iGEM 2021 — 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)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:Nantes/Engineering

### ★ ALERT!

This page is used by the judges to evaluate your team for the [medal criterion](https://2021.igem.org/Judging/Medals) or  [award listed below](https://2021.igem.org/Judging/Awards).

Delete this box in order to be evaluated for this medal criterion and/or award. See more information at  [Instructions for Pages for awards](https://2021.igem.org/Judging/Pages_for_Awards).

# Engineering Success

### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

Please see the [2021 Medals Page](https://2021.igem.org/Judging/Medals) for more information.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

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# Team:Nantes/Experiments

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* [H2S](https://2021.igem.org/wiki/index.php?title=Team:Nantes/h2s)

* [Overview HP](https://2021.igem.org/wiki/index.php?title=Team:Nantes/overviewHP)
* [Integrated HP](https://2021.igem.org/wiki/index.php?title=Team:Nantes/integratedHP)
* [Implementation](https://2021.igem.org/wiki/index.php?title=Team:Nantes/impementation)
* [Education and Public Engagement](https://2021.igem.org/wiki/index.php?title=Team:Nantes/educAndPublicEngagment)

* [Members](https://2021.igem.org/wiki/index.php?title=Team:Nantes/members)
* [Attribution](https://2021.igem.org/wiki/index.php?title=Team:Nantes/attribution)
* [Collaboration](https://2021.igem.org/wiki/index.php?title=Team:Nantes/collaboration)

## Summary

* [Gibson Assembly](#ilk1)
* [Miniprep ADN](#ilk2)
* [Transformation of E.coli bacterias](#ilk3)
* [Expression and dialysis](#ilk4)
* [BugBuster Protein Extraction](#ilk5)
* [FACE](#ilk7)
* [HPAEC-PAD](#ilk8)
* [Migration Gel](#ilk9)
* [PCR](#ilk10)
* [Electrophoresis](#ilk11)
* [Mediums Experiments](#ilk12)

# I- Experiments and protocols

![](https://static.igem.org/mediawiki/2021/4/46/T--Nantes--gibson.png)

## I- Gibson Assembly

### Goal : To slot our genes of interest into pEVOL and pET11 plasmids

Equipment :

* Thermocycle
* P10 micropipet
* P2 micropipet
* Ice
* PCR microtubes

Reagents :

* Plasmids/Vectors:

+ pET11
+ pEVOL

* DNA template containing genes encoding enzymes/Inserts:

+ Degradation enzymes (P30\_PL28, P10\_Plnc, et P31\_GH39)
+ Sulfatases (P18\_S1\_7, P32\_S1\_8, et P36\_S1\_25)
+ FGE

* Gibson Assembly master mix (2X)

Before manipulate, make sure you have :

3 time more inserts (FGE for pEVOL // P10, P18, P30, P31 and P32 for pET11)
than vectors (pEVOL and pER11)
We need to have in 5µL, 25-50ng of vectors and 10-300ng of inserts. Moreover, we need to have equimolar ratios

### To calculate :

pmol of DNA : (ng of DNA / (660 \* number of bases))\*1000
ng of DNA : (pmol of DNA \* (660 \* number of bases))/1000

### Operating mode:

All these steps must be done in ice

- Make the mix of DNA inserts and vector equimolar ratios (final volume=5µL) in pcr tube
- Vortex the 2 times the mix and add 5µL to the DNA preparation
- Up and down the mix
- Launch the PCR cycle called GIBSON (1hour at 50°C)
- Store the reaction at -20°C

## II- Miniprep ADN

Miniprep ADN (Inaara) FICHE FOURNIE PAR PROMEGA, sauf a la fin on fait l’élution en 2 fois pour récupérer un max de produit et on le fait dans le Free nucléase water (kit Wizard) et on la chauffe a 62°C pour qu’elle soit moins visqueuse

![](https://static.igem.org/mediawiki/2021/0/08/T--Nantes--miniprep.png)

Goal : Isolation and purification of our plasmids containing our genes of interest in our E coli BL21 expression strains

### Operating mode:

Everything is done with gloves (to avoid DNase) and in a cold block

Equipment:

* Promega Kit for DNA extraction
* Centrifugal machine
* Bacteria containing the vectors : pEVOL + pET11
* Bacteria containing our genes : pUC

Reagents:

* Ethanol
* Lysis Buffer
* Endotoxin Removal Wash (ERB)
* Column Wash Solution (CWC)
* Elution Buffer

## Preparation

### Step 1 : Prepare Lysate :

1. Add 600μl of bacterial culture to a 1.5ml microcentrifuge tube.
Note: For higher yields and purity use the alternative protocol below to harvest and process up to 3ml of bacterial culture.
2. Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.
3. Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.
4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
5. Transfer the supernatant (~900μl) to a PureYieldTM Minicolumn without disturbing the cell debris pellet.
6. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
7. Discard the flowthrough, and place the minicolumn into the same Collection Tube.

### Step 2 : Wash :

8. Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds.
9. Add 400μl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

### Step 3 : Elute :

10. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl of Elution Buffer or nuclease-free water directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
11. Centrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.

### Bibliographical references :

NucleoSpin® Plasmid Transfection-grad, PureYield Plasmid Miniprep System

## III-Transformation of E.coli bacterias

![](https://static.igem.org/mediawiki/2021/5/54/T--Nantes--eColi.png)

Goal : Transform our E.coli BL21 with our recombinant plasmids

Equipment:

* 500 mL erlenmeyer
* Eppendorfs tubes
* Incubator agitation
* Spectrophotometer
* Centrifuge
* Ice

Reagents:

* Petri dishes with antibiotic (ampicillin and chloramphenicol)
* DH5α
* TSS solution

## Operating mode :

### 1st step : Preculture

-Inoculate 5 mL of LB into a closed 50 mL Eppendorf tube with an isolated colony
-Incubate overnight at 37°C with agitation at 230 rpm

### 2nd step : Transformation

-Inoculate 10 mL of LB with 500 µL of preculture in 50 mL erlenmeyer
-Incubate at 37°C with agitation at 230 rpm
-Monitor bacterial growth by measuring OD=600nm every 30 min
-Stop the culture when the OD is between 0.45 and 0.5
-Place the tubes on ice for 10 min
-Aliquot the culture by 1 mL into the tubes placed in ice
-Centrifuge for 5 min at 4000 g at 4°C\*
-Dispose of the supernatant in a liquid bacterial waste bin
-Resuspend the pellets with 100 µL of cold TSS at 4°C
-Store tubes in the freezer at -80°C

### 3rd step : Testing the competence of cells

-In a cold tube put 100 µL of competent cells with 2 µL of Gibson
-Incubate 30 min in ice
-Heat choc at 42°C for 45 secs
-Put 2 min on ice
-Add 900µL of LB
-Incubate 1h at 37°C at 230 rpm
-Centrifuge 5 min at 3000 g
-Remove the supernatant and resuspend the pellet with 100 ul of this supernatant
-Spread 100 ul of this solution on a Petri dish containing LB agar and the associated antibiotic
-Incubate overnight at 37°C with agitation at 230 rpm

### 4th step : Analysing colonies

-Extract plasmids from bacterias DH5α by miniPrep
-Digest a part of plasmids with our restriction enzymes (Nde1 and BamH1)
-Make an electrophoresis
-Make sure you can see both plasmids and inserts
-Send the other part of plasmid to sequencing
-Make sure you don’t have any mutation
-Insert recombinant plasmids into E.coli BL21

### Bibliographical references :

Gibson Assembly Hifi 1 step kit Manual

## IV- Expression et dialyse

![](https://static.igem.org/mediawiki/2021/b/b3/T--Nantes--expresso.png)

**Goal : Check the activity of the enzymes obtained and remove contaminants**

|  |  |
| --- | --- |
| **Equipment:** | Microtube 3mL   Dialysis membrane |
| **Reagents:** | Transformed E.coli BL21 DE3   Ampicillin (plasmid pET11)   LB medium (for 1L: Bactotryptone (10g), yeast extract (5g), NaCl (10g), agarose (15g)   IPTG   TB medium   HEPES   NaCl   L-arabinose   Phosphate buffer (PBS), 100mM, ph=7.6 for dialysis) |

### Step 1 : Expression

Controls :
Positive: T7 Promoter plasmid + GFP gene
Negative: Prom T7 plasmid without GFP gene= empty

Preparation of bacterial transformation E coli BL 21
E. coli BL21 (DE3) was transformed with plasmid pET11 (degradation enzymes: Plnc + GH39 + PL28
E. coli BL21 (DE3) was transformed with plasmid pET11 (sulfatases: P18 + P32 + P36 +FGE) + 0.5 mM IPTG
1mL ZYP-5052 or TB? with 100µg.mL-1 Ampicillin were inoculated from a O/N culture

Day 1:
-> If colonies are not isolated, subculture 3 colonies on liquid and solid LB medium then let grow over-night.
1st step : Put the culture at 30°C and 180 rpm until OD600nm = 1
2nd step : Place at 20°C for 48h
1st step : Incubation at 37°C at 180 rpm until OD600nm= 0.3 - 0.5
2nd step : Addition of 1.5mM L-Arabinose
3rd step : Incubation 90 min at 37°C
4th step : Put the culture 2h at 18°C
Step 5: Addition of 0.5 mM IPTG and incubation O/N at 18°C
Day 4: Addition of 0.5 mM IPTG and incubation O/N at 18°C
Step 2 : Cell lysis :
See protocol V Bug Buster.
Step 3 : Dialysis
1st step: Put the supernatant in the dialysis membrane.
2nd step: Put 1 ml of 100 mM Phosphate Buffer (PBS), pH 7.6 in a 3mL microtube
3rd step: Dialysis for 2 to 3h at room temperature.
4th step: Change the phosphate buffer and dialyze overnight at 4°C.
5th step: Collect the supernatant in the dialysis membrane.

### Bibliographical references:

"A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan” -  **Lukas Reisky, Aurélie Préchoux.**
[Article link](https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/dialysis-methods-protein-research.html)

## V-BugBuster Protein Extraction

![](https://static.igem.org/mediawiki/2021/4/42/T--Nantes--blockbuster.png)

|  |  |
| --- | --- |
| Equipment: | Tubes of 1,5 mL   Ice |
| Reagents: | Bacterial culture   - 10X BugBuster® kit:   10X BugBuster reagent   Benzonase®Nuclease   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   4X SDS Sample Buffer   SDS-PAGE |

### Operating mode:

**1st step: Bacterial extraction**
-Weigh the tube
-For tubes >1,5ml : Centrifuge bacterial culture at 10 000 g during 10 min
-For tubes ≤1,5ml : Centrifuge bacterial culture at 14 000 g during 10 min
-Withdraw supernatant
-Weigh bacterial pellet

**2nd step: bacterial lysis**
Use 5g of BugBuster reagent for each gram of pellet
-Dilute 10X Bugbuster at 1/10
-Resuspend pellet into diluted Bugbuster by pipetting

**3rd step: Reduce lysate viscosity**
-Add 1µL Benzonase®Nuclease for each mL of Bugbuster

**4th step: Optimise protein extraction**
-Dilute rLysozyme solution using rLysozyme Dilution Buffer
-Add 1 KU rLysozyme for each mL of BugBuster used
-Keep in ice

**5th step: Inhibit potential enzyme degradation**
-Add proteases inhibitors (they must be compatibles with all different reagents used before)

**6th step: Purification**
-Incubate suspended cells during 15 min at room temperature
-Centrifuge at 16 000 g during 20 min at 4°C
-Transfer supernatant in a fresh tube
-In a tube, add 50 µL of supernatant to 50µL of 4X SDS
-Heat the tube at 85°C (bain marie)
-Store in ice until SDS-PAGE analysis
**7th step: Electrophoresis in denaturing condition**

Remark: remaining soluble extract can be directly charged in a purification gel

### Bibliographical references :

[Reference 1](https://static.igem.org/mediawiki/2018/0/07/T--Exeter--BugBusterProteinExtraction.pdf)
[Reference 2](https://www.merckmillipore.com/FR/fr/product/BugBuster-10X-Protein-Extraction-Reagent%2CEMD_BIO-70921#anchor_USP)

![](https://static.igem.org/mediawiki/2021/6/66/T--Nantes--reference.png)

Description

Image source

### Goal: Perform affinity purification of the resulting His-tagged fusion proteins

|  |  |
| --- | --- |
| Equipment: | 1.5mL microtubes   Sample containing His tagged proteins   Magnetic holder   Vortex   Centrifuge |

### Operating mode:

Step 1: Put 40 µL (0.5mg) of HisPur Ni-NTA into a 1.5 mL microtube - Adjust if more beads are desired.
Step 2 : Add 160 µL of equilibration buffer and vortex for 10 seconds
Step 3: Place the tube in a magnetic holder to collect the beads on the side of the tube. Remove and discard the supernatant.
Step 4 : Add 400 µL of equilibration buffer, vortex for 10 seconds and collect the beads on the side of the tube with the magnetic holder. Remove and discard the supernatant.
Step 5 : Prepare the protein extract with the same volume of equilibration buffer.
Step 6 : Add 400 µL of the protein solution, vortex 10 seconds and mix for 30min.
Step 7 : Collect the beads on the side of the tube with the magnetic holder. Remove and discard the supernatant unless you want to keep it for further experiments.
Step 8: Add 400 µL of Wash Buffer and mix well. Collect the beads with the magnetic holder, remove and discard the supernatant.
Step 9: Repeat the washing step once.
Step 10 : Add 25 µL of elution buffer, vortex for 15 seconds. If necessary, centrifuge for 1 min at 700g to ensure that all beads are in the elution solution. Incubate for 15min on a rotating platform. Alternatively, vortex the tube for 15 seconds every 5 min.
Step 11 : Collect the beads with the magnetic holder. Be careful to remove and save the supernatant that contains the His Tagged proteins.
Step 12: Repeat the elution step using 25 µL of elution buffer. Incubate for 10min.
Step 13: Monitor protein elution with the Pierce 660nm Protein Assay Kit (#: 22662). Or it can also be analyzed by SDS PAGE.

### Bibliographical references:

[Manual Reference](https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0011794_HisPur_NiNTA_Mag_Bead_UG.pdf?_ga=2.45221728.576931359.1620127926-8981698.1620127926)

## VII-FACE

![](https://static.igem.org/mediawiki/2021/c/c8/T--Nantes--vivace.png)

Description

Image source

|  |  |
| --- | --- |
| Equipment: | -Freezer at -80°C   -PCR tubes   -Ice   -Gloves   -Generator   -Electrophoresis tank: Mini protean III from Biorad   -Extractor hood   -UV device |
| Reagents | -Ulva   -10X proteinase K   -Hyaluronidase SD   -Chondroitinase ABC   -Heparinase (Flavobacterium heparinum)   -Heparinase I (Flavobacterium heparinum)   -Héparitinase II (Flavobacterium heparinum)   -100 mM Ammonium acetate, pH 7,0   -SDS 10%   -Absolute ethanol   -75%Ethanol   -BSA   -400 mM Tris-acétate solution   AMAC   DMSO   Sodium cyanoborohydride: TOXIC   GLacial acetic acid   GeneRuler 1 kb DNA Ladder   TBE electrophoresis buffer   Acrylamide : Bis Acrylamide 30 : 1   10% APS   TEMED |

### Operating mode:

**1st step: Making reagents**

10x protéinase K : 1 mg/mL with 0,01% SDS
Dissolve 100 mg of PK in 10 mL of 100 mM ammonium acetate during 30 min
Add ammonium acetate to obtain 100 mL
Add 100 µL of 10% SDS
Mix until SDS is soluble
Aliquot 1,5 mL in 2 mL tubes
Keep at -80°C
Hyaluronidase SD : 2,5 mU/µL
Dilute 200 µL of 100 mM ammonium acetate containing 0,1% of BSA to 0,5 U of hyaluronidase
Incubate for 20 min and vortex
Aliquot 10 µL in PCR tubes
Keep at -80°C
Chondroitinase ABC : 25 mU/μL
Dilute 400 µL of 100 mM ammonium acetate containing 0,1% of BSA to 10U of chondroitinase ABC
Incubate for 20 min and vortex
Aliquot 10 µL in PCR tubes
Keep at -80°C
Heparinase (Flavobacterium heparinum) : 1 mU/μL
Dilute 100 µL of 100 mM ammonium acetate containing 0,1% of BSA to 0,1 U of heparinase
Incubate for 20 min and vortex
Aliquot 10 µL in PCR tubes
Keep at -80°C
Heparitinase I (Flavobacterium heparinum) : 1 mU/μL
Dilute 100 µL of 100 mM ammonium acetate containing 0,1% of BSA to 0,1 U of heparinase I
Incubate for 20 min and vortex
Aliquot 10 µL in PCR tubes
Keep at -80°C
Heparitinase II (Flavobacterium heparinum) : 1 mU/μL
Dilute 100 µL of 100 mM ammonium acetate containing 0,1% of BSA to 0,1 U of heparinase II
Incubate for 20 min and vortex
Aliquot 10 µL in PCR tubes
Keep at -80°C

 **2nd step: Digestion of protein K:**
Add 250 µL of 10X Proteinase K for every 100 mg of wet ulvan
Incubate at 60°C until Ulvan is digested, vortex every 30 min

**3rd step: 1st precipitation by ethanol**
-Add 4 volumes of absolute ethanol at -20°C to each sample of digested ulvan
-Incubate overnight at -20°C
-Centrifuge the samples at 14000 g for 10 min at room temperature
-Throw supernatant
-Wash the pellet by adding 4 volumes of 75% ethanol at -20°C
-Vortex
-Centrifuge samples at 14 000 g for 10 min
-Throw supernatant
-Let the pellet dry at ambient temperature for 20 min
-Add 35 µL of 100 mM acetate ammonium
-Incubate at ambient temperature for 20 min
-Vortex
-Incubate the samples at 100°C for 5 min
-Put the samples in Ice

**4th step: Chondroitin sulfate and hyaluronidase digestion**
Add 1 µL of chondroitinase ABC and/or 1µL of hyaluronidase SD to each samples of 35µL
Vortex
Incubate at 37°C for 18 hours

**5th step: 2nd precipitation by ethanol**
Add 160 µL of absolute ethanol at -20°C to each sample
Incubate overnight at -20°C
Centrifuge the samples at 14 000 g for 10 min
Keep the supernatant in a 1,5 mL tube
Wash the pellet with 100 µL of 75% ethanol at -20°C
Vortex
Centrifuge samples at 14 000 g for 10 min
Keep the supernatant
Let the pellet dry at ambient temperature for 20 min
Add 35µL of 100 mM ammonium acetate
Incubate at ambient temperature for 20 min and vortex
Incubate samples at 100°C for 5 min
Put the samples in ice
Freeze-dry the supernatants
**6th step: Heparane sulfate digestion**
Mix in equal proportions Heparinase, heparinase I and heparinase II
Add 1 µL of this mix to each sample of 20 µL
Incubate overnight at 37°C

**7th step: 3rd precipitation by ethanol**
Add 80 µL of absolute ethanol at -20°C to every samples
Incubate overnight at 20°C
Centrifuge samples at 14 000 g for 10 min
Keep the supernatant in a 1,5 mL tube
Wash the pellet with 100 µL of 75% ethanol at -20°C
Vortex
Centrifuge samples at 14 000 g for 10 min
Keep the supernatant
Let the pellet dry at ambient temperature for 20 min
Add 35µL of 100 mM ammonium acetate
Incubate at ambient temperature for 20 min and vortex
Incubate samples at 100°C for 5 min
Put the samples in ice
Freeze-dry the supernatants

**8th step: Marking by fluorophore 2-amino-acridone (AMAC)**
-AMAC solution
Dissolve 25 mg of AMAC in 2 mL of DMSO
Complete to 6,89 mL with DMSO
In a 15 mL tube, add 770 mg of sodium cyanoborohydride to 9,8 mL of water
Incubate for 20 min with an occasional inversion
Add 1,216 mL of acetic acid to the 6,89 of AMAC
Mix by inversion
Mix 8 mL of AMAC solution tu 8 mL of sodium cyanoborohydride in a 50 mL tube
Mix by inversion
Aliquot 120 µL and 480µL
Keep at -80°C

### Marking :

Add 1µL of AMAC solution every cm² of cultivation area
Add 1 µL of AMAC solution for every 2,5 mg of wet tissue
Incubate the samples at 37°C for 18 hours in the dark

### 9th step: Gel preparation and revelation

Manipulate with gloves!

In a 10 mL tube, put 4 mL of H20
Add 1,8 mL of Acrylamide : Bis Acrylamide 30 : 1
Add 1,5 mL of 1,5 MTris-HCL , pH 6,8
Add 25 µL of 10% APS
Add 5 µL of TEMED under fume cupboard
Pour 4,5 mL of the mix between 2 glass plates
Add 1 mL of distilled water above the separation gel
Add the comb
Let the gel polymerize
withdraw the comb
File the samples
Fill the tank with TBE buffer
Place the cover with electrical connections
Set up refrigeration
Turn the generator on
Adjust tension to 140/160 Volts
Wait for the samples to migrate
Turn the generator off
Empty interior and exterior tanks
Separate glass plates to the gel
Analyze under UV

### Bibliographical references

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

## VIII-HPAEC-PAD

![](https://static.igem.org/mediawiki/2021/b/bd/T--Nantes--e-pad.png)

Description

Image source

### Goal: Quantification of ulvan composition in free monosaccharides and sulfates

|  |  |
| --- | --- |
| Equipment | ICS-3000 Dionex   Dionex CarboPac PA10 4 x 250 mm |
| Reagents | Ulvan degraded   Eluent: 1M NaOH   Distilled water or buffer   L-Rhamnose monohydrate   Xylose monohydrate   Glucuronic acid   Iduronic acid |

### Operating mode:

**1st step : Preparation of the eluent :**
To obtain a 0.16 M solution:
Dilute 80 mL of 1M NaOH in 420 mL of distilled water
To obtain a 0.1 M solution:
Dilute 50 mL of 1M NaOH in 450 mL of distilled water

**2nd step: Preparation of standard solutions - 5 mL**
Dissolve L-Rhamnose and xylose powders in distilled water

 **3rd step : Start-up of the column**
Explanation of the equipment and software on site
How to set up the eluent flow (0.10 to 0.16 M concentration gradient) at a flow rate of 1.5 mL/min

**Step 4: Processing of standard samples**
Inject each sample into the column
Note the retention time of monosaccharides
Print the chromatograms

 **Step 5: Processing of the degraded ulvan monosaccharides**
Inject each sample into the column
Compare the retention times with the reference ones

### Bibliographical references:

[Reference](https://www.sciencedirect.com/science/article/pii/S0144861720300266)

## IX-Migration Gel

### Goal: Size exclusions of proteins after Bugbuster and purification

|  |  |
| --- | --- |
| Equipment: | Gloves   Eppendorf Tube   Power supply   Buffer tank : Mini protean III de Biorad   Hot water bath   Fume hood |
| Reagents | Soluble fraction from lysis   Washing fraction from purification step   Fraction d’élution issue de la purification   Acrylamide : Bis Acrylamide 30 : 1   Tris-HCl 1,5M, pH 8,8   Running buffer Tris-glycine SDS 0,1% pH 8,9   Loading buffer SDS (réf. 70607-3)   SDS 10%   APS 10 %   Temed   Size standards gene ruller of 1 kb   Coomassie Brilliant Blue   Cooler |

### Operating mode

1st step: Separating gel preparation 10% (using gloves)

Table of quantities for 10 mL :

|  |  |
| --- | --- |
| H2O | 4 ml |
| Acrylamide : Bis Acrylamide 30 : 1 | 3,3 ml |
| Tris-HCl 1,5M, pH 8,8 | 2,5 ml |
| SDS 10% | 100 µL |
| APS 10% | 100 µL |
| TEMED (only at the end) under fume hood | 4 µL |

Mix acrylamide, Tris-HCL, SDS and APS with water
Add TEMED, and quickly poor 4.5 mL into the two glass plates
Keep the rest in a tube as control
Add on the top of the gel, 1 mL of distilled water
Let the gel polymerize

2nd step: Concentrating gel preparation 4,5% (using gloves)

|  |  |
| --- | --- |
| H2O | 3,4 ml |
| Acrylamide : Bis Acrylamide 30 : 1 | 0,83 ml |
| Tris-HCl 1,5M, pH 8,8 | 0,63 ml |
| SDS 10% | 50 µL |
| APS 10% | 50 µL |
| TEMED (only at the end) under fume hood | 4 µL |

Remove the water on the top of the separating gel, using absorbent paper
Add TEMED, and quickly poor the mix to the top of the two glass plates
place the electrophoresis comb
Wait for polymerization

**3rd step: Préparation des échantillons**
Put 1.5 mL of the soluble fraction from the lysis in an Eppendorf tube.
Put 1.5 mL of the washing fraction and elution fraction in an Eppendorf tube.
Add 50 µL of loading buffer in the tubes
Vortex
Heat the hot water bath during 3 min at 85°C
Place the tubes in ice and let them cool

**4th step: Samples**
Remove the comb from the gel, and fill one well with size standards and the other well with 20 µL of samples
Poor 800 mL of running buffer (1x) in the tank
Place the lid with power supply wires
Turn on refrigeration
Turn on the power supply and set the tension at 140/160 Volts

**5th step: Reveal**
Turn the power supply off and empty the upper and the lower tanks
Separate the two plates, and carefully remove the gel
Colour the gel using Coomassie Brilliant Blue for 2-24 hours
Discolor the gel with a distilled water bath until color bands appear

### Bibliographical references:

Handout of “Travaux Pratiques de Plateaux Technique Licence 3 Chimie-Biologie 2020-2021” by P. Weigel, F. Fleury, E. Camberlein, University of Nantes

## X-PCR

### Goal: Amplified DNA using primers

|  |  |
| --- | --- |
| Equipment | Thermal cycler Microtubes de 0,2 mL Micropipettes p20, p200 |
| Reagents | Distilled water   pET11   pEVOL-1   PCR Buffer (with Tris-HCl (pH 8,8); (NH4)2SO4; KCl; BSA; Triton; MgSO4) 5X (To obtain a 1X buffer)   dNTP mix 10 mM (for Cf= 0,8 mM)   Primers T7 10 µM (for Cf= 1 µM)   Taq proof reading (Phusion) |

### Operating mode (for one pcr reaction):

All reactives need to be fully unfrozen, vortexed, centrifuged, and placed in ice before reaction.

**1st step:**
Put 10 µL of distilled water in a 0,2 mL microtube
Add 5 µL of the plasmid solution pET11 or pEVOL-1
Add 5µL of PCR buffer, containing MgCl2
Add 2µL of dNTP solution
Add 2,5 µL of T7 primer solution
Add 0,5 µL of RNA polymerase TAQ
Mix by pipetting 2-3 times using p200 pipettes set on 25 µL
Do not forget to make a control using water

**2nd step:**
Place the tube in the thermal cycler
Set the thermal cycler:

### Bibliographical references:

Handout of “TP Biologie Moléculaire pour les Biotechnologie, Licence 2” by DEL GATTO-KONCZAK, GALVANI, THYS, GUILBAUD, MOREAU-AUBRY, RIO, JAHAN, GLEMAIN, University of Nantes

Handout of “TP de Biologie Moléculaire 1, Licence 2” by DEL GATTO-KONCZAK, University of Nantes

## Electrophoresis gel

### Goal: Purify PCR products

|  |  |
| --- | --- |
| Equipment | Graduated cylinder de 500 ml et 50 mL   Parafilm   Beaker   Precision balance   Magnetic heat stirrer// Microwave   Magnetic agitator   aluminium foil   UV Light   Electrophoresis tank   16 wells electrophoresis comb   UV protection plate   Micropipettes p20, p100 |
| Reagent | TAE 5X (to obtain 0.5X TAE)   Agarose   Distilled water   Loading buffer   Size standards   GelRed |

### Operating mode:

**1st step: Electrophoresis buffer TAE 0.5X preparation**
Add 50 mL of TAE 5X buffer in a 500 mL graduated cylinder
Fill to 500 mL using 450 mL of distilled water
Cover the top with parafilm
Mix by reverse the graduated cylinder

**2nd step: Agarose gel (1%) preparation**
Gel 1 %: Weight in a beaker, 0.6 g of agarose powder
Add 60 mL of electrophoresis buffer
Put the magnetic agitator in the beaker
Cover the beaker using aluminium foil, and make a small hole in it
Dissolve agarose by heating the beaker under magnetic stirring using the magnetic heat stirrer (the mix need to boil to dissolve all the powder, but be careful to not burn it)
Wait the solution to cool during couples minutes
Cast the solution in the high size gel mold
Place the electrophoresis comb in the mold
Wait one hour to let the gel polymerized

**3rd step: Electrophoresis tank**
Place the gel on the plate in the electrophoresis tank, the wells need to be on the cathode side
Fill the tank using loading buffer to submerge the gel (+1 / 2 cm over the gel)

**4th step: PCR products loading**
Add 10 µL of loading buffer to the PCR products
Homogenized by pipetting couples times
**5th step: Samples deposit**
Put 5 µl of size standards in the first and the last wells
Be sure to make a deposit plan (Take notes and number each wells)
Put 10 µL of PCR products in each wells (except the first and the last, do not put more than 25 µL per well)

 **6th step: Migration**
Close the tank
Set the tension on 100V
Check the position of electrodes
Start migration
Check during the 5 first minute for the good migration of the samples (they should not escape from the gel, and move in the good direction)
Stop the migration when the blue dye reach the third quarter of the gel

 **7ème step : Gel revealing**
Color the gel during 10 to 30 minutes in GelRed solution
Wash the gel 10 min with water to remove the GelRed excess
Place the gel under UV light (UV transilluminator)
Close with the UV protection plate
Turn on the UV lights
Take a picture

### Bibliographical references:

Handout of “TP Biologie Moléculaire pour les Biotechnologie, Licence 2” by DEL GATTO-KONCZAK, GALVANI, THYS, GUILBAUD, MOREAU-AUBRY, RIO, JAHAN, GLEMAIN, University of Nantes

Handout of “TP de Biologie Moléculaire 1, Licence 2” by DEL GATTO-KONCZAK, University of Nantes

## XI-Solid and Liquid mediums

|  |  |
| --- | --- |
| Equipment | Galoves Balance 2 L Flask Magnet 1 mL pipette, or p1000 micropipette + Eppendorf tube 1 L Erlenmeyer Tissu cap Autoclave Thermometer Sterile petri dish |
| Reagents | Distilled water   Tryptone   Yeast extract   NaCl   1N NaOH   Agar ou agarose |

### Operating mode:

Liquid LB for 1 liter

|  |  |
| --- | --- |
| Distilled water | 950 mL |
| Tryptone | 10g |
| Yeast extract | 5g |
| NaCl | 5g |
| 1N NaOH | 1 ml |

Use a 2 L flask
Mix using magnet until everything is dissolve
Put the mix in a 1 L Erlenmeyer and close it using tissue cap
Autoclave the solution during 25 min
Wait the mix to cool to 50°C (you should hold it without burning your hand)
Add nutriments and antibiotics needed in the chilled mix

LB solid medium

|  |  |
| --- | --- |
| Distilled water | 1 liter |
| Tryptone | 10g |
| Yeast extract | 5g |
| NaCl | 5g |
| 1N NaOH | 1 ml |
| Agar or Agarose | 15g |

Place the petri dishes open in a room temperature location, and let them dry during 2 to 3 days, or place them in an incubator during 30 min at 37°C. This prevent from bacterium development in the petri dishes due to humidity
When dry, stock them at 4°C in their packaging
Mix up all the compounds and autoclave it during 25 min
Let it cool to 50°C before adding other compounds (nutrients, antibiotics, etc)
Be aware to stay over 45°C to keep the mix liquid
Poor the mix il petri dishes (about 40 mL per dishes)
1 L allow to make 25 petri dishes

### Bibliographical references:

Handout of “TP de Plateaux Technique Licence 3 Chimie-Biologie 2020-2021” by P. Weigel, F. Fleury, E. Camberlein, University of Nantes

## XII-DNA precipitation

### Goal: Raise the concentration of the DNA samples

|  |  |
| --- | --- |
| Equipment | Pipettes p10 and p200 Centrifuge |
| Reagents | DNA samples   Sodium Acetate Solution (3M, pH 5.2)   Ethanol 95% |

### Operating mode:

Add 0.1 volume of 3M Sodium Acetate Solution for 1 volume of DNA sample.
Add 2 volumes of Ethanol to the mixture
Leave the solution at -80°C (-112°F) for 30 minutes (or at -20°C (-4°F) overnight).
Centrifuge the solution at 10000 rpm for 30 minutes
Let the solution settle and then remove the supernatant
Air-dry the pellet for 10 to 15 minutes until all liquid is evaporated, at room-temperature 65°C
Resuspend the pellet in the desired volume of water or buffer. 10µl à 65°+
Measure the quantity of DNA with the Nanodrop

### Bibliographical references:

Protocol from UFIP laboratory (UMR 6286-CNRS), University of Nantes

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Nantes/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)