure a consistently stable and high alginate production with the mutants. With these modifications, it is possible to achieve relatively stable and high alginate production.

# Growth of *P. putida* on glycerol medium

In order to make our product as competitive as possible, we had the idea of ​​developing a culture medium for our *P. putida* bacteria that was as inexpensive as possible.
Thus, we were able to obtain several liters of 80% raw vegetable glycerin, which are a byproduct from the Saipol company. The composition of this product is:

* 82% glycerol
* 3% ash
* 13% water
* 1.5% sodium
* 0.4% fat

We chose to test this composition as a medium which we have diluted with different amounts of water, in comparison with LB medium and laboratory glycerol.

| Medium | 0h | 1h | 2h | 3h | 4h | 5h | 6h |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5% Saipol glycerol | 0 | 0,04 | 0,09 | 0,18 | 0,19 | 0,17 | 0,17 |
| 10% Saipol glycerol | 0,01 | 0,02 | 0,05 | 0,08 | 0,07 | 0,08 | 0,07 |
| 20% Saipol glycerol | 0,02 | 0,04 | 0,05 | 0,06 | 0,05 | 0,07 | 0,08 |
| 40% Saipol glycerol | 0 | 0,06 | 0,07 | 0,06 | 0,12 | 0,06 | 0,06 |
| 5% Lab glycerol | 0,02 | 0,02 | 0,03 | 0,03 | 0,05 | 0,05 | 0,05 |
| 10% Lab glycerol | 0,03 | 0 | 0 | 0 | 0,01 | 0,01 | 0,01 |
| 20% Lab glycerol | 0,02 | 0,01 | 0 | 0 | 0 | 0 | 0 |
| 40% Labo glycerol | 0,01 | 0,02 | 0,01 | 0,01 | 0,01 | 0 | 0 |
| LB | 0,02 | 0,1 | 0,42 | 1,3 | 3,4 | 4,1 | 4,5 |

![](https://static.igem.wiki/teams/4582/wiki/glycerol-putida-growth.png)

Figure 13: Growth of *P. putida* on glycerol medium

We can notice that the medium with 5% glycerol from Saipol stands out from the others. In fact, this allowed better growth of the bacteria.

![](https://static.igem.wiki/teams/4582/wiki/glycerol-lb-putida-growth.png)

Figure 14: Growth of *P. putida* on glycerol medium compared to growth on LB

On the other hand, in comparison with the growth of *P. putida* in LB medium, the growth in glycerol medium seems insignificant.

But the data obtained must be taken into consideration because there is still growth, although slight, with around 5% Saipol glycerol.

Subsequently, we considered improving this culture medium by providing different carbon sources, amino acids or sugars, always coming from industrial waste, allowing our medium to remain as cheap as possible.

## Sources

1. Chang, W. S., Van De Mortel, M., Nielsen, L., De Guzman, G. N., Li, X., & Halverson, L. J. (2007). Alginate production by *Pseudomonas putida* creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under Water-Limiting conditions. *Journal of Bacteriology, 189(22)*, 8290‑8299.

# Alginate and CaCl2 quantity optimization

We have tried to check how much alginate and CaCl2 would be better to retain the water. This question is significant because eventually we want the cheapest solution achievable so that
we can turn alginate into a commodity. Calcium and alginate won't be free, so we'll want to use the less of them possible for the best result possible.

| raw data | T=0 | T=1h | T=2h | T=3h |
| --- | --- | --- | --- | --- |
| 1% alginate 0.1M CaCl2 | 2 | 1,86 | 1,75 | 1,64 |
| 1% alginate 0.1M CaCl2 | 2 | 1,95 | 1,82 | 1,67 |
| 1% alginate 0.1M CaCl2 | 2 | 1,97 | 1,85 | 1,71 |
| 1% alginate 0.2M CaCl2 | 2 | 2 | 1,89 | 1,78 |
| 1% alginate 0.2M CaCl2 | 2 | 1,81 | 1,71 | 1,57 |
| 1% alginate 0.2M CaCl2 | 2 | 1,92 | 1,79 | 1,64 |
| 1% alginate 0.4M CaCl2 | 2 | 1,86 | 1,77 | 1,64 |
| 1% alginate 0.4M CaCl2 | 2 | 1,9 | 1,8 | 1,68 |
| 1% alginate 0.4M CaCl2 | 2 | 1,9 | 1,75 | 1,61 |
| 1% alginate 0.8M CaCl2 | 2 | 1,97 | 1,89 | 1,77 |
| 1% alginate 0.8M CaCl2 | 2 | 1,89 | 1,77 | 1,64 |
| 1% alginate 0.8M CaCl2 | 2 | 1,97 | 1,85 | 1,72 |
| 0.5% alginate 0.1M CaCl2 | 2 | 1,86 | 1,7 | 1,56 |
| 0.5% alginate 0.1M CaCl2 | 2 | 1,88 | 1,72 | 1,57 |
| 0.5% alginate 0.1M CaCl2 | 2 | 1,92 | 1,75 | 1,6 |
| 0.5% alginate 0.2M CaCl2 | 2 | 1,89 | 1,71 | 1,56 |
| 0.5% alginate 0.2M CaCl2 | 2 | 1,89 | 1,72 | 1,57 |
| 0.5% alginate 0.2M CaCl2 | 2 | 1,92 | 1,75 | 1,62 |
| 0.5% alginate 0.4M CaCl2 | 2 | 1,82 | 1,65 | 1,59 |
| 0.5% alginate 0.4M CaCl2 | 2 | 1,86 | 1,74 | 1,6 |
| 0.5% alginate 0.4M CaCl2 | 2 | 1,88 | 1,77 | 1,68 |
| 0.5% alginate 0.8M CaCl2 | 2 | 1,87 | 1,74 | 1,58 |
| 0.5% alginate 0.8M CaCl2 | 2 | 1,89 | 1,78 | 1,67 |
| 0.5% alginate 0.8M CaCl2 | 2 | 1,88 | 1,81 | 1,7 |

| mean | 0 | 1h | 2h | 3h |
| --- | --- | --- | --- | --- |
| 1% alginate 0.1M CaCl2 | 2 | 1,93 | 1,81 | 1,67 |
| 1% alginate 0.2M CaCl2 | 2 | 1,91 | 1,8 | 1,66 |
| 1% alginate 0.4M CaCl2 | 2 | 1,89 | 1,77 | 1,64 |
| 1% alginate 0.8M CaCl2 | 2 | 1,94 | 1,84 | 1,71 |
| 0.5% alginate 0.1M CaCl2 | 2 | 1,89 | 1,72 | 1,58 |
| 0.5% alginate 0.2M CaCl2 | 2 | 1,9 | 1,73 | 1,58 |
| 0.5% alginate 0.4M CaCl2 | 2 | 1,85 | 1,72 | 1,62 |
| 0.5% alginate 0.8M CaCl2 | 2 | 1,88 | 1,78 | 1,65 |

| standard deviation | 0 | 1h | 2h | 3h |
| --- | --- | --- | --- | --- |
| 1% alginate 0.1M CaCl2 | 0 | 0,06 | 0,05 | 0,04 |
| 1% alginate 0.2M CaCl2 | 0 | 0,10 | 0,09 | 0,11 |
| 1% alginate 0.4M CaCl2 | 0 | 0,02 | 0,03 | 0,04 |
| 1% alginate 0.8M CaCl2 | 0 | 0,05 | 0,06 | 0,07 |
| 0.5% alginate 0.1M CaCl2 | 0 | 0,03 | 0,03 | 0,02 |
| 0.5% alginate 0.2M CaCl2 | 0 | 0,02 | 0,04 | 0,06 |
| 0.5% alginate 0.4M CaCl2 | 0 | 0,03 | 0,06 | 0,05 |
| 0.5% alginate 0.8M CaCl2 | 0 | 0,01 | 0,04 | 0,06 |

![](https://static.igem.wiki/teams/4582/wiki/alginate-cacl2-optimization.png)

Figure 15: Alginate and CaCl2 quantity optimization

As we can see, neither the alginate or the CaCl2 seems to have a significant effect. It's most likely because the protocol is wrong: we should not test how much the soil retain water for a few hours, but rather for a few weeks.

If we can restart these experiences, we may use in the future dedicated hardware such as [Hydrop 2](https://www.metergroup.com/en/meter-environment/products/hyprop-2-soil-moisture-release-curves)

# Project Achievements

During our projects there are some successes:

* We successfully introduced a mutated *mucA* gene in *P. putida*.
* This mutant *P. putida* overproduced alginate compared to the wild type in non-stressed conditions - that was the goal of our project and a big success
* We successfully cultivated *P. putida* on a crude glycerol medium

But also some failures:

* We attempted several *mucA* mutation but only one of them worked
* We did not cultivate our mutant *P. putida* on a glycerol medium. It would have been interesting to quantify the alginate production in this environment.
* In the very last days of our wetlab project, we tried to go one step further by putting mucA wild type in an inducible plasmid to be able to easily modulate and increase the alginate production but unfortunately it did not work. Time constraits prevented us to better analyze this lack of success.

{% endblock %}