"

From 2010.igem.org

Revision as of 17:16, 14 October 2010 (view source) AndreasConstantinou (Talk | contribs) (→Andreas) ← Older edit		Latest revision as of 11:10, 26 October 2010 (view source) Hassanforoughiasl (Talk | contribs)
(2 intermediate revisions not shown)
Line 87:		Line 87:
	**(BL21, pEX.SOD)		**(BL21, pEX.SOD)
	**(BL21, pEX.yCCS)		**(BL21, pEX.yCCS)
		+
		+	==Nina==
		+
		+	===Protein purification===
		+
		+	====Lysis====
		+
		+	I have talked to people in the department that work with protein purifications and they recommended me to start working from a bigger culture such as 40 ml instead of 12 ml.
		+
		+	I used a 40 ml SOD.His (N terminal) culture for this purification.
		+
		+	I performed a batch purification of SOD.His tagg in E.coli BL21. For this I used a 50 ml falcon tube as the column.
		+
		+	*Ni-NTA tube was vortexed to have a homogenized solution of Ni.
		+	*10 ml Ni-NTA was added in the 50 ml falcon tube (column).
		+	*Centrifuged 30 sec at 4000 rpm to remove the supernatant the Ni was dissolved in.
		+	*Added 30 ml lysis buffer without imidazole, DNase and PMSF for equilibration of the Ni-resin. Left on shake in 4 °C for 1 h.
		+	*I prepared the lysis buffer:
		+
		+	For 5 ml pelleted culture use 630 ul lysis buffer
		+
		+	40 ml culture/5 = 8
		+
		+	8 * 630 ul lysis buffer = 5 ml
		+
		+	I double this 5 to 10 ml since I heard at the department that this would be good and there is no harm in using to much of lysis buffer.
		+
		+	Therefore I had 10 ml lysis buffer.
		+
		+	PMSF:
		+
		+	10000 ul/100 = 100 ul (1 mM PMSF) add in lysis buffer
		+
		+	Imidazole:
		+
		+	(10000 ul * 10*10^-3)/2 = 50 ul (10 mM Imidazole) add in lysis buffer
		+
		+	DNase:
		+
		+	10*10^3 ug/ml * Volume = 20 ug/ml * 10 ml
		+
		+	Volume = 20 ul (20 ug/ml) add in lysis buffer
		+
		+	*Mixed the lysis buffer with the pellet from 40 ml culture & transfered to a glas tube to sonicate for 5 min (75%)
		+	*Transfered the sonicated lysate back to a falcon tube and centrifuged for 20 min 8000 rpm in the cold room.
		+	*performed a ultracentrifugation on the supernatant 40 min 50000 rpm (1 ml of supernatant in each 10 centrifugation tubes with the size of 1 ml). I saved the pellet from both centrifugation steps. The first centrifugation removes the unopen cells and the second centrifugation pellets the membranes and debris from the first centrifugation.
		+	*Centrifuged the column and removed the supernatant that had equilibrated the resin.
		+	*Added the supernatant from the second centrifugation (50000 rpm) to the Ni-resin in the falcon tube. Incubated in 4 °C overnight on shake.
		+
		+	===PCR Colony===
		+
		+	I did a PCR colony screen on the transformations from yesterday.
		+
		+	36 tubes
		+
		+	Master Mix:
		+
		+	*MgCl2 18 ul
		+	*Phusion buffer 5X 180 ul
		+	*dNTP 18 ul
		+	*primerF 54 ul
		+	*primerR 54 ul
		+	*polymerase 18 ul
		+	*water 540 ul
		+
		+	Had 24 ul of the mix in each 36 tubes.
		+
		+	Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.
		+
		+
		+
		+
		+
		+	== Mimmi ==
		+
		+	=== clone non-CPP operon ===
		+
		+	==== Digestion ====
		+
		+	{|
		+	! mix
		+	| pC.S
		+	| pC.Sh
		+	| pC.hS
		+	| pA.RyC
		+	| pMA.Sh
		+	| pMA.hS
		+	|
		+	| pA.RyC
		+	|-
		+	| DNA
		+	| 5.2
		+	| 4.3
		+	| 6.6
		+	| 6.0
		+	| 4.78
		+	| X
		+	| 16.0
		+	|-
		+	| dH<sub>2</sub>O
		+	| 10.8
		+	| 11.7
		+	| 9.4
		+	| 10.0
		+	| 15.22
		+	| 11.85
		+	| X
		+	| 0
		+	|-
		+	| 10xbuffer
		+	| 2
		+	| 2
		+	| 2
		+	| 2
		+	| 2
		+	| 2
		+	| X
		+	| 2
		+	|-
		+	| XbaI
		+	| -
		+	| -
		+	| -
		+	| 1
		+	| -
		+	| -
		+	| X
		+	| 1
		+	|-
		+	| SpeI
		+	| 1
		+	| 1
		+	| 1
		+	| -
		+	| 1.5
		+	| 1.5
		+	| X
		+	| -
		+	|-
		+	| PstI
		+	| 1
		+	| 1
		+	| 1
		+	| 1
		+	| 1
		+	| 1
		+	| X
		+	| 1
		+	|-
		+	| tot
		+	| 20µl
		+	| 20µl
		+	| 20µl
		+	| 20µl
		+	| 20.5µl
		+	| 20.5µl
		+	| X
		+	| 20µl
		+	|}
		+	- c = 50ng/µl
		+
		+	*Incubate in 37&deg;C for 1.5h
		+
		+
		+	==== Ligation ====
		+
		+	{|
		+	! mix
		+	|
		+	|-
		+	| vector
		+	| 1
		+	|-
		+	| insert
		+	| 5
		+	|-
		+	| 10xbuffer
		+	| 2
		+	|-
		+	| H<sub>2</sub>O
		+	| 11
		+	|-
		+	| T4 ligase
		+	| 1
		+	|-
		+	| align="right" | tot
		+	| 20µl
		+	|}
		+
		+	*Incubate in 22&deg;C for 15min
		+
		+
		+
		+	==== Gel extraction ====
		+	(pA.RBS.yCCS    806bp)
		+
		+	*Tube 1.01g
		+
		+	*Tube + gelband 1.11g
		+
		+
		+
		+	==== Transformation ====
		+
		+	* 66µl thawed cells + 2µl ligated vector
		+
		+	*Incubate on ice 30min
		+	*Heat shock 55s in 42&deg;C, cool down on ice
		+	*Add 900µl LB, incubate in 37&deg;C for 45min
		+	*Spinn down cells and remove 900µl supernatant
		+	*Resuspend cells and plate
		+
		+	{{Stockholm/Footer}}

---

Latest revision as of 11:10, 26 October 2010

Contents 1 Andreas 1.1 Growth curve assay 1.1.1 Induction 1.1.2 OD590 measurements 1.1.3 ON cultures 2 Nina 2.1 Protein purification 2.1.1 Lysis 2.2 PCR Colony 3 Mimmi 3.1 clone non-CPP operon 3.1.1 Digestion 3.1.2 Ligation 3.1.3 Gel extraction 3.1.4 Transformation

Andreas

Growth curve assay

New attempt to measure growth curves for CPPs.

Induction

• 150 ml side-arm E-flasks
• 15 ml LB
• 150 μl ON culture (1:100)
  • BL21
  • ON cultures set by Mimmi 13/10
• 37 °C, 220 rpm

OD₅₉₀ measurements

	0 min	30 min	60 min	90 min	120 min	150 min	180 min	210 min	240 min	270 min
pEX.SOD⋅His	0.03	0.04	0.10	0.23	0.45	0.78	1.06	1.36	1.56	1.67
pEX.nTra10⋅SOD⋅His	0.03	0.04	0.09	0.19	0.36	0.61	0.94	1.18	1.42	1.56
pEX.nTAT⋅SOD⋅His	0.03	0.05	0.10	0.23	0.49	0.78	1.06	1.35	1.55	1.66
pEX.nLMWP⋅SOD⋅His	0.04	0.05	0.11	0.25	0.52	0.88	1.14	1.40	1.60	1.69

Experiment was canceled after 270 min as I was made aware that the spectrophotometer only makes accurate measurements up to ≈OD₅₉₀ = 0.5.

ON cultures

New ON cultures set (including two for Mimmi)

• 5 ml LB + Amp 100; 37 °C, 225 rpm
  • BL21, pEX.SOD⋅His
  • BL21, pEX.nTra10⋅SOD⋅His
  • BL21, pEX.nTAT⋅SOD⋅His
  • BL21, pEX.nLMWP⋅SOD⋅His
  • (BL21, pEX.SOD)
  • (BL21, pEX.yCCS)

Nina

Protein purification

Lysis

I have talked to people in the department that work with protein purifications and they recommended me to start working from a bigger culture such as 40 ml instead of 12 ml.

I used a 40 ml SOD.His (N terminal) culture for this purification.

I performed a batch purification of SOD.His tagg in E.coli BL21. For this I used a 50 ml falcon tube as the column.

• Ni-NTA tube was vortexed to have a homogenized solution of Ni.
• 10 ml Ni-NTA was added in the 50 ml falcon tube (column).
• Centrifuged 30 sec at 4000 rpm to remove the supernatant the Ni was dissolved in.
• Added 30 ml lysis buffer without imidazole, DNase and PMSF for equilibration of the Ni-resin. Left on shake in 4 °C for 1 h.
• I prepared the lysis buffer:

For 5 ml pelleted culture use 630 ul lysis buffer

40 ml culture/5 = 8

8 * 630 ul lysis buffer = 5 ml

I double this 5 to 10 ml since I heard at the department that this would be good and there is no harm in using to much of lysis buffer.

Therefore I had 10 ml lysis buffer.

PMSF:

10000 ul/100 = 100 ul (1 mM PMSF) add in lysis buffer

Imidazole:

(10000 ul * 10*10^-3)/2 = 50 ul (10 mM Imidazole) add in lysis buffer

DNase:

10*10^3 ug/ml * Volume = 20 ug/ml * 10 ml

Volume = 20 ul (20 ug/ml) add in lysis buffer

• Mixed the lysis buffer with the pellet from 40 ml culture & transfered to a glas tube to sonicate for 5 min (75%)
• Transfered the sonicated lysate back to a falcon tube and centrifuged for 20 min 8000 rpm in the cold room.
• performed a ultracentrifugation on the supernatant 40 min 50000 rpm (1 ml of supernatant in each 10 centrifugation tubes with the size of 1 ml). I saved the pellet from both centrifugation steps. The first centrifugation removes the unopen cells and the second centrifugation pellets the membranes and debris from the first centrifugation.
• Centrifuged the column and removed the supernatant that had equilibrated the resin.
• Added the supernatant from the second centrifugation (50000 rpm) to the Ni-resin in the falcon tube. Incubated in 4 °C overnight on shake.

PCR Colony

I did a PCR colony screen on the transformations from yesterday.

36 tubes

Master Mix:

• MgCl2 18 ul
• Phusion buffer 5X 180 ul
• dNTP 18 ul
• primerF 54 ul
• primerR 54 ul
• polymerase 18 ul
• water 540 ul

Had 24 ul of the mix in each 36 tubes.

Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.

Mimmi

clone non-CPP operon

Digestion

mix	pC.S	pC.Sh	pC.hS	pA.RyC	pMA.Sh	pMA.hS		pA.RyC
DNA	5.2	4.3	6.6	6.0	4.78	X	16.0
dH2O	10.8	11.7	9.4	10.0	15.22	11.85	X	0
10xbuffer	2	2	2	2	2	2	X	2
XbaI	-	-	-	1	-	-	X	1
SpeI	1	1	1	-	1.5	1.5	X	-
PstI	1	1	1	1	1	1	X	1
tot	20µl	20µl	20µl	20µl	20.5µl	20.5µl	X	20µl

- c = 50ng/µl

• Incubate in 37°C for 1.5h

Ligation

mix
vector	1
insert	5
10xbuffer	2
H2O	11
T4 ligase	1
tot	20µl

• Incubate in 22°C for 15min

Gel extraction

(pA.RBS.yCCS 806bp)

• Tube 1.01g

• Tube + gelband 1.11g

Transformation

• 66µl thawed cells + 2µl ligated vector

• Incubate on ice 30min
• Heat shock 55s in 42°C, cool down on ice
• Add 900µl LB, incubate in 37°C for 45min
• Spinn down cells and remove 900µl supernatant
• Resuspend cells and plate