iGEM 2016 — Team Evry

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# Team:Evry/Notebook

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*Let's PLAy project* - Bioproduction of PLA

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* Note book

# Note book

Incubation 1 h at 37 °C and after incubation 20 min at 80 °C.

###### Gel Extraction

* 50 mL of Agarose 1 % with 2.5 µL of midorigreen.
* Migration 25 min at 100 mV.
![2-September_agarose-gel](https://static.igem.org/mediawiki/2016/8/8b/T--Evry--agarose-gel1_2Sept.png)

\*Digestion mixes with EcoRI and PstI were loaded on the gel.

##### 3rd of September

###### DNA extraction from agarose gel

Kit: Wizard SV Gel and PCR Clean-Up System (Ref: A9282, Promega)

* pSB1C3 => 6.4 ng/µL
* LDH2 => 4.8 ng/µL
* PCT2 => 2.9 ng/µL
* PhaC3 => 4 ng/µL
* PhaC4 => 5.8 ng/µL

###### DNA concentration by water evaporation

* pSB1C3 => 7.1 ng/µL
* LDH2 => 6.5 ng/µL
* PCT2 => 5.5 ng/µL
* PhaC3 => 4.0 ng/µL
* PhaC4 => 5.0 ng/µL

DNA concentration still seems not efficient

###### DNA transformation

* Thaw 50 µL chemo-competent DH5-α *E. coli*
* Add 5 µL DNA (mix ligation with pSEVA 2311 or 224)
* Incubation 25 min on ice
* Heat shock: 45 s at 42 °C
* Incubation 3 min on ice
* Add 250 µL LB
* Incubation 1h15 at 37 °C with shaking
* Spread on plates LB Kanamycin (50 µg/mL) (50 µL of bacteria on one plate and 10 µL on another one for each vector)
* Incubation overnight 37 °C

##### 4th of September

###### Ligation in pSB1C3

| - | LDH2 (6.5 ng/µL) | PCT2 (6.5 ng/µL) | PhaC3 (4.0 ng/µL) | PhaC4 (5.0 ng/µL) |
| --- | --- | --- | --- | --- |
| Vector | 4.2 µL | 4.2 µL | 4.2 µL | 4.2 µL |
| Insert (gene) | 2.8 µL | 0.5 µL | 0.5 µL | 0.5 µL |
| Buffer T4 DNA ligase (10X) | 2.39 µL | 2.39 µL | 2.39 µL | 2.39 µL |
| T4 DNA ligase | 3.96 µL | 1.95 µL | 4.02 µL | 1.11 µL |
| H20 (qs 10 µL) | 2.15 µL | 4.15 µL | 2.08 µL | 5.0 µL |

Incubation 1 h at 16 °C

Poor plates LBC (30 µg/mL chloramphenicol)

###### Transformation

* Thaw 25 µL chemo-competent DH5-α *E. coli*
* Add 4 µL DNA (mix ligation)
* Incubation 25 min on ice
* Heat shock: 45 s at 42 °C
* Incubation 3 min on ice
* Add 225 µL LB
* Incubation 1 h at 37 °C with shaking
* Spread 120 µL on plates LBC
* Incubation overnight 37 °C

### Week 10

##### 7th of September

###### Digestion of genes

See complete version of the notebook (pdf) for details on the conditions.

Incubation 1 h at 37 °C and after incubation 20 min at 80 °C.

##### 8th of September

###### Digestion of bacbone Igem

Mix: Enzyme Master Mix E+P

* 0.5 µL NEB 2.1 Buffer
* 0.5 µL BSA (Bovine Serum Albumin)
* 0.5 µL EcoR1-HF
* 0.5 µL Pst1
* 0.5 µL Dnp1
* 18 µL H2O

Mix of 4 µL of linearized backbone and 4 µL of master mix enzyme

Incubation of all the solution at 37 °C during 30 min and then, heat deactivation during 20 min at 80 °C.

###### Ligation

| Genes | length genes | Volume Final | Concentration Final | amount for 1 µL (Cf/Vf) |
| --- | --- | --- | --- | --- |
| pSB1C3 | 2037 bp | 8 µL | 100 ng | 25 ng= 2 µL |
| LDH\_V2 | 1227 bp | 50 µL | 1095 ng | 21.9 ng/µL |
| PCT\_V2 | 1612 bp | 50 µL | 1130 ng | 22.6 ng/µL |
| PhaC\_V3 | 1717 bp | 50 µL | 1118.25 ng | 22.365 ng/µL |
| PhacC\_V4 | 1720 bp | 50 µL | 1101.25 ng | 22.025 ng/µL |

Amount calculation of insert for Ligation with NEBio Calculator:

| Genes | Amount | Volume for deduced |
| --- | --- | --- |
| LDH\_V2 | 15.06 ng | 0.7 µL |
| PCT\_V2 | 19.78 ng | 0.9 µL |
| PhaC\_V3 | 21.07 ng | 0.9 µL |
| PhaC\_V4 | 21.11 ng | 0.96 µL |

**Mix of ligation**

* 2 µL of pSB1C3
* Volume calculate of digested insert
* 1 µL T4 Ligase Buffer
* 0.5 µL T4 Ligase
* qs 10 µL

I put the mix at 16 °C while 30 min and after heat kill for 20 min at 80 °C

###### Transformation by heat shock

With 2 µL of Ligation mix and 25 µL of Competent Cells.

* 2 µL of Ligation with 25 µL of competent cells.
* Put the mix 20 min in the ice.
* Put the mix at 42 °C while 45 seconds
* 2 min on the ice.
* Add 950 µL of LB
* Recovery 1 h at 37 °C with agitation
* 7000 rpm during 1 min
* Suppression of the flow-throw
* Resuspend with the remaining
* Spread on plates (put the simple in the middle of the plates)
* Incubation overnight 37 °C

###### Plates preparation

1 plates = 25mL of LB Agar + 15 µL of chloramphenicol [30 µg/mL]

![](https://static.igem.org/mediawiki/2016/6/63/T--Evry--FormulaLabbook.png)

###### Preculture

3 mL LB liquid medium + 1 µL *E. coli* DH5-α bacteria from stock at -80 °C

##### 9th of September

###### Preparation of chemo-competent *E. coli* DH5-α by heat shock

* WORK STERILE
* Launch a pre-culture of the strain in LB at 37 ° C
* Inoculate 25 mL of LB 1/50 (500 μl) with the pre-culture
* Let it growth at 37 ° C with stirring until OD(600 nm) = 0.5-0.7
* After 1h30, OD= 0.9
* For Spectrometer measurements, 1 mL of LB for the blank
* A dilution was performed to obtain 0.5 OD(600 nm) and the culture was split into 2 falcons with 27.5 mL of culture
* Cells are cooled 10 min on ice
* Centrifugate culture 6 min at 4000 rpm at 4 °C
* Suppression of the flow-through
* Dilution of the pellet in 1/2 volume of cold CaCl2 0.1M (13.75 mL)
* Cells cooled on ice during 20 min
* Culture centrifugated 6 min at 4000 rpm at 4 °C
* Suppression of the flow-through
* Resuspension the pellet in 1 / 50e of final volume (550 μl final volume) of cold CaCl2 + 10 % glycerol
* Vi= 110 µL
* The mix is 440 µL CaCl2+ 110 µL glycerol 50 %
* I multiplied by 2 the doses for have more mix
* 22 aliquots of 50 µL bacteria

##### 11th of September

###### PCR Amplification of LDH2, PCT2, PHAC3, PHAC4 with Q5 DNA polymerase

**For 50 µL reaction**

* 25 µL of Q5 Master Mix
* 2.5 µL of Forward Primer (IG063)
* 2.5 µL of Reverse Primer (IG064)
* 0.5 of Template DNA
* 19.5 of H2O

**PCR program**

* 98 °C 30 s
* (98 °C 30 s, 55 °C 30 s, 72 °C 1 min) 35 times
* 72 °C 2 min
* 10 °C ∞

###### PCR Colonie LDH2 with One Taq

For 25 µL to 1 Reaction --> Vf= 125 µL

**Mix**

* 25 µL of One Taq Standard Reaction Buffer
* dNTPs = 2.5 µL
* 2.5 µL of Forward Primer (IG001)
* 2.5 µL of Reverse Primer (IG002)
* 0.625 µL of One Taq DNA Polymerase
* 91.9 µL of Nuclease Free Water

**PCR program**

* 94 °C 30 s
* (94 °C 30 s, 50 °C 1 min, 68 °C 1 min 45 sec) 30 times
* 68 °C 5 min
* 10 °C ∞

### Week 11

##### 12st of September

###### Miniprep colonies LDH

* 3 µL of LB
* Take one of the colony in the plates
* Incubated at 37 °C overnight

###### Digestion Backbone IGEM pSB1C3 linearized by E+P

**Mix: Enzyme Master Mix E+P for 22 reaction**

* 2 µL NEB 2.1 Buffer
* 2 µL BSA (Bovine Serum Albumin)
* 2 µL EcoRI-HF
* 2 µL PstI
* 2 µL DnpI
* 72 µL H2O

* 16 µL of linearized backbone and 16 µL of the MasterMix enzyme
* Incubation at 37 °C during 30 min
* Enzyme inactivation with 20 min incubation at 80 °C.

###### Precultures

* 5 mL LB medium
* + 5 µL antibiotic (Kanamycin 50 mg/mL for pSEVA 212S, Spectinomycin 50 mg/mL for pSEVA 424 and Gentamycin 10 mg/mL for pSEVA 628S)
* + some bacteria (sent as bacteria aliquots in LB medium by de Lorenzo)
* Incubation 37 °C overnight

##### 13st of September

###### Miniprep of pSEVA plasmids

2 aliquots stocks in 50 % glycerol stored at -80 °C

GenElute Plasmid Miniprep Kit (Sigma, PLN350-1KT)

3 DNA vectors: pSEVA 424 (equivalent to the pSEVA 224 but with spectinomycin resistance gene instead of kanamycin, pSEVA 212S and pSEVA 628S used for genes deletion in genomic DNA in *Pseudomonas putida*)

###### Digestion of the “old” gene by E+P

| Ratio 1 : 2 | | |
| --- | --- | --- |
| Genes | Amount | Volume for deduced |
| LDH\_V2 | 30.12 ng | 1.37 µL |
| PCT\_V2 | 39.57 ng | 1.71 µL |
| PhaC\_V3 | 42.15 ng | 1.88 µL |
| PhaC\_V4 | 42.22 ng | 1.92 µL |

**Ligation mix**

* 2 µL of pSB1C3
* Volume calculate of digested insert
* 1 µL T4 Ligase Buffer
* 0.5 µL T4 Ligase
* qs 10 µL

I put the mix at 16 °C while 30 min and after heat kill for 20 min at 80 °C

###### Minipreps DNA Purification System kit “Wizard Plus”

* Take 2 µL of overnight culture
* Centrifuge at top speed for 1 min
* Suppression of the flow-throw
* Resuspend pellet with 250 µL of Cell Resuspension Solution
* Add 250 µL of Cell Lysis Solution and invert 4 times to mix
* Add 10 µL of Alkaline Protease Solution and invert 4 time to mix
* Incubate 5 min at room temperature
* Add 350 µL of Neutralization Solution and invert 4 times to mix
* Centrifuge at top speed for 10 min at room temperature
* Insert Spin Column into Collection Tube
* Decant cleared lysate into Spin Column
* Centrifuge at top speed for 1 min
* Discard flowthrough
* Add 750 µL of Wash Solution and centrifuge at top speed 1 min
* Discard flowthrough
* Repeat with 250 µL of Wash Solution
* Centrifuge at top speed for 2 min
* Transfert Spin Column to 1.5 mL tube
* Add 100 µL of Nuclease free water to the Spin Column
* Centrifuge at the top speed for 1 min
* Discard column

| Ratio 1 : 5 | | |
| --- | --- | --- |
| Genes | Amount | Volume for deduced |
| LDH\_V2 | 75.29 ng | 3.44 µL |
| PCT\_V2 | 98.92 ng | 4.38 µL |
| PhaC\_V3 | 105.4 ng | 4.71 µL |
| PhaC\_V4 | 105.5 ng | 4.79 µL |

###### Purification of Amplification PCR LDH2, PCT2, PHAC3, PHAC4

###### Nanodrop

**For the genes**

| Genes | Concentration (ng/µL) | Ratio 260/280 |
| --- | --- | --- |
| LDH2 | 62.7 | 1.78 |
| PCT2 | 52.8 | 1.79 |
| PHAC3 | 50.3 | 1.76 |
| PHAC4 | 69.7 | 1.78 |

**For the LDH colonies**

| Colonies | Concentration (ng/µL) | Ratio 260/280 |
| --- | --- | --- |
| LDH\_n°1 | 70 | 1.92 |
| LDH\_n°2 | 31 | 2.06 |

###### Digestion “New” genes LDH2, PCT2, PHAC3, PHAC4

Digestion by EcoR1-HF+PSt1

| Genes | Quantity of DNA (µL) | Restriction Enzymes | Buffer Digest NEB 2.1 (µL) | H2O |
| --- | --- | --- | --- | --- |
| LDH2 (62.7 ng/µL) | 16 | 1 µL EcoR1-HF | 5 | 27 |
| 1 µL Pst1 |
| PCT2 (52.8 ng/µL) | 19 | 1 µL EcoR1-HF | 5 | 24 |
| 1 µL Pst1 |
| PHAC3 (63.9 ng/µL) | 16 | 1 µL EcoR1-HF | 5 | 27 |
| 1µL Pst1 |
| PhAC4 (69.7 ng/µL) | 14.5 | 1 µL EcoR1-HF | 5 | 28.5 |
| 1 µL Pst1 |
| pSB1C3 (240.3 ng/µL) | 4.5 | 1 µL EcoR1-HF | 5 | 38.5 |
| 1 µL Pst1 |

Other digestion

| Genes | Quantity of DNA (µL) | Restriction Enzymes | Buffer Digesr NEB 2.1 (µL) | H2O |
| --- | --- | --- | --- | --- |
| PHAC3 | 16 | 1 µL Xba1 | 5 | 27 |
| 1 µL Pst1 |
| PHAC4 | 14.5 | 1 µL Xba1 | 5 | 28.5 |
| 1 µL Pst1 |
| PCT2 | 19 | 1 µL EcoR1-HF | 5 | 24 |
| 1 µL Spe1 |

Incubation at 37 °C during 30 min and then, enzymes deactivation by heat kill for 20 min at 80 °C.

###### PCR Purification

###### Nanodrop Vf = 50 µL

| Genes | Concentration (ng/µL) | Ratio 260/280 |
| --- | --- | --- |
| LDH\_V2 | 62.7 | 1.78 |
| PCT\_V2 | 52.8 | 1.79 |
| PHAC\_V3 | 50.3 | 1.76 |
| PHAC\_V4 | 69.7 | 1.78 |

##### 14st of September

The promoter region of the vectors pSEVA 224 and pSEVA 2311 were amplified by PCR with Q5 DNA polymerase with primers allowing to add the prefix and suffix regions at both sides of the amplified region.

**Mix**

* 10 µL Q5 reaction buffer 5X
* 1 µL dNTP mix
* 2.5 µL 10 µM Forward Primer (iG063)
* 2.5 µL 10 µM Reverse Primer (iG064)
* 1 µL DNA template
* 0.5 µL Q5 DNA polymerase
* 32.5 µL Nuclease-free water (qs 50 µL)

**PCR program:**

* 98 °C 30 s
* (98 °C 30 s, 55 °C 30 s, 72 °C 1 min) 35 times
* 72 °C 2 min
* 10 °C ∞

After checking on agarose 1 % gel, we determined that the fragments were successfully amplified.

###### Digestion

The amplified fragments were digested by EcoRI and PstI during 1 h at 37 °C. The reaction was performed in NEB CutSmart buffer.

The digested mixes were purified by using the kit Wizard SV Gel and PCR Clean-Up System (Ref: A9282, Promega).

Design Primer for Gibson Assembly -> PR\_IG0075 -> 81

##### 15st of September

###### Ligation

The 2 fragments were then ligated separately in linear pSB1C3 provided by IGEM Headquarters in a ratio 5:1.

The ligation mix was used for transformation in E. coli DH5-alpha.

###### Transformation

- Thaw 25 µL chemo-competent DH5-alpha E. coli
- Add 3 µL DNA (mix ligation)
- Incubation 20 min on ice
- Heat shock: 45 s at 42 °C
- Incubation 3 min on ice
- Add 225 µL of LB medium
- Incubation 1 h at 37 °C with shaking
- Spread 120 µL on plates LBC
- Incubation overnight 37 °C

##### 16th of September

###### PCR colony

**Mix (25 µL total volume reaction):**

- 12.5 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
- 2.5 µL 10X DreamTaq Green Buffer (ThermoScientific)
- 0.25 µL Forward Primer (0.5 µM) iG001
- 0.25 µL Reverse Primer (0.5 µM) iG002
- 9.5 µL H2O filtrated (qs 25 µL)

+ clones

###### Agarose gel 1 %

![16-September_agarose-gel](https://static.igem.org/mediawiki/2016/c/ca/T--Evry--agarose-gel1_16September.png)

###### Precultures

Positive clones selected from the agarose gel results were picked and used to inoculate 3 mL of LBC (25 µg/mL chloramphenicol) medium.

*Observation*

All the precultures have grown

###### PCR colony

**Mix (25 µL total volume reaction):**

- 10 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
- 1.5 µL Forward Primer (0.5 µM) iG001
- 1.5 µL Reverse Primer (0.5 µM) iG002
- 6 µL H2O filtrated (qs 25 µL)

+ clones

###### Migration map

![16-September_map-gel](https://static.igem.org/mediawiki/2016/4/47/T--Evry--map-gel_16September.png)

![16-September_map-gel2](https://static.igem.org/mediawiki/2016/6/61/T--Evry--map-gel2_16September.png)

# October 2016

###### Gel shown the last results corresponding to the genes selected

The promoters isolated from vectors pSEVA2311 and pSEVA224 were also sent to iGEM HQ after checking by sequencing.

![20-October_gel](https://static.igem.org/mediawiki/2016/8/80/T--Evry--map-gel2_20October.png)

##### For information, PhaC3 and PCT2 without RBS were successfully cloned in pSB1C3 and sent to iGEM HQ (verified by sequencing)

[Check the rest of our note book (.pdf)](https://static.igem.org/mediawiki/2016/c/cd/T--Evry--end-labbook.pdf)

* [Genopole](http://www.genopole.fr/?lang=en "Genopole")
* [IDT](http://eu.idtdna.com/site "Integrated DNA technologies")
* [iSSB](http://www.issb.genopole.fr/ "Institute of Systems & Systhetic biology")
* [UEVE](http://www.univ-evry.fr/fr/index.html "University of Evry-Val-d'Essonne")
* [IBISC](https://www.ibisc.univ-evry.fr/en/start "Informatique, Biologie Intégrative et Systèmes Complexes")
* [République Française](http://fr.franceintheus.org/ "French Ambassy in the United-States")
* [Crous](http://www.crous-paris.fr/ "Crous")
* [New-England Biolab](https://www.neb.com/ "New-England Biolabs")
* [Geneinous](http://www.geneious.com/ "Geneious")
* [Mathworks](http://mathworks.com/ "Mathworks")
* [MIE](http://www.paris.fr/services-et-infos-pratiques/famille-et-education/etudier-a-paris/maison-des-initiatives-etudiantes-2521 "Maison des initiatives étudiantes (House of Student initiatives)")
* [Papeterie GPS](http://www.grandparissud.fr/ "Grand Paris Sud")

 **Evry**2016.