Team:IONIS Paris/Notebook

Notebook

MARCH 2015

APRIL 2015

MAY 2015

JUNE 2015

JULY 2015

AUGUST 2015

SEPT. 2015

Results after PCR from 17/09

Expected results

Results

The band for the third well is good. WE GOT IT !!!!!

Induction of BBa_K325909 and BBa_K1159001 with a solution of arabinose 1%, and a solution of IPTG 1 mM for T7-Nanoluc (should correspond to the effect of a constitutive promoter like T7). Under these conditions, light intensity of T7-nanoluc was 1,2 time higher than the BBa_K1159001.

Effect of the concentration of IPTG inducer to the light intensity of T7-nanoluc part. While the concentration of IPTG does not exceed 1 mM, the light intensity remains low.

Effect of the concentration of arabinose inducer to the light intensity of BBa_K1159001 part. Light intensity is linked to inducer concentration however over 1% of arabinose (final concentration), the intensity not rise anymore.

18 Sept. 15

Miniprep of liquid culture (pSB1C3-Nanoluc)

Digestion of miniprep

Mix digestion preparation:

Tube	pSB1C3
H2O MQ	4 µL
Buffer 2.1	1 µL
DNA	3 µL
EcoRI	1 µL
PstI	1 µL

37°C, 1h

Expected results

Results

No profile of double digestion for pSB1C3-nanoluc. The third sample looks like a simple digestion PCR checking
- Biobrick quantification for submission:

• Holin/Endolysin reverse: 115 ng/µl
• CCDB reverse: 73 ng/µl
• HOKD reverse: 10 ng/µl
• VVD-YC: 38 ng/µl
• VVD-YN: 37 ng/µl

PCR pSB1C3-NanoLuc

Mix PCR preparation:

Tube	pSB1C3-NanoLuc (x5)
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer pSB1C3 Fwd	1 µL
Primer pSB1C3 Rev	1 µL
DNA	1 µL
Taq Pol	0,5 µL

37°C, 1h
iGEM TAQ

17 Sept. 15

Miniprep

Liquid culture (pSB1C3-Holin/Endolysin x4 and pSB1C3-pDawn x2)
Liquid culture from the bacterial growth kinetic experiment (pDawn-Holin/Endolysin x2)

Digestion

Mix digestion preparation:

Tube	Miniprep	pDawn-H/E
H2O MQ	2 μL	3 μL
Buffer 2.1	1 μL	1 μL
DNA	5 μL	5 μL
EcoRI	1 μL	1 μL
PstI	1 μL	-

37°C, 1h

Expected results

Results

There were no expected profile of digestion excepted for the first and the third wells with pDawn-Holin/Endolysin.

Digestion

Mix digestion preparation:

Tube	Biobrick
H2O MQ	5 μL
Buffer 2.1	1 μL
DNA	3 μL
EcoRI	1 μL

37°C, 1h

Liquid culture of bacteria transformed with pSB1C3-nanoluc (x5)

Liquid culture on 96 wells plate

When 0,6 < OD₆₀₀ < 0,8, induction with arabinose or IPTG Incubate 5h at 30°C; for BBa_K325909, measure the kinetic during the 5h of induction and take the maximum value
Add 50µL of NanoGlow substrate (Promega) for 50 uL of bacteria
Measure kinetic over 120 min and take maximum value for culture with BBa_K1159001 and T7-nanoluc

16 Sept. 15

Measurement of the OD₆₀₀ of liquid culture

When the OD₆₀₀ has reached 0.6 or more, the volume of culture has been divided into 3 erlenmeyers:

1 erlenmeyer without light exposition, 37°C
1 erlenmeyer with light exposition, 37°C
1 erlenmeyer with light exposition, room temperature

Measurement of the OD₆₀₀ every hour

3 culture on 5have shown a bacterial development

Colony - A6

Colony - A8

Colony - F1

All colonies exhibits same profile under same condition of light exposition and temperature. When the temperature is favorable for a bacterial development, the light activation of pDawn leading to the production of toxin is weak and do not impact a lot the growth of the population of bacteria. But at room temperature (RT°), the induction for protein synthesis is high enough to stop bacterial development

Two things lack to this experiment:

• A longer time to measure if the population of bacteria could really decrease
• A control at room temperature without light exposition to compare it evolution of OD 600 to the grey curve

Digestion

Mix digestion preparation:

Tube	Miniprep	pSB1C3 iGEM
H2O MQ	4 μL	-
Buffer 2.1	2 μL	4 μL
DNA	3 μL	30 μL
EcoRI	0,5 μL	3 μL
PstI	0,5 μL	3 μL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:

Tube	pSB1C3-nanoluc (1:3)	pSB1C3-nanoluc (1:5)
H2O MQ	9 μL	9,5 μL
T4 buffer	2 μL	2 μL
pSB1C3	5 μL	5 μL
NanoLuc	3 µL (35ng.µL-1)	2,5 µl (70 ng.µL-1)
T4 ligase	1 μL	1 μL

Room temperature, 30min heat kill: 65°C, 20min

• Transformation of competent cells with 2 µL of ligation products

Liquid culture

4 colonies from transformation with pSB1C3-Holin/Endolysin
2 colonies from transformation with pSB1C3-pDawn

Transformation

Transformation of competent BL 21(DE3) cells with BBa_K325909, BBa_K1159001 and T7-nanoluc plasmids

15 Sept. 15

PCR Nanoluc

Mix PCR preparation:

Tube	pSB1C3-Gblock
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer Fwd	0,5 µL
Primer Rev	0,5 µL
DNA	1µL
Taq Pol	0,5 µL

PCR cycle – IGEM TAQ NanoLuc

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	58	30 sec
Extension	72	1 min
Final extension	72	5 min	1
Hold	4	Infinite

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:

Tube	pSB1C3-VVD YC + YN (x7)	pSB1C3-VVD YN + YC (x6)
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer Fwd	YC Fwd, 0,5 µL	pSB1C3 Fwd 2, 1 µL
Primer Rev	pSB1C3 Rev 2, 1 µL	YN Rev 1, 0,5 µL
Taq Pol	0,5 µL	0,5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	35
Annealing	51	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Expected results

Results

Not expected bands for every PCR colony Could be due to the choice of primers

• Make another one with primers for pSB1C3 (more simple)

Digestion

Mix digestion preparation:

Tube	pSB1C3-pDawn	pSB1C3 iGEM	Nanoluc
H2O MQ	-	-	14 μL
Buffer 2.1	1,2 μL	1,1 μL	2 μL
DNA	10 μL	8 μL	2 μL
Enzyme 1	XbaI 1 μL	EcoRI 1 μL	EcoRI 1 μL
Enzyme 2	-	PstI 1 μL	PstI 1 μL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:

Tube	pSB1C3-pDawn	pSB1C3-pDawn I-V (1:2)	pSB1C3-pDawn I-V (2:1)	pSB1C3-nanoluc (1:1)	pSB1C3-nanoluc (1:2)
H2O MQ	-	-	-	0,8 μL	4,3 μL
T4 buffer	1,4 μL	-	0,5 μL	0,5 μL	1 μL
pSB1C3	-	1,4 µL	5,5 µL	7 µL	3,5 µL
Insert	12,2 μL	20 μL	22 μL	0,7 μL	0,7 μL
T4 ligase	0,5 μL	1 μL	1 μL	0,5 μL	0,5 μL

Room temperature, 45min heat kill: 65°C, 20min

• Transformation of competent cells with all ligation products using 1 µL or 3 µL

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:

Tube	pSB1C3-VVD YC + YN (x7)	pSB1C3-VVD YN + YC (x7)
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer pSB1C3 Fwd	1 µL	1 µL
Primer pSB1C3 Rev	1 µL	1 µL
Taq Pol	0,5 µL	0,5 µL

Expected results

Results

No differences with previous PCR colony
Bands weight do not correspond to the two possible cases “no insertion” or “insertion” of the part

• Liquid culture of 1 colony of each to analyse the DNA
  

Bacterial growth kinetic of bacteria transformed with pDawn-H/E

Liquid culture of 100 ml LB + 100 µl Kan for 5 interesting colonies from the experiment of the 09/09
37°C, 120 rpm, O/N, no light exposition

14 Sept. 15

Digestion

Mix digestion preparation:

Tube	VVD YC	VVD YN
H2O MQ	1 μL	1 μL
Buffer 2.1	2 μL	2 μL
DNA	15 μL	15 μL
Enzyme 1	EcoRI 1 μL	XbaI 1 μL
Enzyme 2	SpeI 1 μL	PstI 1 μL

37°C, 1h heat kill: 65°C, 20min

Expected results

Results

Ligation

Mix ligation preparation:

Tube	pSB1C3-VVD YC	pSB1C3-VVD YN	pSB1C3
H2O MQ	2,2 μL	3,4 μL	1,8 μL
T4 ligase buffer	1 μL	1 μL	2 μL
Insert	VVD YN, 4,1 µL	VVD YC, 2,9 µL	pDawn (28/07), 7,2 µL
Backbone	2,2 μL	2,2 μL	8 μL
T4 ligase	0,5 μL	0,5 μL	1 μL

37°C, 30min heat kill: 65°C, 20min

• Transformation of competent cells with pSB1C3-VVD YC + VVD YN and pSB1C3-VVD YN + VVD YC (1 µL or 4 µL have been used); 30°C all the weekend long

Ligation pDawn Gblock

Mix ligation preparation:

Tube	pDawn I-II-III	pDawn IV-V
T4 ligase buffer	2 μL	2,3 μL
pDawn I	2,3 μL	-
pDawn II	12 μL	-
pDawn III	2,7 μL	-
pDawn IV	-	13,2 μL
pDawn V	-	6,2 μL
T4 ligase	1 μL	1 μL

Room temperature, 45min heat kill: 65°C, 20min

Purification of pSB1C3-pDawn

20 µL of ligation product + 80 µL of H₂O MQ
Add 1/10 (10 µL) of the volume of NaOAc 3M pH 5.2
Add 2,5 volumes (275 µL) of Ethanol 100%
Mix and stored at -20°C for 1 hour at least
Centrifuge 30 sec, 14000 rpm at 4°C
Discard the supernatant
Resuspend the pellet with 500 µL of ethanol 70%, invert up the tube several time
Centrifuge 5 min, 14000 rpm at 4°C
Discard the supernatant and let dry the pellet
Resuspend the pellet with the right amount of water following the final concentration expected (20 µL here)

Ligation pDawn Gblock

Mix ligation preparation:

Tube	pDawn I-II-III-IV-V
pDawn I-II-III	20 μL
pDawn IV-V	22,3 μL
T4 ligase	1 μL

Room temperature, 45min heat kill: 65°C, 20min

11 Sept. 15

Miniprep of liquid culture (QIAgen kit)

Digestion of miniprep

Mix digestion preparation:

Tube	Miniprep
H2O MQ	14 µL
Buffer 2.1	2 µL
DNA	3 µL
EcorI	0,5 µL
PstI	0,5 µL

37°C, 1h

10 Sept. 15

E.coli DH5 alpha transformation

Transformation of competent cells with pSB1C3-GFP and pSB1C3-H/E (ratio 3:1, 1:1, 1:3) with 3 µl of plasmid.

Liquid cultures on plates from 08/09

Measurements of DO595 and DO405 (wtGFP) all the day long

Typical profile of colonies transformed with pDawn-HOKD under light exposition

Continuous bacterial growth despite light exposure not expected

Typical profile of colonies transformed with pDawn-CCDB under light exposition

Interesting profile with a diminution of the DO595 after it reach 0,6

Typical profile of colonies transformed with pDawn-Holin/Endolysin under light exposition

Continuous bacterial growth in spite of light exposure not expected

But some clones get the following profile

Very interesting profile, the DO600 fall down earlier than for all others clones

Typical profile of colonies transformed with pDawn-GFP under light exposition

Not interesting, all curve are proportional such as there were no production of GFP

PCR colony for pSB1C3-pDawn I-V and pSB1C3-VVD YC-VVD YN

Mix PCR preparation:

Tube	pSB1C3-pDawn I-V	pSB1C3-VVD YC-VVD YN
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer Fwd	pDawn Fwd 2, 0,5 µL	pSB1C3 Fwd 2, 0,8 µL
Primer Rev	pDawn Rev 2, 0,5 µL	pSB1C3 Rev 2, 0,8 µL
Taq Pol	0,5 µL	0,5 µL

Expected results

Results

Expected results

Results

No amplification for pSB1C3-pDawn
Soft amplification for pSB1C3-VVD YC-VVD YN 1 only
Several bands (only one expected) and unexpected molecular length for VVD YN, VVD YC, HOKD and CCDB biobricks

Liquid culture

pSB1C3-pDawn, colonies 1 to 5 (there were nothing with PCR colony but we have to try…)
pSB1C3-VVD YC-VVD YN, colony 1

9 September 15

Digestion

Mix digestion preparation:

Tube	VVD-YC155	VVD-YN155
Buffer 2.1	1.4 µL	1.4 µL
DNA	12 µL	12 µL
Enzyme 1 (0,5 µL)	EcoRI	XbaI
Enzyme 2 (0,5 µL)	SpeI	PstI

Mix digestion preparation:

Tube	pSB1C3	GFP	Holin/Endolysin
Buffer 2.1	1 µL	1,4 µL	1,4 µL
DNA	8 µL	12 µL	12 µL
EcoRI	0,5 µL	0,5 µL	0,5 µL
PstI	0,5 µL	0,5 µL	0,5 µL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:

Tube	pSB1C3-VVD YC-VVD YN
H2O MQ	0,2 μL
T4 ligase buffer	1 μL
VVD-YC	2 μL
VVD-YN	2,6 μL
pSB1C3	3,7 μL
T4 ligase	0,5 μL

Room temperature, 30 min heat kill: 65°C, 20min

• Transformation of bacteria with 1 µL or 4 µL of pSB1C3-VVD YC-VVD YN

Mix ligation preparation:

Tube	pSB1C3-GFP				pSB1C3-H/E
Ratio (backbone:insert)	3:1	1:1	1:3		3:1	1:1	1:3
H2O MQ	6,6 μL	5,7 μL	3 μL		6,4 μL	5 μL	0,7 μL
T4 ligase buffer	1 μL	1 μL	1 μL		1 μL	1 μL	1 μL
Insert	0,5 μL	1,4 μL	4,1 μL		0,7 μL	2,1 μL	6,4 μL
pSB1C3	1,4 μL	1,4 μL	1,4 μL		1,4 μL	1,4 μL	1,4 μL
T4 ligase	0,5 μL	0,5 μL	0,5 μL		0,5 μL	0,5 μL	0,5 μL

Room temperature, 30min heat kill: 65°C, 20min

PCR pSB1C3-Gblock (CCDB, HOKD, VVD YC, VVD YN)

Mix PCR preparation:

Tube	pSB1C3-Gblock
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer pSB1C3 Fwd	1 µL
Primer pSB1C3 Rev	1 µL
DNA	1µL
Taq Pol	0,5 µL

Run of the IGEM TAQ program

Cultures from 07/09

Many colonies for pDawn-H/E, pDawn-CCDB, pDawn-HOKD and pDawn-GFP
Few colonies for pSB1C3-pDawn I-V

PCR colonies for pSB1C3-pDawn I-V

Mix PCR preparation:

Tube	pSB1C3-pDawn I-V
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer pDawn Fwd 2	0,8 µL
Primer pDawn Rev 2	0,8 µL
Taq Pol	0,5 µL

Liquid culture

Liquid culture on 96 wells plates for pDawn-H/E, pDawn-CCDB, pDawn-HOKD and pDawn-GFP:

• 1st plate (for each plasmid) has filled with 250 µL of LB + 25 µL of Kanamycin, 1 well on 2.1 colony / well
  Withdraw 125 µL/well and fill the same well of a 2^nd plate (save)
  Measure the DO₅₉₅ of 1^st plates and let it at 37°C, 120 rpm, O/N with light exposition

8 September 15

Results of transformation from 04/09 (pSB1C3-pDawn I-V)

No colonies

Digestion of pDawn-Gblocks I and V, pDawn and Holin/Endolysin

Mix digestion preparation:

Tube	pDawn I	pDawn V	pDawn	H/E
Buffer (2 µL)	Cutsmart	Buffer 2.1	Buffer 2.1	Buffer 2.1
DNA	12 µL	12 µL	12 µL	12 µL
Enzyme 1 (0,5 µL)	EcoRI	SphI	EcoRI	EcoRI
Enzyme 2 (0,5 µL)	SalI	PstI	NheI	SpeI

Ligation

Mix ligation preparation:

Tube	pDawn I-II-III
T4 ligase buffer	1 μL
pDawn I	1,8 μL
pDawn II	4,6 μL
pDawn III	2 μL
T4 ligase	0,5 μL

Mix ligation preparation:

Tube	pDawn IV-V
T4 ligase buffer	1,1 μL
pDawn IV	5 μL
pDawn V	4,6 μL
T4 ligase	0,5 μL

Mix ligation preparation:

Tube	GFP	Holin/Endolysin	ccdB	HokD
Water	3,7 µL	3 µL	3,3 µL	4 µL
T4 ligase Buffer	1 µL	1 µL	1 µL	1 µL
pDawn (backbone)	3,5 µL	3,5 µL	3,5 µL	3,5 µL
Gblock	1,3 µL	2 µL	1,7 µL	1 µL
T4 ligase	0,5 µL	0,5 µL	0,5 µL	0,5 µL

Room temperature, 30min heat kill: 65°C, 20min

Mix ligation preparation:

Tube	pDawn I-V
pDawn I-II-III	10 μL
pDawn IV-V	10 μL
T4 ligase	0,5 μL

Room temperature, 30min heat kill: 65°C, 20min

Mix ligation preparation:

Tube	pDawn I-V
pDawn I-V	20 μL
pSB1C3	1 μL
T4 ligase	0,5 μL

Room temperature, 30min heat kill: 65°C, 20min

• Transformation pDawn-Gblocks and pSB1C3-pDawn

7 September 15

Results of liquid cultures

After centrifugation and UV exposure, no green light into the pellet or the supernatant

Liquid culture on plates 96 wells of transformed bacteria with pDawn-toxine (Holin/Endolysin, CCDB, HOKD)

1 colonie / well with 200 µL of LB, 32 wells per type of transformation
37°C, 120 rpm without light exposure

• DO₅₉₅ to follow bacterial growth under no light exposure condition (no time to switch to light exposure condition and to follow the evolution of the DO₅₉₅)

Digestion of miniprep from 03/09

Mix digestion preparation:

Tube	Miniprep
H2O MQ	2 μL
Buffer 2.1	2 μL
DNA	10 μL
EcoRI	0,5 μL

37°C, 1h

Expected results

Results

Bands corresponding to pSB1C3-HOKD and pSB1C3-CCDB seem correct (PCR control required for checking)
About constructions with pDawn, bands at 2100 bp were not expected, and highest bands seem to get the same length require additional tests.

Ligation of pSB1C3 and pDawn Gblocks I-V

Mix ligation preparation:

Tube	pDawn I-II-III
T4 ligase buffer	1,3 μL
pDawn I-V	12,2 μL
pSB1C3	0,5 μL
T4 ligase	0,5 μL

Room temperature, 30min heat kill: 65°C, 20min

Transformation of 100 µL of competent cells; 30°C during the weekend

Digestion of miniprep from 03/09, pDawn-Gblocks

Mix digestion preparation:

Tube	Miniprep
H2O MQ	13 μL
Buffer 2.1	2 μL
DNA	4 μL
EcoRI	0,5 μL
EcoRV	0,5 μL

37°C, 1h

Expected results

Results

No differences between the four digestion no parts into pDawn

4 September 15

Miniprep on liquid culture

Cf liquid culture of the 02/09/15

Digestion of pDawn I-V purified

Mix digestion preparation:

Tube	pDawn i-V
Buffer 2.1	1,2 µL
DNA	10 µL
EcoRI	0,5 µL
PstI	0,5 µL

37°C, 1h - heat kill: 80°C, 20min

Liquid culture on plates 96 wells of transformed bacteria with pDawn-GFP

1 colonie / well with 100 µl of LB, 37°C, 120 rpm and light exposition DO405 and DO595 to determine the presence of GFP Liquid culture into 100 ml Erlenmeyer of the 4 most interesting colonies.

3 September 15

Digestion of miniprep from 01/09 and BBa_K1159001

Mix digestion preparation:

Tube	Miniprep (01/09)	BBa_K1159001
MQ Water	12 µL	12 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
EcoRI	0,5 µL	0,5 µL
PstI	-	0,5 µL
BamHI	-	0,5 µL

37°C, 1h

Expected results

Results

Interesting band for Construction with VVD YC and VVD YN, require control test;
BBa_K1159001: we get 2 on 3 expected bands, confirmation of the structure of the plasmid with a pBad promoter (not indicated on the registry) before the nanoluc gene.

Liquid culture

pDawn – H/E 20 mL LB + 20 µL Kan, without light;
pDawn – GFP 20 mL LB + 20 µL Kan, with light;
pDawn – HOKD 20 mL LB + 20 µL Kan, without light;
pDawn – CCDB 20 mL LB + 20 µL Kan, without light;
pSB1C3– CCDB;
pSB1C3 – HOKD;

pDawn Gblock I-V purification

10 µl of ligation product + 40 µl of H2O MQ;
Add 1/10 (5µL) of the volume of NaOAc 3M pH 5.2;
Add 2,5 volumes (137,5 µl) of Ethanol 100%;
Mix and stored at -20°C for 1 hour at least;
Centrifuge 30 sec, 14000 rpm at 4°C;
Discard the supernatant;
Resuspend the pellet with 500 µl of ethanol 70%, invert up the tube several time;
Centrifuge 5 min, 14000 rpm at 4°C;
Discard the supernatant and let dry the pellet;
Resuspend the pellet with the right amount of water following the final concentration expected (10µL here)

2 September 15

Digestion pDawn and pDawn Gblocks (I – V)

Mix digestion preparation:

Tube	pDawn	pDawn I	pDawn II	pDawn III	pDawn IV	pDawn V
Buffer (2 µL)	Buffer 2.1	Buffer 3.1	Cutsmart	Cutsmart	Buffer 2.1	Buffer 2.1
DNA	12 µL	12 µL	12 µL	12 µL	12 µL	12 µL
Enzyme 1 (0,5 µL)	EcoRI	SalI HF	SalI HF	XmaI	SphI	SphI
Enzyme 2 (0,5 µL)	PstI	-	XmaI	SphI	-	-

37°C, 1h heat kill: 80°C, 20min

Expected results

Results

pDawn quantification = 22,8 ng.µL^-1

Ligation of Gblocks (GFP, H/E, CCDB, HOKD) into pDawn

Mix ligation preparation:

Tube	GFP	H/E	ccdB	HokD
Water	5,1 µL	4,6 µL	4,8 µL	5,3 µL
T4 ligase Buffer	1 µL	1 µL	1 µL	1 µL
pDawn	0,9 µL	1,4 µL	1,2 µL	0,7 µL
Plasmid	2,5 µL	2,5 µL	2,5 µL	2,5 µL
T4 ligase	0,5 µL	0,5 µL	0,5 µL	0,5 µL

Room temperature, 30min heat kill: 80°C, 20min

Transformation of E.coli DH5 alpha competent cells

Dephosphorylation of pDawn IV

Mix plasmid dephosphorylation preparation:

Tube	GFP
Cutsmart Buffer	2 µL
Plasmid	15 µL
rSAP enzyme	1 µL

Ligation of pDawn Gblocks

Mix ligation preparation:

Tube	pDawn I-II-III	pDawn IV-V
Water	4,3 µL	3,7 µL
T4 ligase Buffer	1 µL	1 µL
pDawn I	0,9 µL	-
pDawn II	2,3 µL	-
pDawn III	2,3 µL	-
pDawn IV	-	2,5 µL
pDawn IV	-	2,5 µL
T4 ligase	0,5 µL	0,5 µL

Room temperature, 30min heat kill: 65°C, 20 min

1 September 15

Digestion BBa_K1159001

Mix digestion preparation:

Tube	BBa_K1159001
Buffer 2.1	2 μL
DNA	10 μL
Enzyme EcoRI	0,5 μL
Enzyme PstI	0,5 μL

37°C, 1h heat kill: 80°C, 20 min

Expected results

Results

BBa_K1159001: Not expected 1600 bp long band; the sequence of the part did not correspond to information published on the registry
Good amplification of Gblocks fragment (Holin/Endolysin, GFP, VVD YC and VVD YN)
No amplification of HOKD and CCDB fragment

Gel extraction (QIAgen kit)

PCR of Gblocks (CCDB and HOKD)

Mix PCR preparation:

	Gblock
MQ Water	39 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer Fwd 25 µM	0,5µL pSB1C3 Fwd
Primer Rev 25 µM	0,5µL pSB1C3 Rev
DNA	1µL
Taq Pol enzyme	0,5 µL

Run of the IGEM TAQ 2 program

Expected results

Results

Good amplification gel extraction (QIAgen kit)

Liquid culture

2 colonies from culture on plates have been selected for amplification:

VVD YC/YN (x2)
VVD YC (x2)
VVD YN (x2)
GFP (x2)
Holin/Endolysin (x2)
CCDB (x2)
HOKD (x2)

• 20 ml LB + 20 µl Cam

pDawn
pDusk

• 20 ml LB + 20 µl Kan

31 August 15

Results

Result of transformation (26/08):

1 colony onto a gel artefact (no antibiotic) of the control plate;
Few colonies (3 to 20) for others plates excepted H/E plates

• Stored into a fridge for WE, H/E plates stayed at room temperature

Miniprep (kit QIAgen)

Liquid culture of bacteria transformed with BBa_K1159001 plasmid (from iGEM) prepared into 2 columns; final elution 2x15µl/column

PCR of Gblocks (VVD YC, VVD YN, GFP, H/E, CCDB and HOKD)

Mix PCR preparation:

	Gblock
MQ Water	39 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer Fwd 25 µM	1µL pSB1C3 Fwd
Primer Rev 25 µM	1µL pSB1C3 Rev
DNA	1µL
Taq Pol enzyme	0,5 µL

Run of the IGEM TAQ 2 program

28 August 15

Digestion

Mix digestion preparation:

Tube	VVD - YC155	VVD - YN155
Buffer 2.1	2 µL	2 µL
DNA	12 µL	12 µL
Enzyme EcoRI	0,5 µL	-
Enzyme PstI	-	0,5 µL
Enzyme XbaI	-	0,5 µL
Enzyme SpeI	0,5 µL	-

37°C, 1h heat kill: 80°C, 20 min

Ligation of VVD-YC155 and VVD-YN155 into pSB1C3

Mix ligation preparation:

Tube	pSB1C3
Water	3 µL
T4 ligase Buffer	1 µL
VVD-YC155	1,3 µL
VVD-YN155	1,7 µL
Plasmid	2,5 µL
T4 ligase	0,5 µL

Room temperature, 30min heat kill: 80°C, 20 min

Transformation of bacteria with ligation products

27 August 15

Digestion of pSB1C3 (from iGEM) and miniprep

Mix digestion preparation:

Tube	pSB1C3	pSB1C3	Miniprep
Water	6 µL	12 µL	12 µL
Buffer 2.1	4 µL	2 µL	2 µL
Plasmid	8 µL	5 µL	5 µL
Enzyme EcoRI	1 µL	-	0,5 µL
Enzyme PstI	1 µL	-	0,5 µL
Enzyme XbaI	-	0,5 µL	-
Enzyme SpeI	-	0,5 µL	-

37°C, 1h

Expected results

Results

There are only bands corresponding to linearized pSB1C3 and one band, unexpected, corresponding to unknown sequence.

PCR of pDawn (1,2,3,4) and VVD-YN (1,2,1/4)

Mix PCR preparation:

	pDawn (1,2,3,4)	VVD-YN (1,2,1/4)
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer Fwd 50 µM	0,5µL pDawn Fwd I	0,5µL VVD Fwd I
Primer Rev 50 µM	0,5µL pDawn Rev II	0,5µL YN155 Rev II
DNA	1µL	1µL
Taq Pol enzyme	0,5 µL	0,5 µL

Run of the IGEM TAQ 4 program

Ligation of Gblocks into pSB1C3

Mix ligation preparation:

Tube	VVD-YC155	VVD-YN155	GFP rev	H/E rev	CCDB rev	HOKD rev
Water	2,4 µL	1,3 µL	2,2 µL	-	3,5 µL	4,5 µL
T4 ligase Buffer	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL
Gblock	3,6 µL	4,7 µL	3,8 µL	5,8 µL	2,5 µL	1,5 µL
pSB1C3	2,5 µL	2,5 µL	2,5 µL	2,5 µL	2,5 µL	2,5 µL
T4 ligase	0,5 µL	0,5 µL	0,5 µL	0,5 µL	0,5 µL	0,5 µL

Room temperature, 30min

Ligation of pSB1C3 (from iGEM) XbaI-SpeI (circularization)

Mix ligation preparation:

Tube	pSB1C3
Water	3,5 µL
T4 ligase Buffer	1 µL
Plasmid	5 µL
T4 ligase	0,5 µL

Room temperature, 1h

Expected results

Results

Same band for each sample Impossible Fail

Expected results

Results

Very soft bands at 2000 bp (pSB1C3) and 4000 bp (???)
Some bands between them (apparent on the screen of the gel reader, not on the picture…)

Transformation of bacteria with ligation products

26 August 15

Results of liquid cultures

No bacteria into the control, many bacteria everywhere else
Miniprep (QIAgen)

Digestion of pSB1C3 (from iGEM), miniprep and Gblocks

Miniprep for pDawn and VVD-YN155
Gblocks: ordered fragment from IDT (VVD-YC155, VVD-YN155, GFP rev, Holin/Endolysin rev (H/E rev), CCDB rev, HOKD rev)

Mix digestion preparation:

Tube	pSB1C3	Miniprep	Gblocks
Water	6 µL	12 µL	11 µL
Buffer 2.1	4 µL	2 µL	2 µL
Plasmid	8 µL	5 µL	6 µL
Enzyme EcoRI	1 µL	0,5 µL	0,5 µL
Enzyme PstI	1 µL	0,5 µL	0,5 µL

37°C, 1h heat kill: 80°C, 20min

PCR biobrick pSB1C3_VVD-YC155

Mix PCR preparation:

	VVD-YC155
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer Fwd 50 µM	0,5µl VVD Fwd I
Primer Rev 50 µM	1µl pSB1C3_VVD-YC155
Q5 enzyme	0,5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

Amplification of VVD-YC155 validation of the biobrick
VVD-YN: bands at 2000 bp only or 2000 bp and 1000 bp (profile of pSB1C3 with RFP) no part
pDawn: bands at 2000 bp only or 2000 bp and 1000 bp (profile of pSB1C3 with RFP) no part

Ligation of Gblocks into pSB1C3

Mix ligation preparation:

Tube	VVD-YC155	VVD-YN155	GFP rev	H/E rev	CCDB rev	HOKD rev
Water	9,5 µL	11,2 µL	9,5 µL	12,3 µL	7 µL	4,5 µL
T4 ligase Buffer	2 µL	2 µL	2 µL	2 µL	2 µL	2 µL
Gblock	5 µL	3,3 µL	5 µL	2,2 µL	7,5 µL	10 µL
pSB1C3	2,5 µL	2,5 µL	2,5 µL	2,5 µL	2,5 µL	2,5 µL
T4 ligase	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL

Room temperature, 30min

Expected results

Results

Ligation: epic fail

25 August 15

Results of transformations

2 colonies into the negative control plate (satellite colonies?)
Many white and red colonies into other plates (RFP again but not everywhere)
Liquid culture (2 from the negative control, 7 colonies from pDawn plates and 6 from VVD-YN 155 plates).

24 August 15

Expected results

Results

Quantification of pSB1C3: 44,6 ng / µl

Gibson assembly of pDawn and VVD-YN155, NEB kit

Quantification of VVD-YN155 DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume total
pSB1C3	44.55	2 200	1 430 000	0.000000031155	3.66	1.00	0.000000031155	1.00	0.00	1.00
VVD1	15.71	250	162 500	0.000000096698	1.18	3.10	0.000000031155	1.00	2.10	3.10
VVD2	7.65	250	162 500	0.000000047103	2.42	1.51	0.000000031155	1.00	0.51	1.51
YN155 I	8.42	200	130 000	0.000000064805	1.76	2.08	0.000000031155	1.20	1.30	2.50
YN155 II	14.84	200	130 000	0.000000114117	1.00	3.66	0.000000031155	1.20	3.20	4.40

Quantification of pDawn DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume total
pSB1C3	44.55	2 200	1 430 000	0.000000031155	3.61	1.00	0.000000031155	1.00	0.00	1.00
pDawnI	45.70	1045	679 250	0.000000067280	1.67	2.16	0.000000031155	1.40	1.62	3.02
pDawn II	54,45	882	573 300	0.000000094981	1.18	3.05	0.000000031155	1.00	2.05	3.05
pDawn III	119.57	1635	1 062 750	0.000000112511	1.00	3.61	0.000000031155	1.00	2.61	3.61

DNA mix: 1µL of each diluted DNA solution + 6 µL of MQ Water

DNA mix: completed with 10 µL of master mix
50°C, 15min
E.coli transformation: 1 aliquot with 5 µL of assembly product and 1 aliquot with 5 µL of assembly product diluted by a factor 4 for each assembly product, negative control.
30°C during the weekend

21 August 15

Results of transformation with the Gibson assembly products

Red colonies there is still some RFP into the tube with purified/digested pSB1C3
We have to try again

Digestion of pSB1C3

Mix digestion preparation:

Tube	pSB1C3
Water	1,5 µL
Buffer 2.1	5 µL
Plasmid	41,5 µL
Enzyme SpeI	1 µL
Enzyme XbaI	1 µL

37°C, 120 min --> heat kill: 80°C, 20min

Dephosphorylation of pSB1C3

Mix plasmid dephosphorylation preparation:

Tube	pSB1C3
Cutsmart	4 µL
Digested Plasmid	25 µL
rSAP	2 µL

37°C, 30 min heat kill: 65°C, 5min

Expected results

Results

Incomplete digestion
Excision of 2000bp bands for gel extraction (QIAgen kit)

20 August 15

Assembly of pDawn

Gibson assembly, NEB kit

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume total
pSB1C3	91.11	2 200	1 430 000	0.000000063713	1.77	1.00	0.000000031857	1.00	1.00	2.00
pDawnI	45.70	1045.00	679 250	0.000000067280	1.67	1.06	0.000000063713	0.95	0.00	0.95
pDawnII	54.45	882	573 300	0.000000094981	1.18	1.49	0.000000063713	2.00	0.98	2.98
pDawnIII	119.57	1635	1 062 275	0.000000112511	1.00	1.77	0.000000063713	1.00	0.90	1.90

DNA mix: 1µL of each diluted DNA solution or the indicated volume if no dilution is required (pDawnI) + 6,1 µL of MQ Water
Add 10 µL of master mix
50°C,15min
E.coli transformation: 1 aliquot with 5 µL of assembly product, 1 aliquot with 5 assembly product diluted by a factor 4.

18 August15

pDawn amplification and digestion

PCR2 amplification of pDawn III results

Expected results

Results

Little amplification but not enough

PCR pDawn III

Mix PCR preparation:

	pDawn III	pDawn III
MQ water	40 µL	26,5 µL
RB Buffer	5 µL	/
Mg2+	1,5 µL	/
Q5 Buffer	/	10 µL
High GC content Buffer	/	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd VI	0,5 µL pDawn Fwd VI
Primer Rev 50µM	0,5 µL pDawn Rev III-VI	0,5 µL pDawn Rev III-VI
DNA	1µL pDawn III PCR1	1µL pDawn III PCR1
Taq Pol	0.5 µL	/
Q5 Pol	/	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

Very good amplification of pDawn III
Gel extraction (kit from QIAgen)

Digestion of pDawn I, II and III

Mix digestion preparation:

Tube	pDawnI				pDawnII				pDawnIII
Water	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL
Buffer 2.1	2 µL	2 µL	2 µL	/	2 µL	2 µL	2 µL	/	2 µL	2 µL	2 µL	/
Buffer 3.1	/	/	/	2 µL	/	/	/	2 µL	/	/	/	2 µL
DNA	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL
Enzyme EcoRI	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5µL	/	/	/
Enzyme XbaI	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL	/	/
Enzyme SpeI	/	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL	/
Enzyme PstI	/	/	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL

37°C, 60 min

Expected results

Results

Simple digestions have shown that no other unexpected restriction site have appeared during PCR amplification of each fragment

30 July 15

Gel extraction of the 3 bands of pDawn

Miniprep of liquid culture with the red colonies

We used a kit from QIAgen

Digestion of pDawn and pSB1C3

Mix digestion preparation:

Tube	pDawn	Miniprep
Water	12 µL	12 µL
Buffer 2.1	2 µL	2 µL
Plasmid	5 µL	5 µL
Enzyme EcoRI	0,5 µL	0,5 µL
Enzyme SpeI	0,5 µL	0,5 µL

37°C, 60 min

Expected results

Results

We get 2 bands after the digestion of pDawn meaning a non-expected restriction site appeared into the sequence during the PCR useless to answer biobrick standard requirement.
The profile of the Gibson digestion correspond to pSB1C3 with the RFP (totally unexpected since we have purifies the digested backbone).

PCR pDawn III

Mix PCR preparation:

	pDawn III
MQ water	26,5 µL
Q5 Buffer	10 µL
High GC content Buffer	10 µL
dNTPs 10µM	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd VI
Primer Rev 50µM	0,5 µL pDawn Rev III-VI
DNA	1µL pDawn III PCR1
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

There were some amplifications of DNA materials but which one ????

PCR pDawn III

Mix PCR preparation:

	pDawn III
MQ water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTPs 10µM	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd VI
Primer Rev 50µM	0,5 µL pDawn Rev III-VI
DNA	1µL pDawn III PCR1
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	59-60-61	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Digestion of pDawn (PCR and Plasmid)

Mix digestion preparation:

Tube	pDawn PCR	pDawn PCR	pDawn PCR	pDawn	pDawn
Water	12 µL	12 µL	12 µL	12 µL	12 µL
Buffer 2.1	2 µL	2 µL	2 µL	2 µL	2 µL
Plasmid	5 µL	5 µL	5 µL	5 µL	5 µL
Enzyme EcoRI	0,5 µL	/	/	0,5 µL	/
Enzyme SpeI	/	0,5 µL	/	/	0,5 µL
Enzyme XbaI	/	/	0,5 µL	/	/

37°C, 60 min

Expected results

Results

Comparison of digestion profile between the original plasmid and the fragment amplificated by PCR. Confirmation of a new restriction site into pDawn PCR

29 July 15

Results of the last transformation

2 red colonies (which is weird)
Liquid culture for amplification

PCR fragments quantification

Expected results

Results

We get expected bands but, as for second PCRs of VVD and YN155 fragments, bands 2 time heavier than expected bands appeared

Test PCR pDawn (whole fragment)

Mix PCR preparation:

	pDawn (x3)	YC 155
MQ water	26,5 µL	26,5 µL
Q5 Buffer	10 µL	10 µL
High GC content Buffer	10 µL	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd I	0,5 µL YC155 Fwd
Primer Rev 50µM	0,5 µL pDawn Rev III-VI	0,5 µL YC155 Rev
DNA	1µL pDawn	1µL YC155 PCR1
Taq Pol	0.5 µL	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	59-60-61	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

We get an amplification of pDawn independent of the temperature

Expected results

Results

After the last Gibson reaction we have get red colonies that should correspond to bacteria transformed with pSB1C3-RFP (which should be impossible after the gel migration and purification). So we analyzed the content of the gel extraction and we saw that the tube only had linearized backbone. About pDawn III PCR 2, the migration of 5 µl from the gel extraction of PCR products have shown that there is not a high quantity of DNA material, not enough for a Gibson assembly…

28 July 15

PCR pDawn: whole fragment

Mix PCR preparation:

	pDawn	pDawn
MQ water	35,5 µL	35,5 µL
Q5 Buffer	10 µL	10 µL
High GC content Buffer	/	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd I	1µL pDawn Fwd I
Primer Rev 50µM	1µL pDawn Rev III-VI	1µL pDawn Rev III-VI
DNA	1µL pDawn	1µL pDawn
Taq Pol	0.5 µL	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

No amplification

Gibson Assembly

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume total
pSB1C3	44.55	2 200	1 430 000	0.000000031155	3.66	1.00	0.000000031155	1.00	0.00	1.00
VVD1	15.71	250	162 500	0.000000096698	1.18	3.10	0.000000031155	1.00	2.10	3.10
VVD2	7.65	250	162 500	0.000000047103	2.42	1.51	0.000000031155	1.00	0.51	1.51
YN155 I	8.42	200	130 000	0.000000064805	1.76	2.08	0.000000031155	1.20	1.30	2.50
YN155 II	14.84	200	130 000	0.000000114117	1.00	3.66	0.000000031155	1.20	3.20	4.40

DNA mix:
YN155 1: 1,2µl + 1,3µl of MQ water
YN155 2: 1,2µl + 3,2µl of MQ water
VVD1 : 1µl + 2,1µl of MQ water
VVD2 : 1µl + 0,5 µl of MQ water
pSB1C3 : no dilution
mix= 2µl of each

5µl used for the Gibson assembly added to 15µl of master mix
Transformation of 100 µl of competent cells using 5 µl of Gibson product

27 July 15

Results of bacterial transformation

A total of 12 colonies in both plates containing transformed bacteria
No colony into the negative control
“Biofilm” into the positive control, bacteria are alive and competent

Gel extraction from the cut band of the 16th of July

We used the kit from QIAgen to extract the DNA from the gel
Final elution into 50 µL

Digestion of Gibson product (VVD-YC155)

Mix for digestion

Tube	Gibson purified
Water	12 µL
Buffer 2.1	2 µL
Plasmid	5 µL
Enzyme EcoRI	0,5 µL
Enzyme PstI	0,5 µL

37°C, 60 min

PCR pDawn: whole fragment

Mix PCR preparation:

	pDawn
MQ water	35,5 µL
Q5 Buffer	10 µL
dNTPs 10µM	1 µL
Primer Fwd 50µM	1µL pDawn Fwd I
Primer Rev 50µM	1µL pDawn Rev III-VI
DNA	1µL pDawn
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ 4

	T°C	Time	Cycle
Initial denaturation	98	30 sec	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	3 min
Final extension	72	6 min	1
Hold	4	Infinite

Electrophoresis of PCR products

Expected results

Results

We get expected bands for our plasmid
No amplification of pDawn

22 July 15

Results of bacterial transformation

A total of 12 colonies in both plates containing transformed bacteria
No colony into the negative control
“Biofilm” into the positive control, bacteria are alive and competent

Gel extraction from the cut band of the 16th of July

We used the kit from QIAgen to extract the DNA from the gel
Final elution into 50 µL

Digestion of Gibson product (VVD-YC155)

Mix for digestion

Tube	Gibson purified
Water	12 µL
Buffer 2.1	2 µL
Plasmid	5 µL
Enzyme EcoRI	0,5 µL
Enzyme PstI	0,5 µL

37°C, 60 min

17 July 15

Results of bacterial transformation

No colonies
We should try again with a plasmid we already transformed
Bacterial survival has to be tested

Digestion of Gibson product (VVD-YC155)

NEB double digestion: Buffer 2.1
EcoRI:100%, PstI:75%

Mix for digestion

Tube	Gibson	Gibson
Water	12 µL	11,5 µL
Buffer 2.1	2 µL	/
Buffer 3.1	/	2 µL
Plasmid	5 µL	5 µL
Enzyme EcoRI	0,5 µL	0,5 µL
Enzyme PstI	0,5 µL	0,5 µL
Enzyme EcoRV	/	0,5 µL

37°C, 100 min

Electrophoresis of digested products

Expected results

Results

We get confirmation of the presence of pSB1C3 and our part, but there is still the unknown band. We have cut the heaviest band of the fourth wells to isolate our plasmid from the other one.

Test of competent cells: transformation with pSB1C3-RFP

Transformation of 100µl of homemade competent cells with 1 µl of pSB1C3 containing a RFP following our protocol and plated onto two agar plates containing chloramphenicol
100 µl of homemade competent cells have been split and plated onto two agar plates, one with chloramphenicol and the other one without antibiotic

16 July 15

Test of competent cells (transformation with pUC19)

Transformation of 50µl of competent cells with 1 µl of pUC19 following our protocol
50µL of competent cells have been used as negative control
Both of them have been plated onto agar plates containing chloramphenicol

Digestion of Gibson product (VVD-YC155)

NEB double digestion: Buffer 2.1
EcoRI:100%, PstI:75%

Mix for digestion

Tube	Gibson
Water	12 µL
Buffer 2.1	2 µL
Plasmid	5 µL
Enzyme EcoRI	0,5 µL
Enzyme PstI	0,5 µL

37°C, 60 min

Electrophoresis of digested products

Expected results

Results

We get expected bands at approximatively 2000 bp and 750 bp, but it still an extra band at 1500 bp (already present during the previous electrophoresis). That could correspond to another plasmid.

15 July 15

PCR pDawn BioBricks

PCR 2 pDawn I,II and III

Aim: second step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn I	pDawn II	pDawn III
MQ water	39,5 µL	39,5 µL	39,5 µL
Mg2+ 50 mM	1,5 µL	1,5 µL	1,5 µL
Buffer RB	5 µL	5 µL	5 µL
dNTPs 10µM	1 µL	1 µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd I	1µL pDawn Fwd V	1µL pDawn Fwd VI
Primer Rev 50µM	1µL pDawn Rev IV	1µL pDawn Rev V	1µL pDawn Rev VI
DNA	1µL pDawnI PCR1	1µL pDawnII PCR1	1µL pDawnIII PCR1
Taq Pol	0.5 µL	0.5 µL	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn I, II and III

Expected results

Results

Good amplification of pDawnI and II
The amplification of pDawn 3 is unexpected and cannot be used for the following experiment
About the miniprep of bacteria transformed with the Gibson assembly product: two plasmids have been purified (only one expected); additional checking steps will be lead to characterize these plasmids

Gel purification

QIAquick gel extraction
2 bands of each fragment (pDawnI and II) into 1 column
Concentrate into 30 µl of Elution Buffer

PCR amplification of pDawnIII

Aim: step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III	pDawn III
MQ water	35,5 µL	25,5 µL
High GC content Buffer	/	10 µL
Buffer Q5	10 µL	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III	1µL pDawn Rev III
DNA	1µL pDawnIII PCR1	1µL pDawnIII PCR1
Taq Pol	0.5 µL	0.5 µL

PCR cycle – IGEM TAQ 3

	T°C	Time	Cycle
Initial denaturation	98	30 sec	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

Small amplification of pDawnIII

Gel purification

QIAquick gel extraction
4 bands of pDawnIII into 4 columns (due to high volume of buffer)
Concentrate into 25 µl x4 of Elution Buffer

Production of competent cells (3rd step)

Massive liquid culture into an Erlenmeyer of 500 ml (100 ml of LB medium) using few ml of pre-liquid culture
Application of the protocol for the production of competent cells 95 aliquots of 100µL

10 July 15

Production of competent cells (next steps)

Results of the first step: bacterial amplification

Good bacterial amplification --> miniprep with a MOBio kit (20 ml into 2 columns, final elution with 50 µl for each)
The first PCR to amplify pDawn III has been made by our advisor Samuel Juillot.

Second step

Bacteria have grown on the plate; no bacteria into the control
Pre-liquid culture into an Erlenmeyer without selective condition using an isolated colony from the plate; with a control (Erlenmeyer without bacteria)

9 July 15

PCR pDawn BioBricks

Results of the latest transformation

Aim: first step for pDawn amplification and mutagenesis

After 48h, 2 white shapeless colonies have grown --> Liquid culture (20mL of LB + 20µl of Cam)

Production of competent cells (1st step)

Culture of E.coli DH5 alpha from NEB on a non-selective agar plate with a control (plate without bacteria)

8 July 15

PCR pDawn BioBricks

Results of the latest transformation

Aim: first step for pDawn amplification and mutagenesis

No colonies have grown

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of the Gibson product

Expected results

Results

Soft bands: it means that some reactions occured

7 July 15

PCR pDawn BioBricks

PCR pDawn III (3)

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III	pDawn III
MQ water	25,5 µL	24 µL
Mg2+ 50mM	/	1,5 µL
Q5 Buffer	10 µL	10 µL
High GC content Buffer	10 µL	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III	1µL pDawn Rev III
DNA	1µL pDawn	1µL pDawn
Taq Pol	0.5 µL	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

No amplification

Gibson Assembly

Aim: first step of our VVD Biobrick

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume total
pSB1C3	91.11	2 200	1 430 000	0.000000063712	7.75	1.35	0.000000047103	3.50	1.50	5
VVD1	15.71	250	162 500	0.000000096698	5.10	2.05	0.000000047103	1.00	1.00	2
VVD2	7.65	250	162 500	0.000000047103	10.48	1.00	0.000000047103	1.00	0	1
YC155	128.31	400	260 000	0.000000493488	1.00	10.48	0.000000047103	1.00	9.00	10

DNA mix:

• YC155: 1µL + 9µL of MQ water
• VVD1: 1µL + 1µL of MQ water
• VVD2: no dilution
• pSB1C3: 3.5µL + 1.5µL of MQ water

Mix = 2µL of each
5µl used for the Gibson assembly added to 15µl of master mix

E.coli transformation

3 tubes: - 20µL of competent cells + 5µL of Gibson product - Idem - 20µL of competent cells as negative control
Same protocol as used before but only 200µl of LB has been added to resuspend bacteria. Then the final volume of bacteria which has been plated was 50 µl. 37°C, O/N

6 July 15

PCR pDawn BioBricks

PCR pDawn III (2)

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III
MQ water	25,5 µL
Q5 Buffer	10 µL
High GC content Buffer	10 µL
dNTPs 10µM	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

No amplification

3 July 15

PCR pDawn BioBricks

PCR pDawn III

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III
MQ water	39,5 µL
Mg2+ 50 mM	1,5 µL
Buffer RB	5 µL
dNTPs 10µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Gibson Assembly

Aim: first step of our VVD Biobrick

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume totale
pSB1C3	91.11	2 200	1 430 000	0.000000063712	7.75	1.35	0.000000047103	3.50	1.50	5
VVD1	15.71	250	162 500	0.000000096698	5.10	2.05	0.000000047103	1.00	1.00	2
VVD2	7.65	250	162 500	0.000000047103	10.48	1.00	0.000000047103	1.00	0	1
YC155	128.31	400	260 000	0.000000493488	1.00	10.48	0.000000047103	1.00	9.00	10

DNA mix:

• YC155: 1µL + 9µL of MQ water
• VVD1: 1µL + 1µL of MQ water
• VVD2: no dilution
• pSB1C3: 3.5µL + 1.5µL of MQ water

Mix = 2µL of each
5µl used for the Gibson assembly added to 15µl of master mix

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

Soft band at 1500 bp, good but not enough

1 July 15

PCR pDawn BioBricks

PCR YC155, pDawn I, II and III

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	YC155	pDawn I	pDawn II	pDawn III
MQ water	39,5 µL	39,5 µL	39,5 µL	39,5 µL
Mg2+ 50 mM	1,5 µL	1,5 µL	1,5 µL	1,5 µL
Buffer RB	5 µL	5 µL	5 µL	5 µL
dNTPs 10µL	1 µL	1 µL	1 µL	1 µL
Primer Fwd 50µM	1µL YC155 Fwd	1µL pDawn Fwd I	1µL pDawn Fwd II	1 µL pDawn Fwd III
Primer Rev 50µM	1µL YC155 Rev	1µL pDawn Rev I	1µL pDawn Rev II	1 µL pDawn Rev III
DNA	1µL PCR YC155	1µL pDawn	1µL pDawn	1 µL pDawn
Taq Pol	0.5 µL	0.5 µL	0.5 µL	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products YC155, pDawn I, II and III

Expected results

Results

We get expected bands for YC155, pDawn I and II but there is no amplification for pDawn III
First explanation: human error We should make the reaction again

Gel Purification

Aim: purification of fragment from PCR

QIAquick gel extraction
2 bands of each fragment into 1 column
Concentrate into 50 µl of Elution Buffer

PCR YC155, pDawn I, II and III

Mix PCR preparation:

	pDawn III
MQ water	39,5 µL
Mg2+ 50 mM	1,5 µL
Buffer RB	5 µL
dNTPs 10µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

iGEMTAQ program

Electrophoresis

Aim: Migration of PCR product

Gel : 1,2 g agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of PCR products purified YC155 and pDawn III

Expected results

Results

No amplification of pDawn III
YC155 quantification = 128 ng.µL^-1

30 June 15

Digestion of pSB1C3

Tube preparation:

Tube	pSB1C3 I	pSB1C3 II	pSB1C3 III
Water	3 µL	3 µL	8 µL
Buffer 2.1	5 µL	5 µL	5 µL
Plasmid pSB1C3	40 µL	40 µL	35 µL
Enzyme EcoRI	1 µL	1 µL	1 µL
Enzyme PstI	1 µL	1 µL	1 µL

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 65min

Electrophoresis

Aim: check for digestion

Migration of the three digested pSB1C3 samples

Expected results

Results

Gel Purification

QIAquick gel extraction
4 bands (1,175 g) into 1 column
Concentrate into 30 µL of Elution Buffer

Electrophoresis

Aim: quantification of DNA

Migration of digested pSB1C3 samples

Expected results

Results

Quantification by Image J: 91 ng.µl^-1

12 June 15

Result from 10.06.15

Culture liquid

pSB1C3 culture liquid

pSB1C3: medium became red (RFP) and highly cloudy

BBa_I712074 and BBa_K124003 culture liquid

BBa_I712074 and BBa_K124003: cloudy, no contamination in CT-, it still alive bacteria

Miniprep

Protocol from MOBIO Kit

• pSB1C3: 20 ml into 4 columns, eluted with 30 µl (pooled together)

Electrophoresis

Gel: 1,2 g of agarose + 120 ml of TAE 1x + 150 µl of BET
Migration of purified pSB1C3

Expected results

Results

Quantification of pSB1C3 by Image J = 35 – 40 ng.µL^-1
4 µg (min) of pSB1C3 are requiring for digestion of the plasmid to make a Gibson reaction

• 35 ng x 115 µl (remaining) = 4,025 µg all the remaining 115 µL of pSB1C3 should be digested

11 June 15

Miniprep

Protocol from QIAgen, UltraClean® Standard Mini plasmid Prep Kit

• BBa_I712074
• BBa_K124003

Glycerol stock

Stock of BBa_I712074 and BBa_K124003 (cf protocol for glycerol stock), -80°C

Electrophoresis

Migration of purified BBa_I712074 and BBa_K124003

Expected results

Results

Bands do not correspond exactly to expected weights (supercoiled)

Plate preparation

3 plates: 20 mL of LB agar + 20 µL of Cam

Liquid culture

Double click tube: 3 mL of LB broth + 3 µL of antibiotic

Tube preparation

Tube	Antibiotic
BBa_I712074	Cam
BBa_K124003	Cam
Control Negative	Cam

Survival checking of bacteria after a night spent into a -20°C freezer
Erlenmeyer: culture of bacteria transformed with pSB1C3 coming from glycerol stock; 20 mL of LB broth + 20 µL of Cam

10 June 15

Amplification of iGEM parts

Amplification of bacteria

Amplification of bacteria already transformed with BBa_I712074 (Promoter T7) or BBa_K124003 ( Endolysin/Holin)
On petri dish: 20 mL LB Agar + 20µL Amp & 20µL Kan
LB Broth: 2,5 mL of LB + 2,5µL of Amp & 2,5µL of Kan

9 June 15

Miniprep: QIAgen vs MOBIO

Miniprep test using liquid culture of Terminator T7 and RBS to check the efficiency of miniprep kit from QIAgen and MOBIO

Electrophoresis

Aim: Verification of the purification

Migration of miniprep products

Expected results

Results

Bands have more migrated than expected but it could be due to the folding of circular plasmid. Both kits are efficient.

Plasmid digestion

Tube preparation:

Tube	Terminator T7	RBS
Water	12 µL	12 µL
Buffer 2.1	2 µL	2 µL
Plasmid pSB1C3	5 µL	5 µL
Enzyme EcoRI	0.5 µL	0.5 µL
Enzyme PstI	0.5 µL	0.5 µL

NEB double digestion
Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Migration of digested T7 and bJun from 29.05.2015 and digested T7 and RBS from 05.06.2015

Expected results

Results

After digestion we get expected band for T7 terminator and RBS from minipreps of the day

Gibson Assembly

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1.5 µL pSB1C3
• 1 µL PCR.2 VVD1 (diluted 1/100)
• 2 µL PCR.2 VVD2 (diluted 1/100)
• 1 µL YC155 (diluted 1/10)

5 µL of the DNA mix is adding to the Master mix
Put immediately into a hot bath at 50°C for 1 hour

Transformation

Aim: transformation of E.Coli with Gibson Assembly product

20 µL of competent cells + 5 µL of Gibson product
Protocol for bacteria transformation: using 200 µl of LB broth before incubation for 1 h at 37°C and discarding 150 µl of supernatant before plating

5 June 15

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

Into aliquots, added plasmids or nothing (control) as following:

Tube	pSBC3
Buffer 2.1	2 µL
Plasmid pSB1C3	17 µL
Enzyme EcoRI	0.5 µL
Enzyme PstI	0.5 µL

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: electrophoresis for gel purification

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Results

We get expected bands
Extraction of the heaviest band of digested pSB1C3 and all the others expected bands for VVD 1, VVD 2, YC155, YN155 1, YN155 2.

Gel Purification

QIAquick gel extraction

VVD 1	180 mg				VVD 2	590 mg
YN155 1	180 mg				YN155 2	200 mg
YC155	120 mg				pSB1C3	190 mg

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

Electrophoresis for quantification

Aim: quantification of ADN to perform Gibson Assembly

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Results

There is not enough DNA to detect and quantify pSB1C3 and YC155 Try again

Electrophoresis for quantification - 2^nd

Migration of digested pSB1C3, VVD1 PCR2, VVD2 PCR2, YC155, YN155 I PCR2, YN155 II PCR2

Expected results

Results

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution
pSB1C3	0.92	2 200	1 430 000	0.000000000642	177.7	1	0.000000000642
VVD1	15.71	250	162 500	0.000000096698	1.18	151	0.000000000642
VVD2	7.65	250	162 500	0.000000047103	2.42	73	0.000000000642
YC155	2.50	400	260 000	0.000000009631	11.85	15	0.000000000642
YN155.1	8.42	200	130 000	0.000000064805	1.76	101	0.000000000642
YN155.2	14.84	200	130 000	0.000000114117	1	178	0.000000000642

Liquid culture

Double click tube: 3 mL of LB broth + 3 µL of antibiotic

Plasmid	Antibiotic				Plasmid	Antibiotic
Terminator T7	Cam				bFos	Amp
RBS	Cam				bJun	Amp
Control Negative	Cam				Control Negative	Amp

Concentrate into 30 µl of Elution Buffer Concentrate into 30 µl of Elution Buffer

4 June 15

Results from 29.05.2015

Results of transformation with Gibson assembly product

No bacterial growth

Electrophoresis of digestion product

Aim: check for digestion product from 29.05.2015

Expected results

Results

No bands for miniprep samples or for digestion products
The miniprep has failed

2 June 15

Liquid culture results

Results of the liquid culture from 28/05/2015

After 24h of growth:
Terminator T7 bacteria growth strongly into both tubes
bJun bacteria growth into strongly one tube only, the other one is “cloudy”
bFos both tube are “cloudy”
Control negative clear

Glycerol stock

Stock of Terminator T7 and bJun (cf protocol for glycerol stock), -80°C

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from QIAgen, UltraClean® Standard
Mini plasmid Prep Kit
Terminator T7: 2 tubes
bJun: 1 tube

Plasmid digestion (Terminator T7 & bJun)

Terminator T7

Tube	Terminator T7	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme SpeI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• SpeI: 100%

bJun

Tube	bJun	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme PstI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Gel Purification

Aim: purification of fragment from 28.05.2015

Use of a QIAquick gel extraction kit with the protocol of QIAgen
Gel weight = 270 mg

Gibson Assembly

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1 µL pSB1C3
• 2 µL PCR.2 VVD1
• 2 µL PCR.2 VVD2
• 2 µL YC155

5 µL of the DNA mix is adding to the Master mix
Put immediately into a hot bath at 50°C for 1 hour

Transformation

Aim: transformation of E.Coli with Gibson Assembly product

Transformation

Plasmid	Plate
3 µL of plasmid	20 µL on plate
	50 µL on plate
6 µL of plasmid	20 µL on plate
	50 µL on plate
Control negative	100 µL on plate

Incubate at 25°C during 60 hours

29 May 15

Amplification of pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
pSB1A2
	Ampicillin	1	50 µl of transformed cells
		1	20 µl of transformed cells
Control negative
	Amplicilin	1	50 µl of transformed cells

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation
100 µl remaining competent cells have been put at -80°C again (loss of efficiency)

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	pSB1A2	1
2	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the control plate
Many colonies into the other plate

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

Into aliquots, added plasmids or nothing (control) as following:

Tube	pSBC3
Water	16 µL
Buffer 2.1	2 µL
Plasmid pSB1C3	1 µL
Enzyme EcoRI	0.5 µL
Enzyme PstI	0.5 µL

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: check for digestion

1 gel for purification: 110ml TAE 1X + 1,1g agarose + 140µl BET
Sample (purification): 20µl of digestion product+ 2µl of loading dye (6x)
Sample (quantification): 1µl of DNA + 2 µl of loading dye (6x) + 9µl of water MQ
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ
110 V, 45 min

Expected results

Results

Liquid culture

Antibiotics concentration:
Tube 1 & 2: 3 mL LB + 3 µL Cam; colonies from “1µL” plate of Terminator T7 transformation
Tube 3 & 4: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bFos
Tube 5 & 6: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bJun
Tube 7 & 8: 3 mL LB
37°C, 120 rpm, O/N

28 May 15

Results from cell tranformation

Results of the transformation from 25/05/2015:

After 24h of growth:
No colonies into negative controls Many colonies appears into plates with bFos and bJun transformed bacteria No colonies into plates with pSB1A2 transformed bacteria:

• It could be due to the presence of 2 antibiotics (high environmental pressure)

26 May 15

RBS BioBricks

Recover dried DNA sent by iGEM (part BBa_J61100)

Plasmid characteristic:

• Part name: BBa_J61100
• Plasmid: pSB1A2
• Part name: BBa_J61100
• Resistance: AK (Amplicilin/Kanamicin)

Punch a hole without removing the foil
Pipette 10µl of water MQ (up and down)
Let sit for 5 minutes to make sure the dried DNA is fully resuspended

Recovered plasmid have been put into a 50µl eppendorf tube called “BBa_J61100, RBS”

Amplification of bFos, bJun, pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
bFos
	Amplicillin	1	50 µl of transformed cells
		1	100 µl of transformed cells
bJun
	Amplicillin	1	50 µl of transformed cells
		1	100 µl of transformed cells
pSB1A2
	Ampicillin + Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
Control negative
	Amplicilin	1	50 µl of transformed cells
	Kanamycin	1	50 µl of transformed cells

Antibiotics concentration:

• Ampicilin: 50 µg.ml^-1 diluted 1/1000
• Kanamycin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	bFos	1
2	bJun	1
3	pSB1A2	1
4	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 2 control plates
Many colonies into all the other plates
Colonies from pSB1A2 transformation should exhibit red color

25 May 15

PCR VVD BioBricks

PCR.2 Nter (YN155)

Aim: second step of mutagenesis for YN155

PCR.2 YN155 preparation

Component	PCR.2 YN155 I (x2)	PCR.2 YN155 II (x2)	Control - YN155 I (x2)	Control - YN155 II
Primer Fwd YN155 1	1 µl	--	--	--
Primer Rev YN155 3	1 µl	--	--	--
Primer Fwd YN155 4	--	1 µl	--	--
Primer Rev YN155 2	--	1 µl	--	--
PCR.1, YN155 I	1 µl	--	1 µl	--
PCR.1, YN155 II	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix PCR (x8) without primers, DNA and Taq pol
PCR cycle IGEM TAQ program, Tm = 61,3°C

Electrophoresis

Aim: check for digestion

Remaining gel (120ml TAE 1X + 1,2g agarose + 150µl BET) with 8 wells
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 26 min

Expected results

Results

We get expected bands with a good intensity meaning DNA have been highly amplified.

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 2 tubes: “PCR2, YN I, 22/05/2015”, “PCR2, YN II, 22/05/2015”
Stored at -20°C.

Liquid culture

Use of 3 colonies (3 tubes) from pSB1C3 (BBA_B0015) 1µl and pSB1C3 (BBA_B0015) 4µl
3ml LB Broth + 3µl Cam in double click tubes (12ml) + 1 control tube (3ml LB Broth)
37°C, 120rpm, O/N

22 May 15

PCR VVD BioBricks

PCR.1 YFP Cter (YC155) & Nter (YN155)

Aim: amplification of YC155 and first step of mutagenesis for YN155

PCR YC155 and YN155 preparation

Component	PCR YC155 (x2)	PCR.1 YN155 I (x2)	PCR.1 YN155 II (x2)	Control - YC155	Control - YN155
Primer Fwd YC155	1 µl	--	--	--	--
Primer Rev YC155	1 µl	--	--	--	--
Primer Fwd YN155 I	--	1 µl	--	--	--
Primer Rev YN155 I	--	1 µl	--	--	--
Primer Fwd YN155 II	--	--	1 µl	--	--
Primer Rev YN155 II	--	--	1 µl	--	--
Plasmid pBiFC-bFos	1 µl	--	--	1 µl	--
Plasmid pBiFC-bJun	1 µl	--	--	1 µl	--
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Mix dNTPs: 2 µl of each dNTP
Mix PCR without primers, plasmids and Taq Pol
PCR cycle IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 150µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 25 min

Expected results

Results

We get expected bands with a good intensity meaning DNA have been highly amplified

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 3 tubes: “PCR1, YC155, 21/05/2015”, “PCR1, YN155 I, 21/05/2015”, “PCR1, YN155 II, 21/05/2015”
Stored at -20°C.

Backbone & Terminator

Transformation pSB1C3 – double T7 terminator (BBa_B0015)

Aim: plasmid amplification

3 plates with 25 ml LB medium + 25 µl of Cam antibiotic / plate
Recover dried DNA sent by iGEM

• Punch a hole without removing the foil (Plate 3, well F3)
• Pipette 10 µl of water MQ (up and down)
• Pipette 10 µl of water MQ (up and down)
• Protocol for bacteria transformation

Cell transformation

• E.coli DH5 alpha (NEB5 alpha competent coli)
• Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)
1	pSB1C3 – double T7 terminator (BBa_B0015)	1
2	pSB1C3 – double T7 terminator (BBa_B0015)	4
3	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min. Do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 100 µl
Plate bacteria and incubate at 37°C, O/N

Results
Control was cloudy as cultures (lack of antibiotics) should be done again
21 May 15

PCR VVD BioBricks

PCR.2 VVD - 2nd part

Aim: second step for VVD amplification and mutagenesis

End of PCR.2 VVD preparation from 07.05.15

Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative	Control negative
DNA Mix "PCR.1 VVD1"	1 µl	--	1 µl	--
DNA Mix "PCR.1 VVD2"	--	1 µl	--	1 µl
Taq Pol	0.5 µl	0.5 µl	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for digestion

1 gel: 120ml TAE 1X + 1,2g agarose + 160µl BET
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x) only 10 µl loaded.
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ only 10 µl loaded.
110 V, 30 min

Expected results

Results

We get expected bands about 250 bp meaning DNA have been highly amplified

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 1 tube “PCR2, VVD1, 13/05/2015” and another one “PCR2, VVD2, 13/05/2015”
Stored at -20°C.

13 May 15

PCR VVD BioBricks

PCR.1 VVD - 2nd part

Aim: first step for VVD amplification and mutagenesis

PCR.1 VVD preparation

Component	PCR.1 VVD 1 (x2)	PCR.1 VVD 2 (x2)
PCR mix	43,5 µl	43,5 µl
Primer VVD Fwd 1	1 µl	--
Primer VVD Rev 1	1 µl	--
Primer VVD Fwd 2	--	1 µl
Primer VVD Rev 2	--	1 µl
Water	2 µl	2 µl
Plasmid VVD	1 µl	1 µl
Taq Pol	0.5 µl	0.5 µl

Run of the IGEM TAQ program

Electrophoresis

Aim: check for DNA amplification

Low melting point gel
Sample: 10 µl of PCR product+ 2 µl of loading dye (6x)
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl of loading dye (6x) + 9 µl water MQ
110 V, 26 min

Expected results

Results

We get expected bands about 200 bp with a good intensity meaning DNA have been highly amplified without contamination.

Purification of PRC product

Aim: purify and concentrate amplified DNA

Use of a QIAquick PCR Purification kit and using a microcentrifuge.

For step 1: add 5 volumes of PB buffer for 1 volume of PCR product

• 200 µl of PB buffer added into both coupled of tube with 40 µl of remaining PCR product
• Mix of same PCR product together into a new tube
• Put mixes into their own purification column

Then, we followed the protocol from the supplier

At the end of the purification, we get 1 tube “PCR1, VVD1, 07/05/2015” and another one “PCR1, VVD2, 07/05/2015”
Stored at -20°C.

PCR.2 VVD

Aim: second step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR.2 VVD preparation

Component	PCR.2 VVD 1 (x2)	PCR.2 VVD 2 (x2)	Control negative (x2)
Primer VVD Fwd 1	1 µl	--	--
Primer VVD Rev 3	1 µl	--	--
Primer VVD Fwd 4	--	1 µl	--
Primer VVD Rev 2	--	1 µl	--
Water	39.5 µl	39.5 µl	41.5 µl
Buffer RB	5 µl	5 µl	5 µl
dNTPs	0.5 µl	0.5 µl	0.5 µl
Mg2+ 50mM	1.5 µl	1.5 µl	1.5 µl

7 May 15

PCR VVD BioBricks

PCR.1 VVD

Aim: first step for VVD amplification and mutagenesis

dNTP preparation (4µL):
1 µl dATP 100 mM
1 µl dCTP 100 mM
1 µl dTTP 100 mM
1 µl dGTP 100 mM
Store at -20°C

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	1 min / kb
Final extension	72	5 min	1
Hold	4	Infinite

Mix PCR preparation without primers, plasmid and Taq

MQ water	182,5 µl
Mg2+ 50 mM	7,5 µl
Buffer RB	25 µl
dNTPs	2,5 µl

Mix preparation for 5 tubes: 43,5 µl / tube

6 May 15

Results

Results from the liquid cultures: all the cultures have grown

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from MO BIO, UltraClean® Standard
Mini plasmid Prep Kit l
4 plates with Ampicillin

Dilution of the glycerol in water
Percentage in mass not in volume
Ex: 50% glycerol solution 50 g Glycerol + 50 g water (mQ)
Then filtration with vacuum pump

Glycerol preparation & Glycerol Stock

Aim: store the transformed bacteria for later use

Weight 50 g of pure Glycerol
Weight 50 g of distilled water
Mix and homogenize the solution
Filtration with vacuum pump, then close the bottle under PSM
Under PSM class II: put 1ml of liquid culture in a 1,5 ml sterile tube (pDusk, pDawn, psB1C3, VVD)
Centrifugate 1mL of liquid culture 4000 rpm, 5 min
Remove and discard the supernatant
Under PSM: resuspend with 0,5 ml of LB without antibiotics
Add 0,5 ml of glycerol 50% (25% final)
Homogenize
Transfer in an identified cryotube and store at -80°C

1 April 15

Results

Results observation for the transformed cells culture (in Petri dishes): no colonies for the positive control pUC19, other plates and negative controls are as expected.

Liquid culture

Antibiotics concentration:
Use of 3 colonies (3 tubes) from VVD, pDawn, pDusk, pSB1C3 3ml LB Broth + 3µl
Antibiotic in double click tubes (12ml) 37°C, 120rpm, O/N

31 March 15

Amplification of pDusk, pDawn, pSB1C3, VVD, pUC19 into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 50 ml of LB agar into a Falcon tube (50ml) + 50 µl antibiotic
For 25 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
pDusk
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pDawn
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pSB1C3
	Chloramphenicol	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
VVD
	Amplicilin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pUC19	Amplicilin	1	50 µl of transformed cells

Summary:
6 plates with Kanamycin
3 plates with Chloramphenicol
4 plates with Ampicillin

Antibiotics concentration:
Ampicillin: 50 µg.ml^-1 diluted 1/1000
Chloramphenicol: 25 µg.ml^-1 diluted 1/1000
Kanamycin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)			Tube n°	Reagent added	Volume (µl)
1	pDusk	1			5	pSB1C3	1
2	pDawn	1			6	(Amp control)	/
3	VVD	1			7	(Kan control)	/
4	pUC19	1			8	(Cam control)	/

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 3 control plates
Many colonies into all the other plates;
Colonies from pSB1C3 transformation should exhibit red color

30 March 15

Preparation of media

Aim: prepare media for bacteria culture

For 250mL

LB Broth	LB Agar
2.5g tryptone	2.5g tryptone
2.5g NaCl	2.5g NaCl
1.25g yeast extract	1.25g yeast extract
	5.0g agar

Qsp 250 ml distilled water
2 bottles of LB broth and 2 bottles of LB agar (+magnetic stirrer)
Auctoclave 121°C

28 March 15