Team:Sorbonne U Paris/Notebook

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

Calendars 2018

June
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	1

July
M	T	W	T	F	S	S
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	31

August
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	31

September
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

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)

Aurélie

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)

Aurélie

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

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

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)

Victor

Aurélie

Soukeyna

Aim : Extraction of plasmides from yesterday’s cultures 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)

Aurélie

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

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

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)

Aurélie

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)

Aurélie

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

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)

Aurélie

Ursula

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

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)

Aurélie

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)

Aurélie

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

Thurday 10th July

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

Institut de biologie physico-chimique (IBPC)

Aurélie

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)

Ursula

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)

Aurélie

Anaïs

Migration of digestion reaction products from the day before

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

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)

Aurélie

Anaïs

Arrangement in the box iGEM 2018

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

[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)

Aurélie

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)

Aurélie

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)

Aurélie

Dounia

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

2. Plasmid extraction from 𝝱 bacterias transformed the day before ( 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)

Aurélie

Anais

Plasmid extraction from bacteria transformed

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)

Aurélie

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

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

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)

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

>

Tuesday 31th July

DNA sent to sequencage
Digestion reaction using BSaI

Muséum national d'histoire naturelle (MNHN)

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

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)

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

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)

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)

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)

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

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

Tuesday 7th August

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

Aurélie

1. Digestion/ ligation reaction of pSAD 2 Rb intron

see 06/14/2018 protocol

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)

Aurélie

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)

Aurélie

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

Intron Rubisco

Ligation

Cycle

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)

Aurélie

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 :

Lader (2log)	Rb1+	Rb2+	Rb3+	Rb4*

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

Preparation of DNA samples for sequencing

add 2 µl of primers at 10 µL for each tube

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)

Aurélie

Ursula

Add 2µl of primers at 10µM

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)

Aurélie

Soukeyna

1. Digestion/ligation reaction of otsA and otsB

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)

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)

5. Level 1 ligation protocol for the CARGO retrotransposon

Tuesday 4th September

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

Institut de biologie physico-chimique (IBPC)

Aurélie

Soukeyna

1. Digestion of otsA and otsB ligation reaction of 03/08/2018

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

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

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 : 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)

Soukeyna

1. Transformation of B10 bacteria with cloning reaction of 03/09 : 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)

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)

Aurélie

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

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

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)

Aurélie

1. Ligation reaction of p0-iGEM5 (GAGPOL)

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 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)

Aurélie

1. DNA extraction of the 6 bacteria culture prepare on 7/09

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 : expected result : >800bp

4. Transformation of B10 bacteria with ligation product from 07/09

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)

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)

Sara and Parisa

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)

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)

Sara

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)

Sara

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

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