• July
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July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
4. Waiting 30 min in ice
5. Heat shock 30 sec 42°C
6. Then 5min in ice
7. Add 200uL SOC medium
8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control

I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.

July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method

The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)

The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.

In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)

Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.

When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.

Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.

Centrifuge 10min at 4°C.

Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.

Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.

July 30th

Goal 1

PCR my G block and backbone

Procedure

PCR1 = Backbone + 068 and 069 primers, before purify

PCR2 = FINAL + 068 and 069 primers, before purify

Then PCR purification

Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL

Goal 2

Digestion/Ligation/Transformation

Procedure

First, digestion :

PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL

PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL

10 min at 37°C

Then PCR purification

Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7

For the ligation :

Vector PCR1 100 ng = 5 uL; Insert PCR 2 300 ng = 12uL T4 ligase buffer 2uL ; T4 ligase 1 uL ; Total 20 uL

20-40 min on the bench

Nanodrop : L= 270 ng/uL

Then I did a heat shock transformation (following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation )

C + = 10 ng/uL of RFP in pS1C3, 2 uL transformed (10pg/ul RFP Control (pSB1C3 w/ BBa_J04450)

C - = nothing but efficients cells

T = Plasmid construct 2uL transformed

Each with 50 uL of chemically competents cells done yesterday.

Then I did 5 different plates :

5 plates with 25ug/mL chloramphenicol in LB

200 uL control + , 20 uL control +

200 uL T , 20 uL T

200 uL C -

Incubation overnight at 37°C

July 31th

Goal 1

Check the transformations I've done yesterday

Procedure

According to my plates my competent cells are working, red colonies on the C+. Transformation of my construction didn’t worked, I’ll start again all the process : PCR, purify, digest, ligate, integrate (heat shock), overnight culture.

Goal 2

PCR my backbone and my insert

Procedure

PCR1' = Backbone + 068 and 069 primers, before purify

PCR2' = FINAL + 068 and 069 primers, before purify

After PCR and after purification : 1'p = 66,5 2'p =90,5 ng/uL

The gel migration shows a bad result for PCR 1’ (Linearized pSB1C3)

August 1st

Goal

PCR my backbone and my insert again : 1" and 2"

Procedure

PCR then purify

August 5th

Goal

Ligation/transformations (insertion of FINAL block from IDT in the pSB1C3 backbone, FINAL is my Riboswitch associated with an EGFP)

Procedure

Used 3 different insert PCR products

Ligation after digestion and purification of PCR2’ = 94,6ng/uL , PCR2’’= 57,5 ng/uL , PCR2’’’=83,4 ng/uL ; Backbone pSB1C3 = 37,9ng/uL

	'	"	"'
Insert	4	7	5
Backbone	5	5	5
Buffer	2	2	2
Ligase	1	1	1
Water	8	5	7
TOTAL	20uL	20uL	20uL

I did then a transformation using the iGEM protocol (Heat Shock) with 50uL of chemically competent cells + 3 uL of Ligation Product for each (pSB1C3-TOTAL)

C + : pSB1C3-RFP

C - : just competent cells

Plated on Cm 25 ug/mL

5 plates : ‘,’’,’’’,C+,C-

Results

C- : nothing

C+ : lot of red colonies

‘ : lot

‘’ : lot

‘’’ : lot

I should have done a control transformation with just the backbone

August 6th

Goal 1

Cultivate my transformants

Procedure

There is red and white transformant colonies, all resistant to Cm 25

I selected some white colonies overnight culture

On plate LB Cm —> it grew (some red, maybe contamination)

On broth LB Cm —> it grew (some red, maybe contamination)

Inoculated at 4:00 pm in LB Cm 25 bacteria from a culture ( red —> RFP ) with different AdoCbl concentrations in order to detect the minimal amount of B12 viable for e.coli : 5; 10; 20; 40; 80; 160 ng/mL

Goal 2

PCR the pSB1C3 backbone and my FINAL riboswitch construction

Procedure

2 PCR each

I1 and I2 using primers 150 and 151

LP1 and LP2 using primers 152 and 153

Purification then nano drop : good for I2 : 112.4 ng/uL

Results

Goal 3

Do the PCR again

Procedure

	Template	Primers	MasterMix	Water	TOTAL	Annealing T°C	Elongation time
I3	2ng = 2 uL	1 uL	25 uL	22 uL	50uL	56°C	2 min
I4	2ng = 2 uL	2 uL	25 uL	21 uL	50uL
LP3	25ng = 1uL	1 uL	25 uL	23 uL	50uL
LP4	25ng = 1uL	2 uL	25 uL	22 uL	50uL

Results

After PCR purify, nanodrop (ng/uL)

I3	I4	LP3	LP4
30.7	177.6	86.7	93.9

Gel migration
Very good for LP4, good for LP3
Nothing for I3 and I4

August 7th

Goal 1

Digestion of LP4 and I2

Procedure

	LP4	I2
EcoRI	4uL	4uL
PstI	4uL	4uL
FastAP	4uL	-
FD Buffer	12uL	8uL
DNA	2 ug (90 ng/uL) —> 20 uL	2ug (100 ng/uL) —> 20 uL
Water	76 uL	44 uL
TOTAL	120uL	80uL

New nano drop : LP4 = 87,4 ; I2 = 90,4 ng/uL
Waited 15 min at 37°C
After digestion and PCR purification, nanodrop :
38.2 ng/uL for I2 (d. and p.)
31.2 ng/uL for LP4 (d. and p.)

Goal 2

Ligation of LP4dp and I2dp

Procedure

	L*
Vector LP4 dp	100 ng (31.2 ng/uL) —> 3uL
Insert I2 dp	300 ng (38.2 ng/uL) —> 8uL
T4 L,Buffer	2uL
T4 ligase	1uL
Water	6uL
TOTAL	20 uL

Waited 40 min on the bench (25°C)

Goal 3

Transformation of L*

Procedure

	C-	C+	T*
ChemicallyCompetent Cells	50 uL	50 uL	50 uL
L*	-	-	3 uL
pSB1C3 with RFP 100ng/uL	-	3 uL	-

iGEM Transfo protocol
90 sec heat shock
On saturday they were growing but no fluorescence

August 10th

Goal 1

Colony PCR my T*

Procedure

Pick up 16 colonies on my two T* plates
Put some bacteria in PCR tube + 2 uL of 148 and 149 (iGEM verification forward and backward) ;
10uM + 50 uL of Master Mix Phusion (last in the tubes) + 45 uL of water (first in the tube)
Waiting for a 1577 bp fragment on a gel Gel migration with a GR 1kb

It is quiet bad

August 11th

Goal 1

Do a new PCR of FINAL (G-block) and of pSB1C3 associated with RFP

Procedure

	Template	Primers	Master Mix Phusion	Water	Time of elongation (72°C)	T°C annealing	Lenght	Primers	Nanodrop after purify (ng/uL)
LP1*	5ng	2uL	25uL	QSP for 50uL	2 min	56°C	2078	152 - 153	68.3
LP2*	10ng	2uL	25uL	QSP for 50uL	2 min	56°C	2078	152 - 153	133.5
LP3*	2ng	2uL	25uL	QSP for 50uL	2 min	56°C	2078	152 - 153	127.8
F1*	2ng	2uL	25uL	QSP for 50uL	1 min	58°C	1314	150 - 151	98
F2*	3ng	2uL	25uL	QSP for 50uL	1 min	58°C	1314	150 - 151	102
F3*	1ng	2uL	25uL	QSP for 50uL	1 min	58°C	1314	150 - 151	58