iGEM 2020 — Team Nantes

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# Team:Nantes/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Nantes/Engineering

[![](https://static.igem.org/mediawiki/2019/thumb/e/ec/T--Nantes--logo.jpg/661px-T--Nantes--logo.jpg)](https://2020.igem.org/Team%3ANantes)

Team

[Members](https://2020.igem.org/Team%3ANantes/Members)
[Attributions](https://2020.igem.org/Team%3ANantes/Attributions)
[Collaborations](https://2020.igem.org/Team%3ANantes/Collaborations)

Project

[Description](https://2020.igem.org/Team%3ANantes/Description)
[Design](https://2020.igem.org/Team%3ANantes/Design)
[Experiments](https://2020.igem.org/Team%3ANantes/Experiments)
[Safety](https://2020.igem.org/Team%3ANantes/Safety)
[Implementation](https://2020.igem.org/Team%3ANantes/Implementation)

Human Practices

[Integrated](https://2020.igem.org/Team%3ANantes/Human_Practices)
[Education - Engagement](https://2020.igem.org/Team%3ANantes/Education)

Achievements

[Results](https://2020.igem.org/Team%3ANantes/Results)
[Model](https://2020.igem.org/Team%3ANantes/Model)
[Engineering Success](https://2020.igem.org/Team%3ANantes/Engineering)

[Excellence in another area](https://2020.igem.org/Team%3ANantes/Excellence_in_another_area)
[Hardware](https://2020.igem.org/Team%3ANantes/Hardware)
[Contribution](https://2020.igem.org/Team%3ANantes/Contribution)

 [Judging Form](https://igem.org/2020_Judging_Form?team=Nantes)

Engineering Success

**Research**

First of all, our project comes from the realization of a problem that affects our local coasts: **green tides**. News media, global as well as local journals report a recurrent problem with economical and social issues. Deeper investigation reveals that the hazardous phenomenon associated with green tides is due to the apparition of anoxia zones, which induces the switch of bacteria (especially sulfate-reducing bacteria or SRBs) into anoxic metabolism [[1](#ref)]. This leads to the emanation of hydrogen sulfide (H₂S) by the SRBs, which is a dangerous gas.

Two approaches were then proposed :
I) target the green tide phenomena
or
II) target the H2S emanation

Further researches, by consulting scientific resources and data, has allowed us to identify a specificity of these green algae, that could be used as our target: ulvan. It’s the specific polysaccharide and main component of the Ulva cell wall and precursor of H₂S production. The publication [[2](#ref)] on which we based our project presents the degradation of the cell wall by an enzyme group.

**Imagine**

The first step of our project consisted of selecting 3 enzymes to reduce the algal mass. Our first purpose was to pulverize our enzymatic cocktail directly on stranded algae to decompose them. This project was presented to algae experts to review it, especially to [Mrs. Justine Dumay](https://2020.igem.org/Team%3ANantes/Human_Practices) who shed light on ethical and environmental issues with this project. Instead of just degrading the blooms, she suggested valorizing the algae after their collection. Thus, our team began to think about different ways to valorize the algae. Soon, we have oriented our research to optimize H₂S production, which is an economically interesting compound because it can be transformed into sulfuric acid (H₂SO₄), a widely used compound in the chemical industry, especially as a strong acid.

Our scientific method : The DBTL Workflow

As a scientific method, we use the DBTL workflow which can be represented as a cycle. This engineering method is meant to improve our circuit by following a rigorous approach.

![DBTL cycle](https://static.igem.org/mediawiki/2020/6/6e/T--Nantes--DBTL.png)

DBTL cycle, adapted from Waldby [*et al. (2018)*](https://www.researchgate.net/publication/329523466_Synthetic_Biology_in_Australia_an_outlook_to_2030_Australian_Council_of_Learned_Academies_Expert_Working_Group_Horizon_Scanning_Project)

## Design

To reach our goal of transforming H₂S into H₂SO₄, we developed a brand new project based on [our initial one,](https://2020.igem.org/Team%3ANantes/Human_Practices#inttimeline) and this project was what would become the A3 project. The new objective was no longer to solve the problem of green tides but to exploit it. The dangerous gas which was the reason why green tides were so problematic was going to become our strength.
To do this, we decided to stimulate rather than prevent the production of H₂S in the laboratory by introducing a new type of enzymes (in addition to the degradation enzymes) that would allow better access and release of the sulfates during the ulvan degradation. These enzymes are sulfatases and will work in a tandem with bacteria that are present in the natural environment of our algae. Some of the bacteria already present with the algae are called SRBs and are able to use the sulfated sugar of the algae’s cell wall to produce H₂S in anoxic conditions.

## Build

The H₂S production method consists of expressing our enzymes in several plasmids (one enzyme of interest per plasmid) that are transfected in an E. coli chassis. The E. coli BL21 (DE3) strain has been chosen and is widely described as an effective transfected strain.
We selected our enzymes based on the article written by Reisky et al. (2019) which describes the enzymatic cascade degradation of ulvan. Then, we studied our enzymes’ sequences on UniProt and NCBI. After this, we performed the design of our plasmid on SnapGene. We used the Gibson Cloning Assembly technique because this method is already used and well known by one of our supervisors.

For the plasmids, we based the design on the same article [[2](#ref)]. In this publication, they used a pET-type plasmid with an IPTG inducible promoter for the 6 enzymes we have chosen (degradation enzymes and sulfatases) and a plasmid with arabinose inducible promoter for the FGE enzyme.

## Test

First test :

It consists in the verification of the action of the enzymes produced by our E. coli BL21 strain

![](https://static.igem.org/mediawiki/2020/4/4c/T--Nantes--olgaq1.png)

*** FACE: Fluorophore Assisted carbohydrate electrophoresis**

 Principle of the assay: Test of an enzyme cocktail that breaks glycosaminoglycans glycosidic bonds.

The products of enzymatic degradation will be specific to our chosen enzymes.

Electrophoresis allows us to identify the migration of the different disaccharides in several bands.

→ Sugar quantification by fluorescence
→ Informs on the enzyme's action

Second test :

It consists of the verification of the sulfatases action and free sulfate production.

![](https://static.igem.org/mediawiki/2020/e/e5/T--Nantes--logaq2.png)

*** HPAEC-PAD :**
Principle of the assay: Separation of the different sugar by anionic chromatography. Then, we run an analysis of the separated sugars by the PAD detection cell.

→Sugar quantification
→Informs on the sulfatase’s action

Third test:

It consists of the verification of the presence of SO42- ions, by a chemical reaction.

*** Precipitation with BaCl2 :**
Principle of the assay: Identification of SO42- ions by the apparition of a white precipitate of BaSO4.

→SO42- identification

 →Confirms the correct action of enzymes and sulfatases

If the amounts of SO42- - are still too low or null compared to the negative control, these solutions could be considered :

- Changing for enzymes with similar action

- Trying other measurement techniques

## Learn

*- Does it work? If it doesn't, how can it be improved?*
This step consists of conducting troubleshooting our circuit. According to the different information at our disposition, we will proceed by elimination. We will test different conditions for the adjustable parameters (e.g to determine the most optimal temperature, we will repeat the tests with slightly different temperatures than the ones that give the best result). Many variables can be tweaked, and the nature of the issue will give us clues on what could be the cause. We will get back to that in the next part (“improve”).

*- If it works, what results are expected?*
We expect to detect a significant increase in the measurement of SO42- concentration. If it doesn’t work, there will be little to no change relative to the control.

## Improve (redesign)

There can be many different reasons for dysfunction related to our genetic circuitry. Please see **Table 1** for a non-exhaustive list of potential issues concerning our biological constructs and/or hardware and their possible solutio

|  |  |  |
| --- | --- | --- |
| **Potential reasons for the lack of results** | **Potential causes****(Learn)** | **Potential solutions****(for a new cycle of Build-Test-Learn)** |
|  |  | Currently: *E. coli* BL21 (DE3)  For the next DBTL cycle:  * Use an *E. coli* pLysS strain * Use a new resistant strain (e.g. *E. coli* BL21-AI) |
|  | * Increase the concentration of Arabinose (if FGE synthesis is too low) * Increase the concentration of IPTG (if the synthesis of the other enzymes is too low) |
|  | Currently: RBS force: 20k A.U. designed using [Salislab’s RBS Calculator](https://salislab.net/software/)  For the next DBTL cycle:  * Use a stronger promoter (e.g. [BBa\_J64997](http://parts.igem.org/wiki/index.php?title=Part:BBa_J64997)) * Use a stronger RBS (higher than 20k A.U.) |
|  | * Use a pLysS strain * Use a weaker RBS * Use a weaker promoter * Use of a lower copy plasmid |
|  | * Try genomic integration * Use of a strain optimized for genetic stability (e.g. DH5 alpha) |
|  |  | Currently  * Use of a [Codon-Optimization algorithm](http://www.jcat.de/) |
|  | * Non-optimal environment * Low activity * Weak specificity | * Vary the environment (pH, temperature, buffer, etc.) * Test other enzymes with similar target |
| * Presence of residual O2 in the tanks | * Use nitrogen bubbling to eliminate O2 |
|  | * Density of algal mass is too high * Non- homogenous repartition of enzymes or reagents * High level of impurities (sand for example) | * Regular stirring of the medium * Preliminary crushing of algal masses * Addition of a previous purification step for sand removal * Directed mutagenesis to increase the efficiency of the enzyme |
|  | * Elution is not good enough * Eluant effectiveness is too low | * Change the eluent * Increase eluent concentration * Increase the pH value |

**Table 1** Troubleshooting of several potential issues with the system and their respective possible solutions. This list is obviously non-exhaustive as a multitude of parameters have to be taken into account [[3](#ref)].

Sources
[1] Muyzer, G., & Stams, A. J. (2008). The ecology and biotechnology of sulphate-reducing bacteria. Nature reviews microbiology, 6(6), 441-454.

[2] Reisky, L., Prechoux, A., Zühlke, M. K., Bäumgen, M., Robb, C. S., Gerlach, N., ... & Song, T. (2019). A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan. Nature chemical biology, 15(8), 803-812.

[3] Brophy, J. A., & Voigt, C. A. (2014). Principles of genetic circuit design. Nature methods, 11(5), 508-520.

![our mascot olga](https://static.igem.org/mediawiki/2020/7/7e/T--Nantes--olga.png)

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# Team:Nantes/Experiments

[![](https://static.igem.org/mediawiki/2019/thumb/e/ec/T--Nantes--logo.jpg/661px-T--Nantes--logo.jpg)](https://2020.igem.org/Team%3ANantes)

Team

[Members](https://2020.igem.org/Team%3ANantes/Members)
[Attributions](https://2020.igem.org/Team%3ANantes/Attributions)
[Collaborations](https://2020.igem.org/Team%3ANantes/Collaborations)

Project

[Description](https://2020.igem.org/Team%3ANantes/Description)
[Design](https://2020.igem.org/Team%3ANantes/Design)
[Experiments](https://2020.igem.org/Team%3ANantes/Experiments)
[Safety](https://2020.igem.org/Team%3ANantes/Safety)
[Implementation](https://2020.igem.org/Team%3ANantes/Implementation)

Human Practices

[Integrated](https://2020.igem.org/Team%3ANantes/Human_Practices)
[Education - Engagement](https://2020.igem.org/Team%3ANantes/Education)

Achievements

[Results](https://2020.igem.org/Team%3ANantes/Results)
[Model](https://2020.igem.org/Team%3ANantes/Model)
[Engineering Success](https://2020.igem.org/Team%3ANantes/Engineering)

[Excellence in another area](https://2020.igem.org/Team%3ANantes/Excellence_in_another_area)
[Hardware](https://2020.igem.org/Team%3ANantes/Hardware)
[Contribution](https://2020.igem.org/Team%3ANantes/Contribution)

 [Judging Form](https://igem.org/2020_Judging_Form?team=Nantes)

Experiments

[Gibson Assembly Cloning Protocol](#gibson)
[Mini preparation of DNA](#dna)
[XL1 Blue cells and E.coli](#xl1ecoli)
[Expression Protocol and Dialysis](#expression)
[Chemical Lysis BugBuster (Merck)](#lysis)
[Purification Control](#purification)
[FACE Protocol](#face)
[HPAEC-PAD Protocol](#hpaec)
[Claus Protocol](#claus)
[Contact Process](#contact)

Gibson Assembly Cloning Protocol

**Cloning our genes of interest from cloning vectors (pUC) to expression vectors (pET11)**
Source : [New England BioLabs](https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510)

|  |  |
| --- | --- |
| Reagents:   * Agarose gel * Restriction enzymes: NdeI et BamHI * Gibson Cloning Master Mix:  + T5 Exonuclease + Phusion DNA Polymerase + Taq DNA Ligase | Equipment:  * PCR tubes * Thermal cyler * Electrophoresis * Ice |

**Design**

1. Design your plasmid and order primers.
2. Adjacent segments should have identical sequences on the ends. These identical sequences can be created via PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence.
   Avoid strong secondary structures in the homology region.
You can find the details in the [Design](https://2020.igem.org/Team%3ANantes/Design)  Section on our wiki.

**Protocol**3. Generate DNA segments by PCR
4. Run PCR product on an agarose gel
5. Purify DNA segments (Monarch® DNA Cleanup Kit - by NewEnglands BioLabs)
6. Set up the following reaction on ice:

|  |  |  |
| --- | --- | --- |
| **Reagents** | **2-3 Fragment Assembly** | **Positive control** |
| Total Amount of Fragments | 0.02 - 0.5 pmols   X µL | 10µL |
| Gibson Assembly Master Mix (2X) | 10µL | 10µL |
| Deionized H20 | 10-XµL | 0 |
| Total Volume | 20µL | 20µL |

7. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
8. Transform the DNA into bacteria with 2 µL of the assembly reaction (XL1 Blue *E. coli* cells).
9. For Minipreparation of DNA see below
10. Screen for the correct plasmid product by restriction digest (NdeI and BamHI).
11. Sequence the PCR product of your final plasmid.

Mini preparation of DNA

**Minipreparation of DNA : Extraction of DNA from transformed XL1-Blue cells**
Source : McKnight Brain Institute - University of Florida

|  |  |
| --- | --- |
| Reagents:   * XL1-Blue *E. coli* cells * Antibiotic * LB medium * Solution A : 25mM Tris-HCl, pH 8.0, 10mM EDTA * RNAse A * Lysis Buffer : 50 µL 400mM NaOH, 50 uL 2% SDS * Solution K (neutralization buffer) : 5M potassium acetate, pH 5.5 * Isopropanol * 70% ethanol | Equipment:  * Falcon Tubes * Microcentrifuge tube |

1. Inoculate 3 mL LB + ampicillin medium with XL1-Blue cells, culture with vigorous shaking at 37°C for 14-16 hrs.
2. Aliquot 2 x 750 µL culture into a 1.5 mL microcentrifuge tube, harvest the bacteria by spinning at 12000rpm for 1 min. Aspirate supernatant and keep pellet.
3. Resuspend bacterial pellet by complete vortexing in 100 µL Solution A resuspension buffer.
To prepare the working solution A, add 3uL of RNAse A to 1 mL solution A and mix well. The bacteria should be completely resuspended - no clumps should be visible.4. Add 100 µL freshly prepared lysis buffer and mix gently by inverting 5-6 times at room temperature. *Do not allow the lysis to occur for longer than 5 min.*
5. Add 120 µL of solution K and mix gently by inverting 5-6 times.
Incubate the solution at room temperature for 3 min. *The mixture should contain flocculent white precipitate at this point that consists of bacterial DNA and protein*6. Remove bacterial/protein debris by centrifugation at 12000rpm for 15 min. Transfer the supernatant to a fresh microcentrifuge tube.
7. Add 200 µL isopropanol to precipitate plasmid DNA from supernatant.
Mix thoroughly by inverting 10 times.
Incubate at room temperature for 1 min.8. Collect DNA by centrifugation at 14000 rpm for 1 min.
   Immediately pour off isopropanol into beaker, shaking tube after the pour to remove a bit of the excess isopropanol.
9. Add 500 µL 70% ethanol to the tube, mix by inverting or brief vortexing. Re-spin the DNA down at 14000 rpm (full speed) for 1 min.
10. Respin for 1 min (immediately pour off ethanol).
Take off any excess ethanol.
Air-dry DNA for 10 min with caps open on the lab bench.11. Dissolve DNA in 20-35 µL water (volume used depending on whether you saw pellets). *The final concentration of DNA should be 0.5 – 2.0 μg /μL and the OD**260/280* *value should be above 1.8.*

XL1 Blue cells and *E.coli* BL21 (DE3) Transformation Protocol

**Transformation of XL1 blue cells and then, E.coli BL21 (DE3) competent cells with pEVOL and pET plasmids or pET plasmid only**
Source : UFIP (UMR 6286) Nantes

|  |  |
| --- | --- |
| Reagents:   * Culture of XL1 Blue cells or *E.coli* BL21 (DE3) competent cells * LB medium * DNA (pEVOL and pET plasmids) * Ampicillin * Chloramphenicol | Equipment:  * Ice * LB medium plate |

- Add pEVOL and pET or pET plasmids only to the ***E.coli*** BL21 competent cells : 2 µL per 100 µL tube.

**OR**

Add pEVOL or pET plasmids to the **XL1 Blue competent cells**: 2 µL per 100 µL tube.

1. Incubate  30 min at 4°C (in ice)
2. Heat-shock: Incubate 45 sec at 42°C.
3. Put back on ice for 2 min.
4. Add 0.5mL of LB medium.
5. Incubate 1h at 37°C under shaking with the tube in horizontal position.

- Spread out 100 µL of the culture in a LB  + Ampicillin (pET alone) or Ampicillin and Chloramphenicol (pET and pEVOL) plate.

**OR**

Spread out 100 µL of the culture in a LB  + Ampicillin (pET) or Chloramphenicol (pEVOL) plate.

6. Centrifugation of the rest of the culture 4 min at 4000 rpm.
7. Resuspend the pellet in 100 µL of supernatant.

- Spread on a LB + Ampicillin or Ampicillin and Chloramphenicol plate.

**OR**

Spread on a LB + Ampicillin or Chloramphenicol plate.

8. Incubate O/N at 37°C.

Expression Protocol and Dialysis

**Expression of our protein in E. coli BL21 (DE3)**
Source : Reisky and al, 2019

|  |  |
| --- | --- |
| Reagents:   * Culture of *E.coli* BL21 (DE3) transformed cells * LB medium * Ampicillin and Chloramphenicol * IPTG * Arabinose * TB medium * HEPES buffer * NaCl | Equipment:  * Ice * LB medium plate |

**Degradation enzymes**

**Co-expression of FGE and Sulfatases**

1. Harvest cells by centrifugation at 10 000g at 4°C for 20min and store at -20°C.
2. Resuspend the pellet with  500µL 50nM HEPES and 100mM NaCl (pH 7,4).
3. For Chemical lysis Protocol see below.
4. Remove cellular debris by centrifugation at 13 000g à 4°C for 10min.
5. Dialysis

1. Put phosphate buffer (PBS) in a beaker.
2. Dialyze for 2h to 3h  at room temperature.
3. Change the buffer.
4. Dialyse O/N à 4°C.

6. For Purification Protocol see below.

Chemical Lysis BugBuster (Merck)

**Extraction for obtaining the soluble fraction**
Source : Merck

|  |  |
| --- | --- |
| Reagents:   * Transformed *E. coli* BL21 (DE3) cells * BugBuster 10X Protein Extraction Reagent kit  + BugBUster reagent + Benzonase®Nuclease : 1 µL + rLysozyme™ Solution + rLysozyme Dilution Buffer : 100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton® X-100, pH 7.5  * SDS Sample Buffer (Cat. No. 70607-3) * Electrophoresis gel | Equipment:  * Ice * Electrophoresis * 1.5 mL tubes |

1. Harvest cells from liquid culture by centrifugation at 10 000g for 10 min. For small scale extractions (1.5 mL or less), centrifugation can be performed in a 1.5-mL tube at 14000-16000g. Decant and allow the pellet to drain, removing as much liquid as possible. Determine the wet weight of the pellet.

*Note: If using BugBuster 10X Protein Extraction Reagent, dilute to 1X in desired buffer.*

2. Resuspend the cell pellet in room temperature BugBuster reagent by pipetting or gentle vortexing, using 5 mL reagent/ g of wet cell paste
3. To reduce viscosity of the lysate, add 1 μL (25 units) Benzonase®Nuclease/ 1 mL BugBuster reagent (125 Units/gram cell paste) used for resuspension.
4. To improve protein extraction efficiency in non-pLysS and -pLysE hosts, add 1 KU rLysozyme™ Solution per 1 mL BugBuster reagent (5 KU/gram cell paste). rLysoyzme Solution can be diluted using rLysozyme Dilution Buffer (100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton® X-100, pH 7.5). Dilutions should be stored on ice or at 4°C.

*Experiments are performed on ice.*

5. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10-20 min at room temperature.
6. Remove insoluble cell debris by centrifugation at 16.000g for 20 min at 4°C.
7. Transfer the supernatant to a fresh tube.
8. For SDS-PAGE analysis, remove a small sample (25-50 μL) and combine with equal volume of 4X SDS Sample Buffer. Immediately heat for 3 min at 85°C to denature proteins and then store at -20°C until SDS-PAGE analysis.
9. For Purification Protocol see below.

Purification Protocol

**Purification of protein with a Nickel-NTA (NiNTA) column**

Source : UFIP (UMR 6286) Nantes

|  |  |
| --- | --- |
| Reagents:   * NiNTA beads * Cold lysis buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 10mM * Washing buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 20mM * Elution buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 250mM * Bradford reagent * Loading buffer * β-mercaptoethanol * SDS-PAGE 10% | Equipment:  * Ice * Column * Electrophoresis |

1. Add NiNTA beads previously rinsed with the cell lysate and incubate about 1h at 4°C on a wheel.
2. Load the mix on a column. Collect the unbound fraction and keep at 4°C.
3. Wash the column 3 times with 5 to 10 volumes of washing buffer  (NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 20mM) solution per gel volume  by rinsing the sides of the column and then with 10 mL of washing buffer.
Keep an aliquot of the washing step to load on the SDS PAGE.4. Elute the protein with 4 times 1 mL of elution buffer (NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 250mM) and collect 4 fractions of 1 mL.
5. Perform a protein assay on the different elution fractions with the Bradford reagent :

1. 10µL of elution fraction + 790µL water + 200µL of Bradford reagent.

6. Read OD595nm and Compare with a standard curve of BSA.
7. Load 15µL of purified sample (+5µL of loading buffer + β-mercaptoethanol) on a 1% SDS PAGE.

FACE (Fluorophore Assisted Carbohydrate Electrophoresis) Protocol

**Polysaccharide detection following the degradation of ulvan.**

Source :[MyBioSource](https://www.mybiosource.com/learn/testing-procedures/fluorophore-assisted-carbohydrate-electrophoresis-glycosaminoglycans/)

|  |  |
| --- | --- |
| Reagents:   * 100 mM Ammonium Acetate, pH 7.0 * Tris-Acetate Gel Solution * AMAC (2-aminoacridone) Solution * 10x Proteinase K (PK) * Hyaluronidase SD * Chondroitinase ABC * Heparinase (Flavobacterium heparinum) * Heparitinase I (Flavobacterium heparinum) * Heparitinase II (Flavobacterium heparinum) * 1X TBE (electrophoresis buffer) * Ammonium Persulfate (10 g) * TEMED | Equipment:  * Electrophoresis |

**Protocol**

1. **Proteinase K Digestion**:

1. Tissues: Add 250 μl 10X PK per 100 mg wet weight of tissue. Incubate the samples at 60°C for 2 hours or until the tissue is digested. For tissue samples, vortex the samples every 30 min until the tissue is completely dissociated.

2. **First Ethanol Precipitation**:

1. Add 4 volumes of pre-chilled (-20°C) absolute ethanol to each sample and proceed to the next step or incubate at -20°C O/N.
2. Next day, centrifuge samples at 14000g for 10 min at RT.
3. Discard the supernatant by dumping it into a beaker.
4. Wash pellet by adding 4 volumes of pre-chilled (-20°C) 75% EtOH and vortex.
5. Centrifuge samples at 14,000 g for 10 minutes as before.
6. Discard wash as before, but this time you will need to remove residual ethanol with a pipette.
7. Air-dry the pellet at RT for 20 minutes (do not use a vacuum and do not over dry; this will make the pellet more difficult to re-dissolve).
8. Add 35 μL 100 mM ammonium acetate to each sample. Vortex/Spin.
9. Incubate at RT for 20 minutes. Vortex/Spin.
10. Heat inactivate the PK by incubating the samples at 1000 C for 5 min, then chill them on ice for 5 min.

3. **Digestion of Chondroitin Sulfate and Hyaluronan:**

1. Add enzymes of interest 1 μL of chondroitinase ABC and/or 1μL hyaluronidase SD to each 35 μL sample.
2. Vortex/Spin.
3. Incubate at 37°C O/N (at least 18 hrs).

4. **Second Ethanol Precipitation:**

1. Add 160 μL of pre-chilled (–20°C) 200 proof ethanol to each sample.
2. Vortex/Spin.
3. Proceed to next step or incubate O/N at –20°C.
4. Centrifuge samples at 14,000 g for 10 minutes at RT.
5. Save supernatant in a separate 1.5 mL tube
6. Wash the pellet with 100 μL of pre-chilled (-20°C) 75% ethanol and centrifuge as before.
7. Pool this wash with the previous supernatant for each sample.
8. Proceed to step 7 if uninterested in heparan sulfate analysis. Air-dry the pellet for 20 min at RT.
9. Resuspend the pellet in 20 μL of 100 mM ammonium acetate and incubate at RT for 20 min. Vortex/spin the pellet.
10. Heat inactivate the enzymes by incubating the samples at 100o C for 5 min, then chill them on ice for 5 min. Then, proceed to step 5. For the supernatants, lyophilize them (using a “Speed-Vac” or equivalent) and proceed to step 7.

5. **Digestion of Heparan Sulfate**:

1. Mix equal portions of Heparinase, Heparitinase I and Heparitinase II together and add 1 μL of this mixture to each 20 μL sample.
2. Incubate at 37°C O/N.

6. **Third Ethanol Precipitation**:

1. Add 80 μL of pre-chilled (–20°C) 200 proof ethanol to each sample. Vortex/Spin.
2. Proceed to the next step or incubate overnight at -20°C.
3. Centrifuge samples at 14000g for 10 min at RT.
4. Save supernatant in a separate 1.5 mL tube.
5. Wash the pellet with 100 μL of pre-chilled (-20°C) 75% ethanol and centrifuge as before.
6. Pool this wash with the previous supernatant for each sample.
7. Air dry the pellet for 20 min at RT. Resuspend the pellet in 20 μL of 100 mM ammonium acetate and incubate at RT for 20 min.
8. Vortex/spin the pellet.
9. Heat inactivate the enzymes by incubating the samples at 100°C for 5 min, then chill them on ice for 5 min. If desired, DNA levels can be measured from this resuspended pellet.

7. **Labeling with AMAC: Lyophilize supernatants**

1. Add 1 μL of the AMAC solution per cm2 of culturing area (for both cell layer and conditioned media),
2. Add 1 μL of the AMAC solution for every 2.5 mg of tissue wet weight.
3. Add less AMAC to dilute down later since concentration is not an option.
4. Incubate the sample at 37°C for 18 hrs in the dark.

**Gel preparation**

1. **Gel Casting** :

1. Transfer the amount of gel solution you need to a clean tube
2. You will need about 5 mL/gel for a gel with 0.75 mm spacers
3. For each 5 mL, add 25 μL ammonium persulfate (10%) and 5 μL TEMED
4. Mix by inversion and add to your pre-assembled casting plates.

2. **Chilling the Gels**:

1. Place the gels you just cast into their gel apparatus.
2. Add electrophoresis buffer to the inner/outer chamber
3. Place the gel box in a secondary container.
4. Surround the gel apparatus with ice up to the top of the apparatus.
5. Pour water into the ice to create a slurry until the water level reaches about halfway up the gel apparatus.
6. Cool to 4°C before running the gel.

3. **Loading the Samples:**

1. Add 2-5 μL of sample per lane.
2. Add them directly to the well.
3. Taking the ratio of the IOD of the sample disaccharides to the standard disaccharides will enable an estimation of the concentration of disaccharides in your samples.
4. Add an AMAC “blank” to one of your wells.

4. **Electrophoresis**

1. Run the gel at 500 V constant voltage.

HPAEC-PAD Protocol

**Characterization of the ulvan chemical composition**
Source :  [Elsevier Ltd (2020).](https://www.sciencedirect.com/science/article/pii/S0144861720300266) Composition and structure of cell wall ulvans recovered from Ulva spp. along the Swedish west coast. *[Carbohydrate Polymers,](https://www.sciencedirect.com/science/journal/01448617)* Volume 233, 1 April 2020, 115852.

|  |  |
| --- | --- |
| Reagents:   * 5 mM Tris-HCl * 0.1M NaCl * 0.5 mM CaCl2 * Eluent : NaOH * Standard solution : Rhamnose, xylose, glucuronic acid and iduronic acid * TFA | Equipment:  * HPAEC-PAD : ICS-3000 Dionex * CarboPac PA1 column : 4 x 250 mm |

**HPAEC-PAD**

1. The eluent was pumped at 1.5 mL/min using a gradient program starting with 0.10 M NaOH and increasing to 0.16 M NaOH and 0.19 M sodium acetate during the run.
2. A mixture of arabinose, rhamnose, galactose, glucose, xylose, mannose, glucuronic acid and iduronic acid was used as the reference.
3. The data were processed and analyzed.

**Sulfate content**

1. Sulfate groups on the ulvan backbones were released by hydrolysis in 2M Trifluoroacetic acid (TFA) at 3.3−4 mg/mL for 3h at 100 °C.
2. Released sulfate was measured by a turbidimetric sulfate assay kit (product number MAK132, Sigma-Aldrich) with 0.2 M TFA in the standard curve to fit the dilution of samples.
3. Absorbances at 600 nm were measured with a SPECTROstarNano plate reader (BMG LABTECH).
4. Free sulfate in the solid material was determined by dissolving 3.3−4 mg ulvan in 1 mL Milli-Q water and heating as for samples, 3h at 100 °C.
5. Aliquots of the samples were centrifuged at 20 000g for 10 min and sulfate was analyzed on the supernatants by the Sigma kit above (standards without TFA).
6. The degree of sulfation was calculated by adjusting the sulfate determined after TFA hydrolysis with the free sulfate level.

Claus Protocol

**Oxidation of H2S to SO2**
Source : [ScienceDirect](https://www.sciencedirect.com/topics/engineering/claus-process)- Claus Process

|  |  |
| --- | --- |
| Reagents:   * H2S * O2 from the air | Equipment:  * Burner * Reactor * Boiler * Condenser |

**Thermal step :**

1. Adjust the reactor at a pressure between 130 and 180 kPa and Temperature between 975 and 1300°C.
2. O2 from the air in the reactor in stoichiometric quantities.
3. Inject H2S in the reactor.
4. A third of the gas is burned to produce SO2.
5. Cooling of the gas in the boiler up to 250°C.
6. Sulfur condensation in liquid forms in the condenser.
7. Ejection of the water vapor created.

Contact Process

**Production of liquid sulfuric acid**
Source : [L’Elémentarium](https://www.lelementarium.fr/product/acide-sulfurique/) - Sulfuric Acid

|  |  |
| --- | --- |
| Reagents:   * SO2 * Vanadium pentoxide : V2O5 * Water | Equipment:  * Purification unit * Converter consisting of an alumina support (4 catalytic beds) |

1. Purifying the sulfur dioxide in a purification unit.
2. SO2 is washed with water and dried by sulfuric acid.
3. Adding an excess of oxygen to [sulfur dioxide](https://en.wikipedia.org/wiki/Sulfur_dioxide) in the presence of the catalyst [vanadium pentoxide](https://en.wikipedia.org/wiki/Vanadium%28V%29_oxide) at 440 °C and 100-200 kPa in the converter.
4. The hot [sulfur trioxide](https://en.wikipedia.org/wiki/Sulfur_trioxide) formed is dissolved in concentrated H2SO4 in the absorption tower to form oleum.

H2SO4 (l) + SO3 (g) → H2S2O7 (l)

1. The oleum is then added to water to form sulfuric acid about 98% concentrated.

H2S2O7 (l) + H2O(l) → 2 H2SO4 (l)

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# Team:Nantes/Results

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Team

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Project

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Achievements

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