iGEM 2018 — Team Sorbonne_U_Paris

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# Team:Sorbonne U Paris/Notebook

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![logo of iGEM Sorbonne U Paris team. It is a sugar. It is a Rubik's cube shaped sugar ](https://static.igem.org/mediawiki/2018/c/c8/T--Sorbonne_U_Paris--Sugarevolution.png)

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* Biology

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* Notebook

  [Notebook](https://2018.igem.org/Team%3ASorbonne_U_Paris/Notebook)
  [Protocols](https://2018.igem.org/Team%3ASorbonne_U_Paris/Protocols)
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* Team

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* Awards

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* [Site map](https://2018.igem.org/Team%3ASorbonne_U_Paris/Sitemap)

# Laboratory Notebook

A laboratory notebook is our real time record of our expriments. It porvides a detailled record of exactly what we do in the laboratory to obtain our experimental results. It documents our hypotheses, experiments and initial analysis or interpretation of our experiments. Moreover, we also mentionned the name of exprimenters and the place of our experiments. The protocols that we follow are available on protocols page:  [Protocols](https://2018.igem.org/Team%3ASorbonne_U_Paris/Protocols)

## Calendars 2018

| June | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **M** | **T** | **W** | **T** | **F** | **S** | **S** |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | [12](#jun12 "Tuesday 12th June") | [13](#jun13 "Wednesday 13th June") | [14](#jun14 "Thurday 14th June") | 15 | 16 | 17 |
| 18 | 19 | [20](#jun20 "Wednesday 20th June") | 21 | 22 | 23 | 24 |
| 25 | 26 | [27](#jun27 "Wednesday 27th June") | 28 | [29](#jun29 "Friday 29th June") | 30 | 1 |

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **M** | **T** | **W** | **T** | **F** | **S** | **S** |
| [2](#jul2 "Monday 2nd July") | 3 | 4 | [5](#jul5 "Thurday 5th July") | 6 | 7 | 8 |
| [9](#jul9 "Monday 9th July") | [10](#jul10 "Tuesday 10th July") | 11 | 12 | [13](#jul13 "Friday 13th Jult") | 14 | 15 |
| [16](#jul16 "Monday 16th July") | [17](#jul16 "Tuesday 17th July") | 18 | [19](#jul19 "Thurday 19th July") | 20 | 21 | 22 |
| [23](#jul23 "Monday 23th July") | [24](#jul24 "Tuesday 24th July") | [25](#jul25 "Wedneday 25th July") | [26](#jul26 "Thurday 26th July") | [27](#jul27 "Friday 27th July") | 28 | 29 |
| 30 | [31](#jul31 "Tuesday 31th July") |  |  |  |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **M** | **T** | **W** | **T** | **F** | **S** | **S** |
|  |  | [1](#a1 "Wednesday 1st  August") | [2](#a2 "Thurday 2nd August") | [3](#a3 "Friday 3th August") | 4 | 5 |
| [6](#a6 "Monday 6th August") | [7](#a7 "Tuesday 7th August") | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | [21](#a21 "Tuesday 21th August") | [22](#a22 "Tuesday 22th August") | 23 | 24 | 25 | 26 |
| 27 | [28](#a28 "Tuesday 28th August") | [29](#a29 "Tuesday 29th August") | [30](#a30 "Tuesday 30th August") | 31 |  |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **M** | **T** | **W** | **T** | **F** | **S** | **S** |
|  |  |  |  |  | 1 | 2 |
| [3](#s3 "Monday 3th September") | [4](#s4 "Tuesday 4th September") | [5](#s5 "Wednesday 5th September") | [6](#s6 "Thurday 6th September") | [7](#s7 "Fridat 7th September") | 8 | 9 |
| [10](#s10 "Monday 10th September") | [11](#s11 "Tuesday 11th September") | [12](#s12 "Wednesday 12th September") | [13](#s13 "Monday 13th September") | 14 | 15 | 16 |
| [17](#s17 "Monday 17th September") | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |

| October | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **M** | **T** | **W** | **T** | **F** | **S** | **S** |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 |  |  |  |  |  |

## June

## Tuesday 12th June 2018

#### PCR of rubisco intron, paromomycin CDS and pSAD promoter

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Jimmy

|  | Quantity in µL | Mix\*8 |
| --- | --- | --- |
| Plasmid DNA | 1 |  |
| Phusion enzyme | 0.25 | 2 |
| HF buffer (5X) | 5 | 40 |
| dNTPs (10µM) | 0.5 | 4 |
| Fw Primer (10µM) | 1.25 |  |
| Rv Primer (10µM) | 1.25 |  |
| H20 | 15.75 | 126 |

21.5µL of mix by tube + 1µL of DNA + 2\*1.25µL of primer.

pSAD promoter : primers Ig3/Ig4, template p0-07 : Expected length : 807 pb

Paromomycin CDS : primers Ig11/Ig12, template p0-12: Expected length : 1652 pb

Rubisco intron : primers Ig5/Ig6, template p0-02: Expected length : 177 pb

1. negative control : puc 19 + Ig5/Ig6
2. Rubisco intron : p0-02 + Ig5/Ig6
3. negative control : puc 19 + Ig3/Ig4
4. pSAD promoter : p0-07 + Ig3/Ig4
5. negative control : puc 19 + Ig11/Ig12
6. paromomycin CDS : p0-12 + Ig11/Ig12
7. negative control : H20 + Ig5/Ig6
8. negative control : H20 + Ig11/Ig12

Cycle  : 98°C, 35\*(98°C - 1min, 50°C - 2min, 72°C - 3min), 72°C - 3min, 12°C - hold

## Wednesday 13th June

#### Migration and purification of PCR product “paromomycin”, “pSAD”, “rubisco intron”

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

+ 5µL of loading buffer 6X in PCR product
15 µL/well, 1% agarose gel
Migration : 100V, 20 min

| Position | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Content | Ladder | puc19  Ig5/Ig6  rubisco | p0-2  Ig5/Ig6   rubisco | puc19  Ig3/Ig4   pSAD | p0-7  Ig3/Ig4  pSAD | puc19  Ig11/Ig12 paromycine R | p0-12  Ig11/Ig12  paromycine R | H2O  Ig5/Ig6 | H2O  Ig11/Ig12 |
| Expected lenght (pb) |  | 0 | 177 | 0 | 807 | 0 | 1 652 | 0 | 0 |

##### Gel analysis

![Notebook](https://static.igem.org/mediawiki/2018/b/b6/T--Sorbonne_U_Paris--180613GelelectrophoresisPCR.jpg)

Figure 1 : Gel electrophoresis of PCR products rubisco (2&3) intron,pSAD (4&5), paromomycin(6&7)

![Notebook](https://static.igem.org/mediawiki/2018/b/b6/T--Sorbonne_U_Paris--180613GelelectrophoresisPCR.jpg)

Rubisco and pSAD : Ok for rubisco intron and pSAD, visible bands
Paromycin: the main band does not have the expected length, the band at 1.6 kb is still purifed
Cutting of the bands corresponding to the expected bands (photobenchling)

##### DNA purification

Kit used: Nucleospin Gel 2 PCR Clean up- gel extraction (NT1= 800µl)
Elution in 15µl , mesure of the DNA extracts concentration using a nanodrop

| Fragment | Notation | Measured concentration (ng/µL) |
| --- | --- | --- |
| Rubisco intron | i0- IGEM1 | 26,9 |
| pSAD | i0-IGEM3 | 19,3 |
| paromycin | i0-ApHVIII | 7,8 |

Note : We should re-do the PCR of “ paromycine”

##### Launch of 2 bacterial cultures overnight (4ml with spectinomycine)

- p0universal = pAGM9121
- p0B3 pICH41258

## Wednesday 14th June

#### Plasmid purification & Reaction of ligation/digestion

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Victor

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

Aim : Extraction of plasmides from [yesterday’s cultures](#jun13)  and reactions of digestion/ligation p0universal/ pSAD and p0-B3/intron rubisco.

##### Plasmid purification

(p0- B3 = p0B3- pICH41258)

Purification of p0universel and p0- B3 plasmides (kit used: Nucleopsin Plasmid) : Elution volume= 40µL

Measure of DNA extracts concentration using Nanodrop (2 extracts per plasmid)
- p0-universel= 71,5 ng/µl , 64,9ng/µl
- p0-B3= 92,8ng/µl, 90,7ng/µL

##### Reaction of ligation/digestion

Mix DNA

| Tube number | 2 | 6 | 1 | 5 |
| --- | --- | --- | --- | --- |
| µl plasmid | 0,68 | 0,68 | 0,97 | 0,97 |
| µl insert | 2,733 |  | 1,98 |  |
| µl H2O | 10,56 | 13,32 | 11,06 | 13,07 |
|  | pSAD | pSAD | Rubisco | Rubisco |

2: plasmide uni+ pSAD+ enzymes
6: plasmide uni + enzymes
1: plasmides B3+ introns enzymes
5: plasmides B3+ enzymes

Mid enzyme (6µl/tube) number of ligations : 4.5

| Reaction mix | µL | master mix |
| --- | --- | --- |
| BS/HF (µl) | 1 | 4,5 |
| T4 ligase(µl) | 1 | 4,5 |
| Buffer NEB CS (µl) | 2 | 9 |
| ATP 10mM(µl) | 2 | 9 |

Cycle:
- 37°C- 10min \* 3
- 16°C- 10min \*3
- 37°C- 10min
- 65°C- 20min
- 16°C- ∞

## Wednesday 20th June

#### PCR of the “ paromycine” fragment Purification of “p0- IGEM1” and “p0- IGEM3” plasmides

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

##### PCR reactions

| Product | Quantity (µL) | Quantity (µL) |
| --- | --- | --- |
| Plasmide de p0-12 | 1 | ↗ |
| Enzyme Phusion | 0.25 | 2 |
| HF buffer (5X) | 5 | 40 |
| dNTPs (10mM) | 0.5 | 4 |
| Primer F Ig11 (10µM) | 1.25 | 10 |
| Primer F Ig12 (10µM) | 1.25 | 10 |
| DNA | 0 | 1 |
| H20 | 15.75 | 126 |
| Total quantity | 25 | 25 |

|  | DNA (template) | Hybridation temperature |
| --- | --- | --- |
| Tube 1 | p⦽-12 | 50°C |
| Tube 2 | p⦽-12 | 52°C |
| Tube 3 | p⦽-12 | 53°C |
| Tube 4 | p⦽-12 | 54°C |
| Tube 5 | p⦽-12 | 55°C |
| Tube 6 | p⦽-12 | 157°C |
| Controle E | p⦽-19 | 50°C |

Cycle:
98°C
98°C - 1min \*30
50-57°C- 2 min \*30
72°C - 3 min \*30
72°C - 3 min
12°C - ∞

- Addition of 5µl loading buffer 6\* (5\*µL in 25µl)
- Deposit of 30µl of extract on 1% agarose gel
- Migration at 90V for 40 min

Missing: PCR photo
- PCR ok with paromycine at 800pb different from the expected 1657 pb expected before
- Insert Paro stocked at -20°C

##### Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids

Kit used: Gene JET Plasmid-MiniprepKit- Thermo Scientific
Centrifugation 13000 RPM
Digestion to check the size of the inserts (using BsaI)

| Plasmid(µl) | 5 | 5 |
| --- | --- | --- |
| Cut Smart Buffer (10X) (µl) | 11 | 9C |
| Enzyme Bsa (µl) | 0.5 | 4.5 |
| Water (µl) | 3.5 | 31.5 |

Addition of 2µl of loading buffer 6\* (2µl in 10µl)
Deposit of 12µl on 1% agarose gel
Migration at 90V, for 40min

![Notebook](https://static.igem.org/mediawiki/2018/b/bd/T--Sorbonne_U_Paris--180620GelelectrophoresisPCR.jpg)

Figure : Gel electrophoresis : Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids

![Notebook](https://static.igem.org/mediawiki/2018/b/bd/T--Sorbonne_U_Paris--180620GelelectrophoresisPCR.jpg)

We keep : 1a= rubisco intron= p⦽-IGEM1(1b too is conserved)
2a= pSAD- p⦽-IGEM3
Stock in glycerol￫ -30°C (DH5𝛂)mycologue

Note: Transplanting of the microalguae strain that was previously blocked by FedEX

## Wednesday 27th June

#### Bacterial culture

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

Launch of 2\* 3.5 mL of bacterial culture → +spectromycine of pICH4 1264 which contains the destination plasmid of the “paromicycn fragment)

## Wednesday 29th June

#### Purification of pICH41264 plasmid using the Nucleospin Plasmid K Plasmid concentration measures using nanodrop

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

|  |  |  |  |
| --- | --- | --- | --- |
| Culture 1 | 26,7ng/µl | 1.92 | 1.73 |
| Culture 2 | 25,7ng/µl | 1.92 | 2.02 |

The pICH41264 extracts are stocked at -20°C

![Notebook](https://static.igem.org/mediawiki/2018/8/8b/T--Sorbonne_U_Paris--180629Calculsheet1.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/8/8b/T--Sorbonne_U_Paris--180629Calculsheet1.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/5/5e/T--Sorbonne_U_Paris--180629Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/5/5e/T--Sorbonne_U_Paris--180629Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/4/4c/T--Sorbonne_U_Paris--180629Calculsheet3.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/4/4c/T--Sorbonne_U_Paris--180629Calculsheet3.jpg)

## july

## Monday 2 July

#### Transformation of 𝝱-10 bacterias with p⦽ pICH4 1264 + insert paroR Production of p⦽ plasmids for 3’LTR and 5’LTR parts.

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Dounia

##### Transformation of 𝝱-10 bacterias with p⦽ pICH4 1264 + insert paroR

-𝝱-10 bacteria : 30µl
Ligation product (paro): 4µl (insert or negative control)
30min\_ 4°C
55 sec\_ 42°C
2 min\_ 4°C
+700µL of LB medium
1h\_ 37°C
Staggering of 350 on LB+spec+Xgal dish
37°C O/N

##### Production of p⦽ plasmids for 3’LTR and 5’LTR parts.

Resuspension of IDT1 3’LTR in 31,03µL
Resuspension of IDT2 5’LTR in 27,56µL
[C]= 100 fmol/µL

![Notebook](https://static.igem.org/mediawiki/2018/8/8b/T--Sorbonne_U_Paris--180629Calculsheet1.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/1/1f/T--Sorbonne_U_Paris--180702Calculsheet1.png)

![Notebook](https://static.igem.org/mediawiki/2018/3/3d/T--Sorbonne_U_Paris--180702Calculsheet2.png)

![Notebook](https://static.igem.org/mediawiki/2018/3/3d/T--Sorbonne_U_Paris--180702Calculsheet2.png)

![Notebook](https://static.igem.org/mediawiki/2018/1/1c/T--Sorbonne_U_Paris--180702Calculsheet3.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/1/1c/T--Sorbonne_U_Paris--180702Calculsheet3.jpg)

Sub cloning of 2 white colonies of the day before. These colonies are supposed to contain the pICH4 1264 plasmid and the “PARO” insert.

## Thurday 5 July

#### Transplanting of single clones from each subcloning of the 3rd of july Transformation of 𝝱-10 bacterias with p⦽ pUniversal, containing either LTR5’ or LTR3’ as insert

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Dounia

𝝱-10 Bacteria \_ 30µL
Ligation product\_ 4µL (insert or control)
- 30 min\_4°c
- 55 sec\_ 42°C
- 2 min \_ 4°C
+ 700µl of LB
Spreading of 250µl on a dish containing LB + SPEC+ Xgal at 37°C

## Thurday 9 July

#### Lauch of miniprep Verifcation of Paro1 and Paro 2 clones Assembly of GAG-Pol

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

##### Lauch of miniprep of 4 clones of each LTD in media spec+ Xgal

|  |  |
| --- | --- |
| LTR3’1 | LTR5’1 |
| LTR3’2 | 2LTR5’2 |
| LTR3’3 | LTR5’3 |
| LTR3’4 | LTR5’4 |

##### Verifcation of Paro1 and Paro 2 clones

Purification of plasmids (methode for digestion of verification)\*? , using Nucleospin Plasmid Kit

##### Assembly of GAG-Pol ⇨ 6 parts

1) Dilution at 10fmol/µl of each fragment received

| Fragment received | volume of H2O mQ to add (µl) |
| --- | --- |
| IDT3\_L0\_B1 | 1578/10 ⇒ 157,8 |
| IDT4\_L0\_B2 | 3216/10⇒321,6 |
| IDT6\_L0\_B3 | 900/10 ⇒ 90 |
| IDT4\_L0\_A3 | 1098/10⇒109,8 |
| IDT4\_L0\_A1 | 2078/10⇒207,8 |
| IDT4\_L0\_A2 | 1171/10⇒ 117,1 |

2) Reaction of digestion/ligation

![Notebook](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--180709Calculsheet1.png)

![Notebook](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--180709Calculsheet1.png)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/e/e9/T--Sorbonne_U_Paris--180709Calculsheet3.png)

![Notebook](https://static.igem.org/mediawiki/2018/e/e9/T--Sorbonne_U_Paris--180709Calculsheet3.png)

## Thurday 10th July

#### Transformation of 𝝱10 with digestion/ligation products of GAG POL ([experiences of 07/09/18](#jul9))

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

- 30µl of 𝝱+ 4µl of ligation product
- 30min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
- + 700µL of LB

Spreading of 250µL on 2 petri dishes Xgal+ Spec : control/ Gagpol
Incubation at 30°C over night

## Friday 13th July

#### Launch of minipreps for 6 clones of GAG-Pol

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Asmaa

##### Launch of minipreps for 6 clones of GAG-Pol in Xgal+spec media GAG\_Pol 1, GAG\_Pol 2, GAG\_Pol 3, GAG\_Pol 4 , GAG\_Pol 5, GAG\_Pol 6

Purification of PAGM9121 plasmide using Nucleopsin Plasmid Kit

##### Measure of DNA concentration at Nanodrop

| Fragment | Concentration (ng/µl) |  |  |
| --- | --- | --- | --- |
| LTR3’1 | 42,2 | 1.97 | 1.85 |
| LTR3’2 | 38,4 | 1.98 | 1.86 |
| LTR3’3 | 37,7 | 1.95 | 1.64 |
| LTR3’4 | 94,1 | 1.91 | 2.06 |
| LTR5’1 | 48,4 | 1.95 | 1.86 |
| LTR5’3 | 120 | 1.89 | 1.94 |
| Paromycin 1 | 55,8 | 1.91 | 1.89 |
| Paromycin 2 | 49,1 | 1.91 | 1.87 |
| GAG\_Pol 1 | 78,7 | 1.89 | 1.92 |
| GAG\_Pol 2 | 25,3 | 1.97 | 2.85 |
| GAG\_Pol 3 | 49,0 | 1.94 | 2.62 |
| GAG\_Pol 4 | 17,7 | 2.00 | 3.90 |
| GAG\_Pol 5 | 44,0 | 1.97 | 2.68 |
| GAG\_Pol 6 | 21,2 | 2.01 | 3.65 |

##### Digestion to check the inserts size ( using BsaI)

| Component | 10 µl reaction |
| --- | --- |
| DNA | 200 ng (except for GAG pol 4 & 6 : DNA=150ng) |
| 10X CutSmart Buffer | 1µl |
| BsaI | 0.2µ |
| Nuclease-free Water | to 50 µl |

## Monday 16th July

#### Migration of digestion reaction products

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anaïs

##### Migration of digestion reaction products from the day before

![Notebook](https://static.igem.org/mediawiki/2018/2/24/T--Sorbonne_U_Paris--180716MigrationDigestion.jpg)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size

![Notebook](https://static.igem.org/mediawiki/2018/2/24/T--Sorbonne_U_Paris--180716MigrationDigestion.jpg)

Agarose gel 1%
2µL loading buffer 6X per 10µl sample + BET
Migration 100V, 20 min

## Tuesday 17th July

#### Arrangement in the box iGEM 2018 Preparation of all the inserts in order to send them to sequencing

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anaïs

##### Arrangement in the box iGEM 2018

##### Preparation of all the inserts in order to send them to sequencing

![Notebook](https://static.igem.org/mediawiki/2018/5/50/T--Sorbonne_U_Paris--180717Inserts1.png)

![Notebook](https://static.igem.org/mediawiki/2018/5/50/T--Sorbonne_U_Paris--180717Inserts1.png)

![Notebook](https://static.igem.org/mediawiki/2018/3/34/T--Sorbonne_U_Paris--180717inserts2.png)

![Notebook](https://static.igem.org/mediawiki/2018/3/34/T--Sorbonne_U_Paris--180717inserts2.png)

[C] Nanodrop
PSAD 2a: 107.9 ng/µL
1.97
3.81
PSAD 2d: 150.6 ng/µl
1.94
3.12
Intron Rb= 103 ng/µL
1.99
3.82

[AMG 9121]: 71.5 ng/µl
Samples sent to sequencing

## Thursday 19th July

#### Analysis of sequencing results

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anaïs

| Fragment | Comments |
| --- | --- |
| Paromycine | ok |
| Gagpol (GG40 and GG41)   Rubisco intron   pSAD | Absurd results   Re-do the digestion reaction whith different BsaI enzymes |

## Monday 23th July

#### Transformation to redo the stock of each plasmid

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anaïs

- 10µL of 𝝱10 bacterias
- Gag Pol 5 : 1.5 µL
Gag Pol 3 : 0.5 µL
Igem 2, 4 and 6: 2µL
Igem 3, 2 : 2.5 µL
↳ plasmids to transfect

- 30 min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
+ 700 µl of LB

Spreading of 250µL on plates containing LB+ spec

## Thurday 24th July

#### Transplanting of a clamydomonas clone Plasmid extraction from 𝝱 bacterias transformed Concentration measurements

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Dounia

##### 1. Transplanting of a clamydomonas clone in 3ml of LB+ Spec

##### 2. Plasmid extraction from 𝝱 bacterias transformed [the day before](#jul23) ( directly from dish).

Kit used = Nucleopsin Plasmid
Elution volum= 40µL

##### 3. Concentration measurements using Nanodrop

| Fragment | Concentration(ng/µl) |  |  |
| --- | --- | --- | --- |
| LTR3’2 | 241.3 | 1.90 | 2.17 |
| ParoII | 196.0 | 1.90 | 2.09 |
| LTR5’ | 166.2 | 1.89 | 2.15 |
| ParoI | 160.9 | 1.91 | 2.11 |
| LTR3’4 | 232.8 | 1.89 | 2.21 |
| Gag5 | 393.6 | 1.91 | 2.16 |
| Rubisco | 31.3 | 2.04 | 2.09 |
| pSAD | 133.8 | 1.90 | 2.12 |
| Gag3 | 370.6 | 1.91 | 2.11 |

##### 4. Replanting of Chlamydomonas

## Wednesday 25th July

#### Plasmid extraction from bacteria transformed Concentration measurement Digestion

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anais

##### Plasmid extraction from [bacteria transformed](#jul24)

Put on liquid media the 24/07
 kit used : Nucleopsin plasmid

##### Concentration measurement using Nanodrop

| Fragment | Concentration(ng/µl) |  |  |
| --- | --- | --- | --- |
| Paro II | 110.4 | 1.89 | 2.21 |
| PSAD | 62.1 | 1.89 | 2.21 |
| GagPol 5 | 231.6 | 1.89 | 2.21 |
| ParoI | 96.4 | 1.89 | 2.18 |
| LTR5’ | 65.4 | 1.90 | 2.17 |
| LTR3’2 | 102.3 | 1.91 | 2.26 |
| LTR3’4 | 111.0 | 1.88 | 2.29 |
| Gag Pol3 | 189.8 | 1.90 | 2.21 |
| Intron Rubisco | 73.9 | 1.89 | 2.19 |

##### Digestion

|  | Intron | PSAD | LTR5’1 | LTR3’4 | LTR3’2 | Uni | GagPol3 | GagPol5 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Enzyme | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 | 0.2 |
| Buffer | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| ADN | 2 | 2 | 4 | 2 | 5 | 3 | 3 | 3 |
| H2O | 6.8 | 6.8 | 4.9 | 6.8 | 3.9 | 5.9 | 5.9 | 5.9 |
|  | BstEII   SaeI | BstEI   StuI | BstEII   BamHI | BstEII   BamHI | BstEII   BamHI | BstEII   BamHI | BstEII | BstEII |

Digestion at 37°C/ 1H , conservation at -20°C overnight

## Thurday 26th July

#### Migration of digestion samples Digestion Migration on gel electrophoresis

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Anais

##### Migration of digestion samples from the day before

1% agarose gel, deposit of 10µ+2µL of loading buffer 6X, ladder “2log”
 Gel plan

| Rb intron | pSAD | LTR3’2 | LTR3’4 | LTR5’4 | 9121 | Gag3 | Gag5 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 | 1 3 | 1 3 |
| 3 bands   1714 pb   620 pb   66 pb | 2 bands   1662 pb   1182 pb | 1 band  2000-3000pb   (2270 pb) | | 1 band   2270 pb | 2 band   11831pb  828 pb | 2 bands   4242 pb   2260 pb | |

![Notebook](https://static.igem.org/mediawiki/2018/f/fd/T--Sorbonne_U_Paris--M180726Migrationsample.jpg)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size

![Notebook](https://static.igem.org/mediawiki/2018/f/fd/T--Sorbonne_U_Paris--M180726Migrationsample.jpg)

There seems to be no inserts

##### Digestion

- NDE I, 1h, 37°C
- Tampon 10X : 1µl
- ADN : 3µL
- Nde I : 0.2 µL
- H2O : 5.8 µ

##### Migration on gel

| Rb Intron | pSAD | LTR3’2 | LTR3’4 | LTR5’1 | GagP3 | Gag P5 | Paro I | Paro II |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 2 | 1 2 | 1 2 | 1 2 | 1 2 | 1 | 1 | 1 2 | 1 2 |

Non informative, forgotten time of incubation

## Friday 27th July

#### Enzymatic digestion reaction

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### Enzymatic digestion reaction ( fast digest enzyme)

|  | 1 reaction | mix \*20 |
| --- | --- | --- |
| Bcl Enzyme | 0.2 µL | 4 µL |
| Buffer FD (10X) | 1 µL | 20µL |
| DNA | 3 µL | ↗ |
| H2O | 5.8 µL | 116 µL |

↪7µl mix/ per tube
+ 3µL DNA
+ 0.2 µl XmaII in pSAD

Incubation 2H at 37°C
Migration in 0.1 % agarose gel, ladder “2log”, deposit of 10µL of reaction sample + 2µl de loading buffer 6X

![Notebook](https://static.igem.org/mediawiki/2018/7/79/T--Sorbonne_U_Paris--180727GelPlan.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/7/79/T--Sorbonne_U_Paris--180727GelPlan.jpg)

>

## Tuesday 31th July

#### DNA sent to sequencage Digestion reaction using BSaI

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. DNA sent to sequencage

pSAD
 AYL0014041 ← GG40
 042 ← GG41

##### Digestion reaction using BSaI

|  | 1 reaction | mix \*12 |
| --- | --- | --- |
| Bcl Enzyme | 0.2 µL | 24 µL |
| Buffer Cutsmart (10X) | 1 µL | 12µL |
| DNA | 4 µL | ↗ |
| H2O mq | 4.8 µL | 57.6µL |

Incubation 3h 37°C
Migration on 1% agarose gel of 10µl sample + 2µL loading buffer
Ladder 2Log
90V, 20 minutes

![Notebook](https://static.igem.org/mediawiki/2018/8/89/T--Sorbonne_U_Paris--180731Gelectrophoresis1.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/8/89/T--Sorbonne_U_Paris--180731Gelectrophoresis1.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/1/16/T--Sorbonne_U_Paris--180731Gelectrophoresis2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/1/16/T--Sorbonne_U_Paris--180731Gelectrophoresis2.jpg)

OK for gag pol 3’ and 5’ LTR, Rb+ paro Sequencing
pSAD not ok → replanting of colonies

## August

## Wednesday 1st August

#### Sending samples to sequencing Cloning of pSAD

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. Sending samples to sequencing

- GAG 3 (10µL DNA+ 10µL H2O)
- GAG 5 (10µL DNA+ 10µL H2O)
- 3’ LTR (3’4) (10µL DNA+ 5 µL H2O)
- 5’ LTR (10µL DNA+ 5 µL H2O)
- Rb (10µL DNA+ 5 µL H2O)
- GG40 (50µL) and GG41 (10µL)
-Ig18 (20µL), Ig19, Ig20 (10µL)
Stock from 07/25/18

![Notebook](https://static.igem.org/mediawiki/2018/7/7b/T--Sorbonne_U_Paris--180801Sequencingname.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/7/7b/T--Sorbonne_U_Paris--180801Sequencingname.jpg)

##### Cloning of pSAD from 07/23/2018 experiment, in solid LB+ spec

## Thurday 2nd August

#### Replanting of PSAD and Rb intron

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

Replanting of PSAD and Rb intron in liquid media+ spec .

## Friday 3th August

#### Miniprep of PSAD and Rb

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

Miniprep of PSAD and Rb (5 clones of each)
Kit used: Nucleopsin Plasmid

## Monday 6th August

#### Digestion of the 5 new clones of pSAD and Rb

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

|  | 1 reaction | mix \*12 |
| --- | --- | --- |
| BSAI Enzyme | 0.2 µL | 2.4 µL |
| Buffer Cut Smart (10X) | 1 µL | 12µL |
| DNA | 4 µL | ↗ |
| H2O mq | 4.8 µL | 57.6µL |

6µl of mix per tube + 4µl of DNA
Digestion 2H at 37°C+ 2µL of loading buffer 6X, 4µL of 2log ladder
Migration 90V, 40min

![Notebook](https://static.igem.org/mediawiki/2018/f/f5/T--Sorbonne_U_Paris--180806Gelectrophoresis.jpg)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size : 5 new clones of pSAD and Rb

![Notebook](https://static.igem.org/mediawiki/2018/f/f5/T--Sorbonne_U_Paris--180806Gelectrophoresis.jpg)

## Tuesday 7th August

#### Digestion/ ligation reaction of pSAD 2 Rb intron Transformation Preparation X-GAL

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. Digestion/ ligation reaction of pSAD 2 Rb intron

[see 06/14/2018 protocol](#jun14)

##### 2. Transformation

-90µl of DH5α +Ligation product +10µl CMR
30 min 4°C
90 sec at 42°C
2 min on ice
700µL LB
1h at 37°C
spreading on 2 dishes

##### 3. Preparation X-GAL

40mg ⇨ 2mL DMSO

## Tuesday 21th August

#### New PCR of “pSAD” and “rubisco” intron

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

Reaction Mix

| Solutions | Volume for 1 tube (uL) | Volume for 7 tubes (uL) |
| --- | --- | --- |
| Plasmid DNA | 1 | - |
| Phusion Enzyme | 0.25 | 1.75 |
| HF Buffer (5X) | 5 | 35 |
| dNTP | 0.5 | 3.5 |
| Forward Primer | 1.25 | - |
| Reverse Primer | 1.25 | - |
| H2O | 15.75 µL | 110.25 |

- 21.5 uL of Mix/Tube
- 1 uL of DNA
- 2 X 1.25 uL Primer

pSAD = Ig3/Ig4 primers + template p0.07 → 807 bp
Rb intron = Ig5/Ig6 primers + template 0.07 → 177 bp

1- p007 + Ig3/Ig4
2- pUC19 + Ig3/Ig4
3- H2O + Ig3/Ig4
4- p0-02 + Ig5/Ig6
5- pUC19 + Ig5/Ig6
6- H2O + Ig5/Ig6

## Tuesday 22th August

#### Migration of the PCR products and the fragments of GAG Pol Purification of the product the gel of the 2 PCRs: Cloning of pSAD

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

##### 1. Migration of the PCR products and the fragments of GAG Pol sent by IDT

Products of the PCR à OK
IDT4 L0A1 + IDT4 L0A2 + IDT4 L0A3 + IDT3 L0B1 à OK

No bands for IDT4 L0B2 Were the samples mixed during the loading?
Plusieurs bands for IDT6 L0B3

##### 2.Purification of the product the gel of the 2 PCRs

Elution in 20 uL
Used kit: Nucleospin Gel and PCR clean up

Concentration measurements using the Nanodrop:
- PSAD= 39.8 ng/uL
- Rb= 55.1 ng/uL

##### 3. Cloning of pSAD

![Notebook](https://static.igem.org/mediawiki/2018/f/ff/T--Sorbonne_U_Paris--180822Cloningpsad.png)

![Notebook](https://static.igem.org/mediawiki/2018/f/ff/T--Sorbonne_U_Paris--180822Cloningpsad.png)

##### Intron Rubisco

![Notebook](https://static.igem.org/mediawiki/2018/f/f3/T--Sorbonne_U_Paris--180822Intronrubisco.png)

![Notebook](https://static.igem.org/mediawiki/2018/f/f3/T--Sorbonne_U_Paris--180822Intronrubisco.png)

##### Ligation

![Notebook](https://static.igem.org/mediawiki/2018/4/41/T--Sorbonne_U_Paris--180822Ligation.png)

![Notebook](https://static.igem.org/mediawiki/2018/4/41/T--Sorbonne_U_Paris--180822Ligation.png)

##### Cycle

![Notebook](https://static.igem.org/mediawiki/2018/6/6a/T--Sorbonne_U_Paris--180822Cycle.png)

![Notebook](https://static.igem.org/mediawiki/2018/6/6a/T--Sorbonne_U_Paris--180822Cycle.png)

## Tuesday 28th August

#### Plasmidic extraction of bacteria transformed Measure of the concentration Digestion of the plasmid by BstEII and SalI Preparation of DNA samples for sequencing

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

##### Plasmidic extraction of bacteria transformed by plasmid containing “Rubisco intron”

Used kit : NucleoSpin Plasmid (Macherey-Nagel)

##### Measure of the concentration at Nanodrop

Rb 1 : 109.9 ng/µl
Rb 2 : 96.8 ng/µl
Rb 3 : 77.2 ng/µl
Rb 4 : 88.7 ng/µl

##### Measure of the concentration at Nanodrop

Digestion of the plasmid by BstEII and SalI
Goal : Check if the plasmid effectively contain the insert “Rubisco intron”

Expected result :

![Notebook](https://static.igem.org/mediawiki/2018/b/b4/T--Sorbonne_U_Paris--180828Expectedresults.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/b/b4/T--Sorbonne_U_Paris--180828Expectedresults.jpg)

| Lader (2log) | Rb1+ | Rb2+ | Rb3+ | Rb4\* |
| --- | --- | --- | --- | --- |

![Notebook](https://static.igem.org/mediawiki/2018/3/30/T--Sorbonne_U_Paris--180828Gelelectrophoresis.png)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size

![Notebook](https://static.igem.org/mediawiki/2018/3/30/T--Sorbonne_U_Paris--180828Gelelectrophoresis.png)

##### Preparation of DNA samples for sequencing

![Notebook](https://static.igem.org/mediawiki/2018/5/50/T--Sorbonne_U_Paris--180828.png)

![Notebook](https://static.igem.org/mediawiki/2018/5/50/T--Sorbonne_U_Paris--180828.png)

add 2 µl of primers at 10 µL for each tube

![Notebook](https://static.igem.org/mediawiki/2018/8/8f/T--Sorbonne_U_Paris--180828DNAsequence2.png)

![Notebook](https://static.igem.org/mediawiki/2018/8/8f/T--Sorbonne_U_Paris--180828DNAsequence2.png)

##### Concentration measurement on Nanodrop of recieved parts for trehalose

Provider : DC biosciences

| Part | DNA concentration (ng/µl) |
| --- | --- |
| pUC-SP VS7 otsA\_B21 | 165.3 |
| pUC-SP VS8 otsA\_B22 | 103.9 |
| pUC-SP VS9 otsA\_B23 | 107.6 |
| pUC-SP VS10 otsB\_B41 | 242.7 |
| pUC-SP VS11 otsB\_B42 | 81.2 |

## Tuesday 29th August

#### Sample preparation to send “rubisco intro” at sequencing

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Ursula

![Notebook](https://static.igem.org/mediawiki/2018/3/3e/T--Sorbonne_U_Paris--180829Rubiscointrosequencing1.png)

![Notebook](https://static.igem.org/mediawiki/2018/3/3e/T--Sorbonne_U_Paris--180829Rubiscointrosequencing1.png)

Add 2µl of primers at 10µM

![Notebook](https://static.igem.org/mediawiki/2018/8/86/T--Sorbonne_U_Paris--180829RubiscoIntrosequencing2.png)

![Notebook](https://static.igem.org/mediawiki/2018/8/86/T--Sorbonne_U_Paris--180829RubiscoIntrosequencing2.png)

## Tuesday 30th August

#### Digestion/ligation reaction of otsA and otsB Transformation of B10 bacteria with ligation product PlCH47811 plasmid extraction Replanting of 𝝱10 clones transformed Checking of DNA content of IDT4 sample for Gag Po

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

##### 1. Digestion/ligation reaction of otsA and otsB

![Notebook](https://static.igem.org/mediawiki/2018/0/0a/T--Sorbonne_U_Paris--180830Calculsheet1.png)

![Notebook](https://static.igem.org/mediawiki/2018/0/0a/T--Sorbonne_U_Paris--180830Calculsheet1.png)

![Notebook](https://static.igem.org/mediawiki/2018/b/b1/T--Sorbonne_U_Paris--180830Calculsheet2.png)

![Notebook](https://static.igem.org/mediawiki/2018/b/b1/T--Sorbonne_U_Paris--180830Calculsheet2.png)

![Notebook](https://static.igem.org/mediawiki/2018/5/5a/T--Sorbonne_U_Paris--180830Calculsheet3.png)

![Notebook](https://static.igem.org/mediawiki/2018/5/5a/T--Sorbonne_U_Paris--180830Calculsheet3.png)

![Notebook](https://static.igem.org/mediawiki/2018/7/7d/T--Sorbonne_U_Paris--180830Calculsheet4.png)

![Notebook](https://static.igem.org/mediawiki/2018/7/7d/T--Sorbonne_U_Paris--180830Calculsheet4.png)

##### 2. Transformation of B10 bacteria with ligation product

- 25µl of B10 bacteria + 4µl of ligation product (4 different transformations)
- 30 min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
+700µl of LB medium
1 h at 37°C

Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal

##### 3. PlCH47811 plasmid extraction from overnight culture

kit used: Machinery Nagel easypure kit plasmid DNA extraction

##### 4. PlCH47811 plasmid DNA concentration measurement on Nanodrop

a : 52.8 ng/µl 1.91 1.98
b : 39.8 ng/µl 1.90 1.84

##### 5. Replanting of 𝝱10 clones transformed the day before on LB media + spectinomycine

2 clones of OTSA : OTSA1 OTSA2 (one culture per clone)
2 clones of OTSB : OTSB1 OTSB2 (one culture per clone)
1 clone of pGAM 1276
1 clone of pAGM1299

the 6 cultures previously described are placed on a will for over week/end culture

##### 6. Checking of DNA content of IDT4 sample for Gag Pol

The tube is vortexed upside down then centrifuged (to check if the DNA is not stuck on the tube’s lid)
DNA concentration measurement on nanodrop:
(blank : H2O)
1.5ng/µL
1.6ng/µL
1.5ng/µl

## September

## Monday 3th September

#### Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R Miniprep of otsA, otsB and pAGM1276 and pAGM1299 Concentration measurement Level 0 ligation protocol for GAGPOL parts and Level 1 ligation protocol for the CARGO retrotransposon

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R received today according to Eurofins instructions

Then, hybridation during 15 minutes (there are in fact, the missing DNA fragment of GAGPOL)
.

##### 2. Miniprep on the over week-end culture of otsA, otsB and pAGM1276 and pAGM1299

Used kit : NucleoSpin Plasmid (Macherey-Nagel)

##### 3. Concentration measurement at Nanodrop

- pAGM1276 : 56ng/µl
- pAGM1299 : 76.6ng/µl
- otsA 1 : 78.4ng/µl
- otsA 2 : 75.6ng/µl
- otsB 1 : 73.1ng/µL
- otsB2 : 78.2ng/µl

##### 4. Level 0 ligation protocol for GAGPOL parts : [(see 09/07/2018)](jul9)

![Notebook](https://static.igem.org/mediawiki/2018/e/eb/T--Sorbonne_U_Paris--180903Gagpolparts.png)

![Notebook](https://static.igem.org/mediawiki/2018/e/eb/T--Sorbonne_U_Paris--180903Gagpolparts.png)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/4/4a/T--Sorbonne_U_Paris--180903Gagpolpartscycle.png)

![Notebook](https://static.igem.org/mediawiki/2018/4/4a/T--Sorbonne_U_Paris--180903Gagpolpartscycle.png)

##### 5. Level 1 ligation protocol for the CARGO retrotransposon

![Notebook](https://static.igem.org/mediawiki/2018/a/ad/T--Sorbonne_U_Paris--180903Cargoretrotransposon.png)

![Notebook](https://static.igem.org/mediawiki/2018/a/ad/T--Sorbonne_U_Paris--180903Cargoretrotransposon.png)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/2/2b/T--Sorbonne_U_Paris--180709Calculsheet2.jpg)

## Tuesday 4th September

#### Digestion of otsA and otsB ligation reaction Transformation of B10 bacteria with cloning reaction

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

##### 1. Digestion of otsA and otsB ligation reaction of [03/08/2018](#a3)

Digestion reaction of otsA :

- bstEII-HF : 0.2µl
- CutSmart buffer (10X) : 1 µl
- DNA plasmid : 3µl
- H20: 5.8µl

Digestion reaction of otsB :

- bstEII-HF : 0.1µl
- NotI-HF : 0.1µl
- CutSmart buffer (10X) : 1µl
- DNA plasmid : 3µl
- H20 : 5.8µl

Electrophoresis on 0.1% agarose, with 2 log ladder and 12µl of reaction

![Notebook](https://static.igem.org/mediawiki/2018/thumb/a/ad/T--Sorbonne_U_Paris--180904Expectedresults.jpg/800px-T--Sorbonne_U_Paris--180904Expectedresults.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/thumb/a/ad/T--Sorbonne_U_Paris--180904Expectedresults.jpg/800px-T--Sorbonne_U_Paris--180904Expectedresults.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/9/91/T--Sorbonne_U_Paris--180904Gelelectrophoresis.png)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size

![Notebook](https://static.igem.org/mediawiki/2018/9/91/T--Sorbonne_U_Paris--180904Gelelectrophoresis.png)

The band on the gel are correct but there is a lot od DNA on the top of the gel and the secnd band is very weak. Morover, the sequence we ordered for otsB is wrong, so we need to correct it by PCR. So we will ordered primers and we’ll send to sequence after the PCR. For otsA, we’ll make an other transformation to select only one pure clone

##### 2. Transformation of B10 bacteria with cloning reaction of [03/09](#s3) : p1-iGEM1 and p0-iGEM5

25µl of B10 bacteria + 4µl of ligation product (4 different transformations)
30 min at 4°C
55 sec at 42°C
2 min at 4°C
+700µl of LB medium
1 h at 37°C

Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal for p0-iGEM5
Spreading of 250 µl in plates containing LB+ampicilin+X-gal for p1-iGEM2

## Wednesday 5th September

#### Transformation of B10 bacteria with cloning reaction p1-iGEM1 and p0-iGEM5 Replanting of white colonies from p0-iGEM-GAGPO Re-do of L1 ligation of p1-iGEM2 (Cargo)

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

##### 1. Transformation of B10 bacteria with cloning reaction of [03/09](#s3) : p1-iGEM1 and p0-iGEM5

Observation of plates from the day before
- p1-iGEM2 (Cargo) : only blue colonies for + and - controle
- p0-iGEM5- GagPol: only blue colonies for the positive (+) experiment , and both white and blue colonies for the negative (-) control

##### 2. Replanting of white colonies from p0-iGEM-GAGPOL (-)on liquid media

Composition of the media :Amp+ Spec+ Xgal
4 colonies were replanted

##### Re-do of L1 ligation of p1-iGEM2 (Cargo) [(same experiment as the 09/03/2019)](#s3)

## Tuesday 6th September

#### PDNA extraction of 4 differents clones p0-iGEM5 Concentration measurement Verification digestion of p0-iGEM5 plasmid Transformation of bacteria with the ligation produc

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Soukeyna

##### 1. DNA extraction of 4 differents clones p0-iGEM5

Kit utilisé : NucleoSpin Plasmid (Macherey-Nagel)

##### 2. Concentration measurement at Nanodrop

p0-iGEM5-1 : 97.8ng/µl
p0-iGEM5-2 : 89.5ng/µl
p0-iGEM5-3 : 123.4ng/µl
p0-iGEM5-4 : 67.6ng/µL

##### 3.Verification digestion of p0-iGEM5 plasmid

|  | Clone 1 and 3 | Clone 2 and 4 |
| --- | --- | --- |
| bstEII enzyme | 0.2µl | 0.2µl |
| Plasmid DNA | 2µl | 3µ |
| NEB cutsmart buffer (10X) | 1µl | 1µ |
| H20 | 6.8µl | 5.8µl |

![Notebook](https://static.igem.org/mediawiki/2018/d/da/T--Sorbonne_U_Paris--180906expectedresults.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/d/da/T--Sorbonne_U_Paris--180906expectedresults.jpg)

![Notebook](https://static.igem.org/mediawiki/2018/c/cc/T--Sorbonne_U_Paris--180906Gelelectrophoresis.png)

Figure : Gel electrophoresis of digestion reaction products to check the inserts size

![Notebook](https://static.igem.org/mediawiki/2018/c/cc/T--Sorbonne_U_Paris--180906Gelelectrophoresis.png)

##### 4. Transformation of bacteria with the ligation product of the 05/09

25µl of bacteria + 3µl of ligation product
30min at 4°C
55sec at 42°C
2min at 4°C
+700µl of LB medium on each tube
1h at 37°C
Spreading of 200µl of each transformation on LB + Xgal + amp + IPTG plate
Oovernight at 37°C

## Friday 7th September

#### Ligation reaction of p0-iGEM5 (GAGPOL) otsB PCR

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. Ligation reaction of p0-iGEM5 (GAGPOL)

![Notebook](https://static.igem.org/mediawiki/2018/8/88/T--Sorbonne_U_Paris--180907p0-iGEM5.png)

![Notebook](https://static.igem.org/mediawiki/2018/8/88/T--Sorbonne_U_Paris--180907p0-iGEM5.png)

![Notebook](https://static.igem.org/mediawiki/2018/8/88/T--Sorbonne_U_Paris--180907p0-iGEM5_Mix.png)

![Notebook](https://static.igem.org/mediawiki/2018/8/88/T--Sorbonne_U_Paris--180907p0-iGEM5_Mix.png)

![Notebook](https://static.igem.org/mediawiki/2018/f/f9/T--Sorbonne_U_Paris--180907p0-iGEM5cycle.png)

![Notebook](https://static.igem.org/mediawiki/2018/f/f9/T--Sorbonne_U_Paris--180907p0-iGEM5cycle.png)

##### 2. otsB PCR

- p0-iGEM8 (otsB) : 1µl at 2ng/µl
- Enzyme Phusion : 0.25µl
- HF Buffer (5X) : 5 µl
- dNTP : 0.5µl
- Primer OTSB-v2-F : 1.25 µl
- Primer OTSB-v2-R : 1.25µl
-H20 : 15.75 µl

|  | \*30 | | |  |  |
| --- | --- | --- | --- | --- | --- |
| 98°C | 98°C | 50°C | 72°C | 72°C | 12°C |
| 1min | 1 min | 2 min | 3 min | 3 min | Hold |

##### 3. 6 colonies [from yesterday](s6) transformation p1igem2 in LB medium + amp

## Monday 10th September

#### DNA extraction of the 6 bacteria culture Concentrations measurement Digestion reaction to check p1-iGEM2 cloning reaction Transformation of B10 bacteria

###### Institut de biologie physico-chimique (IBPC)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Aurélie

##### 1. DNA extraction of the 6 bacteria culture prepare on [7/09](#s7)

Used kit : NucleoSpin Plasmid (Macherey-Nagel)

##### 2. Concentrations measurement at Nanodrop

#1 : 101.5ng/µl
#2 : 90.0ng/µl
#3 : 97.7ng/µL
#4 : 75.1ng/µl
#5 : 61.5ng/µl
#6 : 115.6ng/µl

##### 3. Digestion reaction to check p1-iGEM2 cloning reaction

| NcoI | 0.1µl | 6.5µl |
| --- | --- | --- |
| NdeI | 0.1µl | 6.5µL |
| NotI | 0.1µl | 6.5µl |
| Cutsmart buffer 10X | 1µl | 6.5µl |
| ADN | 2.5µl |  |
| H2O | 6.2µl | 40.3µl |

Used kit : NucleoSpin Plasmid (Macherey-Nagel)

##### Concentrations measurement at Nanodrop

7.5µl of mix by tube + 2.5µl of DNA

On 1% agarose gel, with otsB PCR from [7/09](#s7) : expected result : >800bp

![Notebook](https://static.igem.org/mediawiki/2018/thumb/8/82/T--Sorbonne_U_Paris--180910Expectedresults.png/800px-T--Sorbonne_U_Paris--180910Expectedresults.png)

![Notebook](https://static.igem.org/mediawiki/2018/thumb/8/82/T--Sorbonne_U_Paris--180910Expectedresults.png/800px-T--Sorbonne_U_Paris--180910Expectedresults.png)

##### 4. Transformation of B10 bacteria with ligation product from [07/09](#s7)

B10 bacteria + 3.5µl of ligation product
- 30 min at 4°C
- 55sec at 42°C
- 2min at 4°C
- +700µl of LB medium
- 1h at 37°C
- Spreading of 200µl on plate with LB+spec+Xgal

## Tuesday 11th September

#### Digestion of p1-IGEM2 with bbsI Transformation of bacteria with Gagpol

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Sara and Parisa

##### 1. Digestion of p1-IGEM2 with bbsI

| Components | 1 reaction | Mix (\*6) |
| --- | --- | --- |
| bbsI | 0.5µL | 3 µL |
| Cutsmart buffer (10X) | 1 µL | 6 µL |
| DNA | 2.5 µL | 6.5µl |
| H2O | 6 µL | 36µL |

##### 2. Transformation of bacteria with Gagpol

2 white colonies were observed on the plate containing bacterias transformed with GAGPOL
- Replanting on liquid media
- Replanting on plate (not isolable)

## Wednesday 12th September

#### Migration of the digestion products from the day before (pI-IGEM2) Plasmid extraction from the GAG clone culture Purification of DNA contained in the migration gel for OtsB PCR products Digestion reaction for Gag POL Level 0 Otsb (digestion/ligation reaction)

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Sara and Parisa

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Victor

##### 1. Migration of the digestion products from the day before (pI-IGEM2)

(0.1% agarose gel, 2-log ladder, 100V)
We were expecting a 4.5kb band for the plasmid and a 2.6kb band for the insert.
We do have the expected band for the plasmid but, we observe a band at 0.6kb for the insert (Lac Z maybe?)

##### 2. Plasmid extraction from the GAG clone culture launched the day before

Kit used Nucleospin Plasmid

##### 3. Purification of DNA contained in the migration gel for OtsB PCR products [( from 09/07/2018)](#s7)

Kit used : Nucleospin gel to PCR clean up

##### 4. DNA concentration measurement on Nanodrop

Insert OtsB= 4ng/µL
GAG palamsid = 48.1 ng/µl

##### 5. DNA concentration measurement on Nanodrop

Insert OtsB= 4ng/µL
GAG palamsid = 48.1 ng/µl

##### 6. Level 0 Otsb (digestion/ligation reaction)

→ purification of PCR band and Launch of miniprep
(To do next level 1 pI-Igem2)

## Tuesday 13th September

#### Transformation of 𝛃10 bacterias with OtsB ligation products DNA plamsid extraction of pO-IGEM5 Digestion reaction

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Sara

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Victor

##### 1. Transformation of 𝛃10 bacterias with OtsB ligation products obtained the day before

𝛃10+ 4µL of OtsB ligation reaction products
- 30min at 4°C
- 55 sec at 42°C
- 2 min at 4°C
- Addition of 70µL of LB

1 Hour at 37°C
Spreading of 200µL on LB+Xgal + Spec plate
37°C O/N

##### 2. DNA plamsid extraction of pO-IGEM5 launch on liquid media yesterday

Kit used Nucleospin Plasmid

##### 3. Nanodrop measurement for p0-IGEM5

26.4 ng/µL

##### 4.Digestion reaction

bbsI =0.5µL
buffer= 2µL
DNA =10µL
H2O= 6.5µL
Expected result: 4,4kb + (?)

##### 5. Gel migration of digestion reaction products

ast well = digestion reaction product of GagPOL from the day before
→ Does not correspond to the expected lenght

## Monday 17th September

#### Ligation Migration of the Gag Pol sample Preculture of OTSB (7 white colonies)

###### Muséum national d'histoire naturelle (MNHN)

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Sara

![Person](https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png)
Victor

##### 1. Ligation

3 ligation tubes are prepared , T-, G, pre-mix of E, T-
- Tube 1 : T- 1.2µl of blackbone +12.8 µL of H2O (14µL in total)
- Tube 2 : G 2 µL of the 6 sub-parts for GagPol)+.0.8µl of H2O +1.2µL of backbone ( 14µl in total)
Tube 3: Master mix of enzymes

| Components | 1 reaction |
| --- | --- |
| ATP | 4 µL |
| Cutsmart buffer (10X) | 4 µL |
| T4 ligase | 2 µL |
| bbstI | 2 µL |

12µL of mix in total dispatched in tubes 1 and 2 (6µL each)

##### 2. 1% agarose gel preparation, in order to migrate the Gag Pol sample

- 0.5g of agarose
- 50ml of TEA 1X
- Cyber green
Gel plan: well 1 : 3µl of DNA ladder, well 2 and 3 10µl of Gagpol

##### Preculture of OTSB (7 white colonies)

For each :
- 4ml of LB media
- 2µl of spectinomycin (100mg/ml) (in order to obtain 10µg/ml)
- 1 white clone
Incubation at 37°C overnight

##### 4. To do next

- Otsb miniprep + digestion
- Gag digestion
- Gag transfo)

## October

[Back to top](#backtotop)

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# Team:Sorbonne U Paris/Experiments

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* [Home page](https://2018.igem.org/Team%3ASorbonne_U_Paris)
* Project

  [Description](https://2018.igem.org/Team%3ASorbonne_U_Paris/Description)
  [Design](https://2018.igem.org/Team%3ASorbonne_U_Paris/Design)
  [Model](https://2018.igem.org/Team%3ASorbonne_U_Paris/Model)
  [Demonstrate](https://2018.igem.org/Team%3ASorbonne_U_Paris/Demonstrate)
  [Improve](https://2018.igem.org/Team%3ASorbonne_U_Paris/Improve)
* Biology

  [Experiments](https://2018.igem.org/Team%3ASorbonne_U_Paris/Experiments)
  [Results](https://2018.igem.org/Team%3ASorbonne_U_Paris/Results)
  [Parts](https://2018.igem.org/Team%3ASorbonne_U_Paris/Parts)
  [Interlab](https://2018.igem.org/Team%3ASorbonne_U_Paris/InterLab)
  [Safety](https://2018.igem.org/Team%3ASorbonne_U_Paris/Safety)
* Human practices

  [Human Practices](https://2018.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
  [Integrated HP](https://2018.igem.org/Team%3ASorbonne_U_Paris/Integrated_Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3ASorbonne_U_Paris/Public_Engagement)
  [Meet up](https://2018.igem.org/Team%3ASorbonne_U_Paris/Meet_up)
* Notebook

  [Notebook](https://2018.igem.org/Team%3ASorbonne_U_Paris/Notebook)
  [Protocols](https://2018.igem.org/Team%3ASorbonne_U_Paris/Protocols)
  [Brainstorming](https://2018.igem.org/Team%3ASorbonne_U_Paris/Brainstorming)
* Team

  [Members](https://2018.igem.org/Team%3ASorbonne_U_Paris/Team)
  [Collaborations](https://2018.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ASorbonne_U_Paris/Attributions)
  [Sponsors](https://2018.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

* Awards

  [Judging form](https://igem.org/2018_Judging_Form?team=Sorbonne_U_Paris)
  [Medals](https://2018.igem.org/Team%3ASorbonne_U_Paris/Medals)
  [Giant Jamboree](https://2018.igem.org/Team%3ASorbonne_U_Paris/Giant_Jamboree)
* [Site map](https://2018.igem.org/Team%3ASorbonne_U_Paris/Sitemap)

# Experiments

* Index
* [I-Synthetic retrotransposon](#1)
* [II-Retrotransposon functionality(MoClo)](#2)
* [III- Trehalose pathway](#3)
* [IV-Protocols](#4)

## Construction of the synthetic retrotransposon

#### Cloning of the parts from the Chlamydomonas MoClo Kit

Some of the parts (intron, paromomycin, PpSAD, 5’UTRpSAD, PpNIT promoter, 5’UTRpNIT, 3’UTR) we used to construct our synthetic retrotransposon were taken from the MoClo Kit. The way we designed our system is different from a classical transcription unit (TU) assembly. Almost every part we use is in a “different position”. That means the Level 0 (L0) parts stored in the kit were PCR-amplified (and sequenced) with primers that allow the modification of the Fusion sites. Then the PCR products were cloned by Golden Gate cloning in new L0 backbones resistant to spectinomycine: pICH41258, pICH41264 or pAGM9121. In some cases we could use acceptor plasmids with fusion sites that already match our requirements (pICHX). Most of the time we had to use a universal plasmid from the original MoClo kit (pAGM9121). This acceptor plasmid works as the Phytobrick universal acceptor plasmid (BBa\_P10500).

#### Cloning of the parts with genes synthesis

We could not get genomic DNA from *Volvox carterii* to amplify the specific sequences of the *Osser* retrotransposon. *Osser* is quite big: 4.8 kb and contains more than seven illegal restrictions sites for the GoldenGate assembly. We chose to synthetize all the retrotransposons parts by using the IDT 20 kb program. We used the same PCR cloning strategy to add the restrictions sites and fusion sites in 5’ and 3’ of the synthetized sequences.

5’LTR and 3’LTR were quite easy as they are quite short: 197-bp and no restriction sites were in those two sequences. Even if the LTRs are the same, we made two synthesises because we truncated 5’LTR and we did not want to make a PCR from 3’LTR to avoid resequencing

The Gag/Pol design for synthesis was quite challenging! First we had to remove all the illegal restriction sites (*BpiI* and *BsaI*). Then, this part is huge: 4.3 kb. In fact, it was planned to order it in three g-block fragments and assemble them in pL0 through scarless GoldenGate cloning using the same type IIS restriction enzyme (*BpiI*). However, IDT’s algorithms did not validate this strategy because of various repeat sequence and high GC clusters

Finally, we managed to find a design with 6 different g-block with sizes between 90-bp and 1.3 kb. All of it was supposed to be assembled in a one-pot reaction in the universal acceptor plasmid.

**Unfortunately, this was the biggest mistake we did this year.** The complexity and the number of fragments were too high to get this cloning done. Because of that we could not construct the whole synthetic retrotransposon. We should have ordered them in a plasmid with quality control even if it would have been very expensive.

We are working now on cloning every fragment in its own L-1 plasmid and then make a final L0 assembly using donor plasmids instead of linear fragments. To do so, we need to order primers for the 6 fragments to add the L-1 restriction site, and sequence everything. However, we will not have enough time to do this before the Giant Jamboree.

## Characterisation of the retrotransposons' functionality

#### Paromomycine assay

Because microalgae do not bear plasmids, the Chlamydomonas MoClo kit is designed to integrate TUs inside the genome. To do so, once the construction is done, a cassette containing the TUs and the resistance gene for hygromycin is linearized by digestion with a restriction enzyme. The linear DNA is transformed in *C. reinhardtii D66* by electroporation and plated on TAP agar with hygromycine to select mutants after 5 days of incubation at 25 °C.

To assay the functionality of our synthetic retrotransposon, it was planned to induce our system for 6 days by changing the nitrogen source: ammonium 7.5 mM (NH4)Cl for non-inducing condition or nitrate 4 mM KNO3 for inducing the PNIT promoter as describe in Crozet et al 2018. Then the algae will be plated on TAP agar with hygromycin and paromoycine. By counting the number of resistant colonies we can deduce if our system is functional.

Further information about the design of our part are available on Design page

[Design](https://2018.igem.org/Team%3ASorbonne_U_Paris/Design)

#### Screening for the biggest in vivo library.

Our model allows to calculate the library size with two simple parameters. Therefore, need to measure the number of cells while inducing the system. At the end, we need to count the number of transposons per clones.

Further information about the modeling of our project are available on Modeling page.

[Model](https://2018.igem.org/Team%3ASorbonne_U_Paris/Model)

The OD600 can be easily measured in a 96-well plate reader using microplate. For example, every validated construction correctly inserted can be put in culture in a well until OD600 =1. After centrifugation of the microplate, when suspend the cells with the inducing media. OD600 will me measure during the whole experiment.

At the end of the induction, genomic DNA of all the cells inside the wells can be extracted by automation with the magnetic deck provides by Opentron. This will allow to do a qPCR normalized with the OD600

## Construction of the trehalose pathway

The two CDS OTSA and OTSB genes used to produce trehalose were codon-optimized for *C. reinhardtti* which has a high GC codon usage bias. Moreover, we removed all the MoClo illegal restriction sites. We ordered those two sequence from DS BIOSCIENCE lDT because IDT’s algorithm could not synthetize the two 1 kb genes even if it was split in smaller fragment. OTSA and OTSB were split in three fragments with a scarless GolgenGate strategy and ordered as plasmid with quality controls. Then following the MoClo kit procedure they were cloned in L0 acceptor plasmids.

## Protocols

You can find all the protocols we used during our experiments on the Protocols page

[Protocols](https://2018.igem.org/Team%3ASorbonne_U_Paris/Protocols)

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##### iGEM Sorbonne U Paris

4 place Jussieu
 75005 Paris France

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# Team:Sorbonne U Paris/Results

[![](https://static.igem.org/mediawiki/2018/c/c8/T--Sorbonne_U_Paris--Sugarevolution.png)](https://2018.igem.org/Team%3ASorbonne_U_Paris)
![logo of iGEM Sorbonne U Paris team. It is a sugar. It is a Rubik's cube shaped sugar ](https://static.igem.org/mediawiki/2018/c/c8/T--Sorbonne_U_Paris--Sugarevolution.png)

* [Home page](https://2018.igem.org/Team%3ASorbonne_U_Paris)
* Project

  [Description](https://2018.igem.org/Team%3ASorbonne_U_Paris/Description)
  [Design](https://2018.igem.org/Team%3ASorbonne_U_Paris/Design)
  [Model](https://2018.igem.org/Team%3ASorbonne_U_Paris/Model)
  [Demonstrate](https://2018.igem.org/Team%3ASorbonne_U_Paris/Demonstrate)
  [Improve](https://2018.igem.org/Team%3ASorbonne_U_Paris/Improve)
* Biology

  [Experiments](https://2018.igem.org/Team%3ASorbonne_U_Paris/Experiments)
  [Results](https://2018.igem.org/Team%3ASorbonne_U_Paris/Results)
  [Parts](https://2018.igem.org/Team%3ASorbonne_U_Paris/Parts)
  [Interlab](https://2018.igem.org/Team%3ASorbonne_U_Paris/InterLab)
  [Safety](https://2018.igem.org/Team%3ASorbonne_U_Paris/Safety)
* Human practices

  [Human Practices](https://2018.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
  [Integrated HP](https://2018.igem.org/Team%3ASorbonne_U_Paris/Integrated_Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3ASorbonne_U_Paris/Public_Engagement)
  [Meet up](https://2018.igem.org/Team%3ASorbonne_U_Paris/Meet_up)
* Notebook

  [Notebook](https://2018.igem.org/Team%3ASorbonne_U_Paris/Notebook)
  [Protocols](https://2018.igem.org/Team%3ASorbonne_U_Paris/Protocols)
  [Brainstorming](https://2018.igem.org/Team%3ASorbonne_U_Paris/Brainstorming)
* Team

  [Members](https://2018.igem.org/Team%3ASorbonne_U_Paris/Team)
  [Collaborations](https://2018.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ASorbonne_U_Paris/Attributions)
  [Sponsors](https://2018.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

* Awards

  [Judging form](https://igem.org/2018_Judging_Form?team=Sorbonne_U_Paris)
  [Medals](https://2018.igem.org/Team%3ASorbonne_U_Paris/Medals)
  [Giant Jamboree](https://2018.igem.org/Team%3ASorbonne_U_Paris/Giant_Jamboree)
* [Site map](https://2018.igem.org/Team%3ASorbonne_U_Paris/Sitemap)

# Results

Here we are presenting the progression of our lab work, the things we managed to archive and the ones that we could not record, the problems we faced and our new perspectives.

## Our progression in cloning

As of october 16 2018, by using the Moclo tool kit for Chlamydomonas Reinhardtii developed by Pierre Crozet and Stephane Lemaire, we were able to clone most of the Level 0 parts for our retrotransposon tool except for the Gag/Pol part. We believe we will be able to complete the Gag/Pol cloning, to create our Level 1 plasmids as well as the level M in the near future. For each Level 0 plasmid we cloned, we systematically checked the sequence, by sending samples to Eurofin Genomics.

![](https://static.igem.org/mediawiki/2018/4/46/T--Sorbonne_U_Paris--Overview_parts.jpg)

**Figure 1: Overview of our progress in the cloning experiments** In blue: parts that we have managed to clone or are available. In purple: parts we have yet to clone or we have difficulties cloning. The name of the plasmid backbones are referring to the Crozet et al. MoClo tool kit. The available parts are also from that kit. Spec is a short word for Spectinomycin and Amp for Ampicillin.

## Details on our currently available parts

#### 1) Rubisco intron ( RBCS2i1): Bba\_K2703000 - Backbone: piCH41258

This plasmid contains the intron for the rubisco. Based on the retrotransposon tool designed by Crook et al., an RT-PCR experiment will help us determine whether or not this intron was spliced in our microalgae and therefore if an event of retro transposition took place. We designed and cloned this part ourselves, then checked its sequence.

#### 2) Paromycin CDS: Bba\_K2703008 - Backbone: pICH41264

This plasmid contains the coding region of the Paromomycin, a resistance gene we use in our construction, and that we are placing upstream from the intron so that it would be transcribed whether or not the intron is spliced. We designed and cloned this part ourselves, then checked its sequence.

#### 3) pSAD promoter: Bba\_K2703002 - Backbone: pAGM9121

This plasmid contains the promotor of pSAD gene and is necessary for the transcription of pSAD. We designed and cloned this part ourselves, then checked its sequence.

#### 4) 3’LTR : Bba\_K2703003 - Backbone: pAGM9121

This part, as well as the 5’LTR is necessary to insure a single copy, site specific reintegration of the transposable element. We designed and cloned this part ourselves, then checked its sequence.

#### 5) 5’LTR : Bba\_K2703004- Backbone: pAGM9121

This part, as well as the 5’LTR is necessary to insure a single copy, site specific reintegration of the transposable element. We designed and cloned this part ourselves, then checked its sequence.

#### 6) pNIT promoter-5UTR- Backbone: pICH41233

This plasmid contains an inducible promoter pNIT, and a 5’UTR region. This part was given to us by Crozet et al., and is available on their MoClo tool kit.

#### 7)TPSAD (Terminator+3’UTR)- Backbone:pICH41276

This plasmid contains an inducible promoter pNIT, and a 5’UTR region. This part was given to us by Crozet et al., and is available on their MoClo tool kit.

#### 8) OtsA - Backbone:pAGM1276

This plasmid contains the OtsA sequence, which when fused with OtsB, will be responsible our trehalose production. We designed and cloned this part ourselves, then checked its sequence.

#### 9) Trehalose biosensor from Addgene #79754 - Backbone: pAGM1276

This plasmid will be transfected into C.reinardtii. When this biosensor will be expressed, it will enable us to identify the clones producing the most important quantities of trehalose. This will be determined by the reading of a fluorescence signal emitted by the biosensor when it binds trehalose.

![](https://static.igem.org/mediawiki/2018/9/92/T--Sorbonne_U_Paris--trehalosel.jpg)

**Figure 2: Representation of a trehalose compound (4)**

## Details on the parts we have yet to create:

#### 1)Gag/Pol- Backbone: pAGM9121

This plasmid contains the Gag/Pol part encoding for all the necessary factors for the transposition (RNA binding protein for gag, reverse transcriptase, integrase and RNAseH for pol). We designed and cloned this part ourselves, but the cloning experiments did not succeed due to the problems we faced.

#### 2) OtsB- Backbone: pAGM1299

This plasmid contains the OtsB sequence, which when fused with a 2A peptide OtsA will be responsible for trehalose production. We designed and cloned this part ourselves, then checked its sequence.

## Parts characterization

We characterized the paromomycin resistance genes. To see more details about this, please refer to the characterization rubric.

[Experiments](https://2018.igem.org/Team%3ASorbonne_U_Paris/Experiments%20)

## We assess the functionality of the Trehalose biosensor (TBMP):

Before starting the cloning of this gene we wanted to make sure it was working as described. This synthetic biosensor was already under the control of the IPTG inducible promoter Ptrc on a kanamycin resistance backbone. We transformed this plasmid in E.coli DH5 alpha and induced the promoter without or with three different concentrations of trehalose. We used the minimal media M63 without glucose. Trehalose is imported inside the cell to be metabolized by *E.coli*. We collected GFP-fluorescence data with a microplate reader at a rate of one measure every 10 minutes for 12 hours.

![](https://static.igem.org/mediawiki/2018/b/be/T--Sorbonne_U_Paris--caracterisation_plan.jpg)

**Figure 3: Plate plan for the characterization experiments of the Trehalose biosensor**The trehalose concentration changes every 3 columns: 1-3: 0µM of trehalose, 4-6: 10µM, 7-9: 1Mm, 10-12: 10mM. Each well contains 200µL of media. For each row, the strain is different. A: Untransformed DH5α, B: DH5-alpha ttransformed with a pl1, C: DH5α transformed with the biosensor (Addgene plasmid), Kanamycin resistant strain (KR) without IPTG, D: DH5α transformed with the biosensor (Addgene plasmid) (KR) + IPTG, E: Replicate of D.

![](https://static.igem.org/mediawiki/2018/e/e5/T--Sorbonne_U_Paris--coube_alpha_resultsl.png)

**Figure 4: A: Ratio fluo/OD of strains cultured with 10 uM of trehalose
B: Relative standard variation of the ratio fluo/OD of strains cultured with 10 uM of trehalose
(expressed in %).**A/ Fluorescence values of E.coli DH5alpha normalized by the OD 600 , data were collected every 10
minutes over 12 hours in triplicate. Yellow is the Addgene #79754 plasmid with IPTG (1 M), and in gray
the same strain without IPTG. There is a leaky expression because we did not use a tightly regulated
system. In blue is the negative control, it is the strain without plasmid. In orange and cyan E.coli DH5
alpha transformed with a MoClo plasmid that should contain the TBMP CDS. After further verification of
the plasmid was not correct. B/ Relative standard deviation of the normalized fluorescence values
(expressed in %).

The biosensor TBMP appears to work as described in the paper of Nadler DC et al., with trehalose at 10 uM (4).
Here we presented some of our results. And we are still continuing the experiments, so new results will be generated.

##### References

* [1] Crozet, P. et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth. Biol. 7, 2074–2086 (2018).
* [2] Crook, N. et al. In vivo continuous evolution of genes and pathways in yeast. Nat. Commun. 7, 13051 (2016).
* [3] rehalose Compounds | Synthose. Available at: http://www.lcsci.com/chemicals/trehalose.cfm. (Accessed: 4th October 2018)
* [4] . Rapid construction of metabolite biosensors using domain-insertion profiling | Nature Communications. Available at: https://www.nature.com/articles/ncomms12266. (Accessed: 4th October 2018)

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##### iGEM Sorbonne U Paris

4 place Jussieu
 75005 Paris France

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* [![Official website of Solidarity fund for the development of student initiatives of Sorbonne Université http://sciences.sorbonne-universite.fr/fr/vie_des_campus/vie_associative_projets_individuels/initiatives_etudiantes_fsdie/aide_aux_projets_fsdie.html](https://static.igem.org/mediawiki/2018/8/80/T--Sorbonne_U_Paris--Logo_FSDIE.png)](http://sciences.sorbonne-universite.fr/fr/vie_des_campus/vie_associative_projets_individuels/initiatives_etudiantes_fsdie/aide_aux_projets_fsdie.html " This is the official logo of Solidarity fund for the development of student initiatives of Sorbonne Université. The link leads to the official website of this solidarity fund:http://sciences.sorbonne-universite.fr/fr/vie_des_campus/vie_associative_projets_individuels/initiatives_etudiantes_fsdie/aide_aux_projets_fsdie.html ")
* [![Official website of  Molecular and Cellular biology master at Sorbonne Universite http://www.master.bmc.upmc.fr/fr/index.php](https://static.igem.org/mediawiki/2018/7/7f/T--Sorbonne_U_Paris--Logo_Master_BMC.png)](http://www.master.bmc.upmc.fr/fr/index.php "This is the official logo of Molecular and Cellular Biology Master at Sorbonne Université. The link lead to the official website of this master : http://www.master.bmc.upmc.fr/fr/index.php")
* [![Website  of Crous of Paris http://www.crous-paris.fr/](https://static.igem.org/mediawiki/2018/a/a9/T--Sorbonne_U_Paris--Logo_CROUS.png)](http://www.crous-paris.fr/ "This is the official logo of Culture ActionS commission of the Crous of Paris. The link leads to the official website of this commission : http://www.crous-paris.fr/")* * [![Official website of Embassy of France in the United States https://fr.usembassy.gov/fr](https://static.igem.org/mediawiki/2018/1/1c/T--Sorbonne_U_Paris--Logo_Embassy_USA.png)](https://fr.usembassy.gov/fr "This is the official logo of Office for Science & Technology  (OST) of the Embassy of France in the United States. The link lead to the website site of this office:https://fr.usembassy.gov/fr ")
    * [![Official website of Education and Research Unit for Life Science at Sorbonne Université http://biologie.sorbonne-universite.frfr/index.html]( https://static.igem.org/mediawiki/2018/5/5f/T--Sorbonne_U_Paris--Logo_UFR_SDV.png)](http://biologie.sorbonne-universite.frfr/index.html "This is the official logo of Education and Research Unit for Life Sciences at Sorbonne Université . The link lead to the official website of this unit :http://biologie.sorbonne-universite.frfr/index.html ")
    * [![Official website of Foundation of House of Chemystry http://www.maisondelachimie.com](https://static.igem.org/mediawiki/2018/5/5c/T--Sorbonne_U_Paris--Logo_Maisondelachimie.png)](http://www.maisondelachimie.com "This is the official logo of Foundation of the House of Chemistry. The link leads to the official website of this foundation : http://www.maisondelachimie.com ")
    * [![Official website of city of Aubervilliers http://www.aubervilliers.fr]( https://static.igem.org/mediawiki/2018/c/c7/T--Sorbonne_U_Paris--Logo_Aubervilliers.png)](http://www.aubervilliers.fr "This is the official logo of the city of Aubervilliers. The link lead to the official website of the city:http://www.aubervilliers.fr ")
    * [![Official website of city of Goussainville http://www.ville-goussainville.fr]( https://static.igem.org/mediawiki/2018/9/9f/T--Sorbonne_U_Paris--Logo_Goussainville.png)](http://www.ville-goussainville.fr "This is the official logo of the city of Goussaivile . The link lead to the official website of the city of goussainville:http://www.ville-goussainville.fr ")
    * [![Official website of New England Biolabs https://www.neb-online.fr/](https://static.igem.org/mediawiki/2018/f/f9/T--Sorbonne_U_Paris--Logo_NEB.png)](https://www.neb-online.fr/ "This is the offficial logo of New England Biolabs (NEB). The link lead to the official website of NEB :https://www.neb-online.fr/ ")
    * [![Official website of IDT https://eu.idtdna.com/pages](https://static.igem.org/mediawiki/2018/f/f0/T--Sorbonne_U_Paris--Logo_IDT.png)](https://eu.idtdna.com/pages "This is the official logo of Integrated DNA Technologies (IDT). The link lead to the official website of IDT: https://eu.idtdna.com/pages")
    * [![Logo of Macherey Nagel http://www.mn-net.com/](https://static.igem.org/mediawiki/2018/9/96/T--Sorbonne_U_Paris--Macherey_Nagel.png)](http://www.mn-net.com/ "This is the official logo of Macherey-Nagel. The link lead to the official website of Macherey-nagel: http://www.mn-net.com/")

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