Team:Kyoto/Notebook/Construction

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LastModified: 2010-10-17

Notebook: Construction for Lysisbox

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Incubation	Result
<partinfo>J23100</partinfo>	1-18-C	1 µl	20	21	LB (Amp+)	At 37℃, 7/20 20:50 - 7/21 17:00	○
<partinfo>J23105</partinfo>	1-18-M	1	20	21			○
<partinfo>J23116</partinfo>	1-20-M	1	20	21			○
<partinfo>R0011</partinfo>	1-6-G	1	20	21			○
<partinfo>E0840</partinfo>	1-12-O	1	20	21			○
<partinfo>J06702</partinfo>	2-8-E	1	20	21			○
<partinfo>pSB4K5</partinfo>	1-5-G	1	20	21			×
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kan+)		×

A vector of <partinfo>pSB4K5</partinfo> is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture <partinfo>B0015</partinfo> despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>.

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Incubation	Result
<partinfo>pSB4K5</partinfo>	1-5-G	1 µl	20	21	LB (Kan+)	At 37℃, 7/21 20:50 - 7/22 16:30	○
<partinfo>B0015</partinfo>	1-23-L	1	20	21	LB (Kan+)	At 37℃, 7/21 20:50 - 7/22 16:30	○

PCR for SRRz and S

No.	Water	MgSO4	dNTPs	10xBuffer	Template DNA	Primer Fwd.	Primer Rev. (SRRz)	Primer Rev. (S)	KOD Plus ver.2	Total
1	28 µl	3	5	5	5	1.5	1.5	-	1	50
2	28	3	5	5	5	1.5	1.5	-	1	50
3	28	3	5	5	5	1.5	-	1.5	1	50
4	28	3	5	5	5	1.5	-	1.5	1	50
5	28	3	5	5	5	1.5	1.5	-	1	50
6	28	3	5	5	5	1.5	1.5	-	1	50
7	28	3	5	5	5	1.5	-	1.5	1	50
8	28	3	5	5	5	1.5	-	1.5	1	50

94℃	2min
98℃	10sec	30 cycles
55℃	30sec
68℃	4min
4℃	forever

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

No.	Name	Length(bp)	Result
1	SRRz	1386	○
2	SRRz	1386	○
3	S	442	○
4	S	442	○
5	SRRz	1386	○
6	SRRz	1386	○
7	S	442	○
8	S	442	○

Marker: 100bp, 1kb, 1kb, 100bp.

Miniprep

Name	Concentration
<partinfo>J23100</partinfo>	18.5 (ng/µl)
<partinfo>J23105</partinfo>	12.5
<partinfo>J23116</partinfo>	14.6
<partinfo>R0011</partinfo>	8.6
<partinfo>E0840</partinfo>	12.1
<partinfo>J06702</partinfo>	14.7

The concentration of all samples was very week. Probably our shaking incubation was week.

Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>

Friday, July 23 By: Wataru, Tomo, Makoto

Miniprep

Name	Concentration
<partinfo>pSB4K5</partinfo>	79.2 (ng/µl)
<partinfo>B0015</partinfo>	-

We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.

PCR Purification

No.	Name	Concentration	New Name
1	SRRz	18.6 ng/µl	-
3	S	77.6	S_Sam7(1)
5	SRRz	33.6	-
7	S	65.4	S_Sam7(2)

The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.

Standard PCR for SRRz

No.	Water	MgSO4	dNTPs	10xBuffer	Template DNA	Primer Fwd. (SRRz)	Primer Rev. (SRRz)	KOD plus ver.2	Total
1	28 µl	3	5	5	5	1.5	1.5	1	50
2	28	3	5	5	5	1.5	1.5	1	50
3	26.5	4.5	5	5	5	1.5	1.5	1	50
4	26.5	4.5	5	5	5	1.5	1.5	1	50
5	25	6	5	5	5	1.5	1.5	1	50
6	25	6	5	5	5	1.5	1.5	1	50

94℃	2min
98℃	10sec	30 cycles
55℃	30sec
68℃	4min
4℃	forever

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme

No.	Name	Sample	10xBuffer	BSA	Enzyme		MilliQ	Total	Incubation
1	<partinfo>J06702</partinfo>	5 µl	1	0.1	EcoRI	0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	<partinfo>J06702</partinfo>	5	1	0.1	XbaI	0.1	3.6	10
3	<partinfo>J06702</partinfo>	5	1	0.1	SpeI	0.1	3.6	10
4	<partinfo>J06702</partinfo>	5	1	0.1	PstI	0.1	3.6	10
5	<partinfo>J06702</partinfo>	5	1	0.1	-		3.7	10

Marker: 1kb. Comparison to No. 5 (control, circular DNA), the bands of No. 1, 2, 3, and 4 was shifted. The DNA of them was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

Restriction Digestion and Ligation to insert S gene to <partinfo>E0840</partinfo>

Name	Sample	10xBuffer	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
S_Sam7(1)	11 µl	5	EcoRI	0.2	SpeI	0.2	33.6	50	At 37℃ for 2h
S_Sam7(2)	11	5	EcoRI	0.2	SpeI	0.2	33.6	50
<partinfo>E0840</partinfo>	45	5	EcoRI	0.2	XbaI	0.2	0	50

After PCR Purification, evaporated them and diluted 3ul.

Name	Vector		Insert		Ligation High	Total
S_Sam7(1)-E0840	<partinfo>E0840</partinfo>	0.5µl	S_Sam7(1)	0.5	1	2
S_Sam7(2)-E0840	<partinfo>E0840</partinfo>	0.5	S_Sam7(2)	0.5	1	2

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

No.	Name	Length(bp)
1	SRRz	1386
2	SRRz	1386
3	SRRz	1386
4	SRRz	1386
5	SRRz	1386
6	SRRz	1386

Marker: 1kb. At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), SRRz is amplified very much. So we decided to use them.

PCR Purification

No.	Name	Concentration	New Name
4	SRRZ	51.6 ng/µl	SRRz_Sam7(1)
5	SRRZ	59.3
6	SRRZ	59.6	SRRz_Sam7(2)

Transformation

Name	Well	Sample	Competent Cell	Total	Plate	Incubation	Result
<partinfo>E0240</partinfo>	1-12-M	1 µl	20	21	LB (Amp+)	At 37℃ 7/26 - 7/27	×
<partinfo>I20260</partinfo>	2-17-F	1	20	21	LB (Kan+)		×
<partinfo>J04450</partinfo>	1-5-E	1	20	21	LB (Kan+)		×

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Colony PCR of S_Sam7-E0840 (Electrophoresis for 35min)

No.	Name	Length	Result
1	S_Sam7(1)-E0840	1522	○
2	S_Sam7(1)-E0840	1522	×
3	S_Sam7(1)-E0840	1522	○
4	S_Sam7(1)-E0840	1522	×
5	S_Sam7(1)-E0840	1522	○
6	S_Sam7(1)-E0840	1522	◎ (Use as S_Sam7(1)-E0840)
7	S_Sam7(2)-E0840	1522	×
8	S_Sam7(2)-E0840	1522	×
9	S_Sam7(2)-E0840	1522	×
10	S_Sam7(2)-E0840	1522	×
11	S_Sam7(2)-E0840	1522	◎ (Use as S_Sam7(2)-E0840)
12	S_Sam7(2)-E0840	1522	○
13	S_Sam7(2)-E0840	1522	○
+	<partinfo>E0840</partinfo>	1116
-	None

Marker: 1kb, 100bp

Miniprep

Name	Concentration
<partinfo>R0011</partinfo>	26.9 ng/µl
<partinfo>B0015</partinfo>	120.0
<partinfo>E0840</partinfo>	120.1

Restriction Digestion

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
<partinfo>B0015</partinfo>	30 µl	5	0.5	EcoRI	0.4	XbaI	0.3	13.7	50	At 37℃ 16:45 - 18:00
SRRz_Sam7(1)	40	5	0.5	EcoRI	0.4	SpeI	0.4	3.8	50
SRRz_Sam7(2)	40	5	0.5	EcoRI	0.4	SpeI	0.4	3.8	50

Ligation

Transformation

Name	Sample	Competent Cells	Total	Plate	Incubation	Result
SRRz_Sam7(1)-B0015						○
SRRz_Sam7(2)-B0015						○

Wednesday, July 28 By:

Miniprep

Name	Concentration
S_Sam7(1)-E0840	95.5 ng/µl
S_Sam7(2)-E0840	98.6

Diluted S_Sam7(1)-E0840 and S_Sam7(2)-E0840 20 times with water, and used as template DNA.

Deletion PCR to delete a functional domain of S gene

	Water	MgSO4	dNTPs	10xBuffer	Primer Fwd.	Primer Rev.	S_Sam7(1)-E0840	S_Sam7(2)-E0840	KOD Plus ver.2	Total
S_Sam7,ΔTMD1(1)-E0840 (1)	28	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(1)-E0840 (2)	28	3	5	5	1.5	1.5	5	-	1	50
S_Sam7,ΔTMD1(2)-E0840 (1)	28	3	5	5	1.5	1.5	-	5	1	50
S_Sam7,ΔTMD1(2)-E0840 (2)	28	3	5	5	1.5	1.5	-	5	1	50

94℃	2min
98℃	10sec	35 cycles
55℃	30sec
68℃	4min
4℃	forever

Restriction Digestion to check the function of DpnI

Name	Sample	fast digestion buffer	DpnI	MilliQ	Total
S_Sam7,ΔTMD1(1)-E0840 (1)	3	1	0.1	5.8	10
S_Sam7,ΔTMD1(2)-E0840 (2)	3	1	0.1	5.8	10

Electrophoresis for 35min

No.	Name	Length
1	Not digested S_Sam7(1)-E0840	3363
2	Not digested S_Sam7(2)-E0840	3363
3	Digested S_Sam7(1)-E0840	1021, 933, 402, 341, 258, 105, ...
4	Digested S_Sam7(2)-E0840	1021, 933, 402, 341, 258, 105, ...

Marker: 1kb, 100bp DpnI works correctly.

Thursday, July 29 By:

Restriction Digestion

Name	Sample volume	Fastdigestion Buffer	Enzyme 1		MilliQ	Total	Incubation
S_Sam7,ΔTMD1(1)-E0840 (1)	50 µl	6	DpnI	0.2	3.8	60	07/29 09:40 - 07/29 11:00
S_Sam7,ΔTMD1(2)-E0840 (1)	50	6	DpnI	0.2	3.8	60	07/29 09:40 - 07/29 11:00

Ligation and Phosphorylation

Name	Sample	MilliQ	Ligation High	T4 Kinase	Total	Incubation
S_Sam7,ΔTMD1(1)-E0840 (1)	2 µl	7	5	1	15	07/29 11:30 ~ 07/29 13:00
S_Sam7,ΔTMD1(1)-E0840 (2)	2	7	5	1	15	07/29 11:30 ~ 07/29 13:00

Transformation

Name	Sample Volume	Competent Cell	Total	Plate	Incubation	Result
S_Sam7,ΔTMD1(1)-E0840 (1)	3 µl	30	33	LB Amp+	07/29 ~ 07/30	○
S_Sam7,ΔTMD1(1)-E0840 (2)	3	30	33	LB Amp+	07/29 ~ 07/30	○

Monday, August 2 By: Wataru, Ken

Miniprep

Name	Concentration(ng/µL)
S_ΔTMD1-E0840-1	52.7
S_ΔTMD1-E0840-2	54.4
S_ΔTMD1-E0840-3	89.5
<partinfo>pSB4K5</partinfo>	50.7
<partinfo>R0011</partinfo>	18.6

Standard PCR of <partinfo>E0240</partinfo>

E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.

Name	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template E240	KOD Pllus ver.2	Total
E0240₁	28	3	5	5	1.5	1.5	5	1	50
E0240₂	28	3	5	5	1.5	1.5	5	1	50

94℃	2min
98℃	10sec	35 cycles
55℃	30sec
68℃	4min
4℃	forever

Electrophoresis

PCR Purification

Sample number	Concentration(ng/µL)
E0240₁	42.6
E0240₂	55.3

Restriction Digestion for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
E0240₁(X-P)	30	5	0.5	XbaI	0.2	PstI	0.2	14.1	50
E0240₂(X-P)	30	5	0.5	XbaI	0.2	PstI	0.2	14.1	50

PCR Purification

Name	Concentration(ng/µL)	Volume(µL)
E0240₁(X-P)	21.8	40
E0240₂(X-P)	32.4	45

Stored at -20℃.

Error PCR

Name	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template Δ1	Template	Template	KOD Pllus ver.2	Total
S_ΔTMD1-E0840₁-1	32	3	5	5	1.5	1.5	1	-	-	1	50
S_ΔTMD1-E0840₁-2	32	3	5	5	1.5	1.5	-	1	-	1	50
S_ΔTMD1-E0840₂	32	3	5	5	1.5	1.5	-	-	1	1	50

94℃	2min
98℃	10sec	20 cycles
68℃	4min	20 cycles
4℃	forever

Transformation

Name	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
S_ΔTMD1-E0840₁-1	2	20	22	rowspan="3"	rowspan="3"	○
S_ΔTMD1-E0840₁-2	2	20	22	}
S_ΔTMD1-E0840₂	2	20	22	○

Tuesday, August 3 By:

Culture of each two colonies of S_ΔTMD1-E0840₁-1 and S_ΔTMD1-E0840₂ for 37℃ 08/03-08/04

Miniprep for Construction of Measure(lacP) and Measure(Standard)

Sample number	Concentration(ng/µL)
<partinfo>pSB4K5</partinfo>	60.7
<partinfo>R0011</partinfo>	26.8

Restriction Digestion

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
R0011	50	6	0.6	EcoRI	0.2	SpeI	0.2	3	60
pSB4K5(E-P)	50	6	0.6	EcoRI	0.2	PstI	0.2	3	60
E0240₁(X-P)	50	6	0.6	XbaI	0.2	PstI	0.2	3	60
E0240₂(X-P)	50	6	0.6	XbaI	0.2	PstI	0.2	3	60

PCR Purification

Sample number	Concentration(ng/µL)
pSB4K5(E-P)	39.5
E0240₁(X-P)	21.8
E0240₂(X-P)	32.4

pSB4K5(E-P) is concentrated 10µL and E0240₁(X-P), E0240₂(X-P) are concentrated 1µL.

Ethanol Precipitation

Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ

Ligation

	Vector	Insert 1		Insert 2		Ligation High	Total	Incubation
R0011-E0240₁[Low]	pSB4K5(E-P)	1	R0011(E-S)	1	E0240₁(X-P)	1	3	15	17:30 - 20:20
R0011-E0240₂[Low]	pSB4K5(E-P)	1	R0011(E-S)	1	E0240₂(X-P)	1	3	15	17:30 - 20:20

Standard PCR of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU

Name	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template J23101-E0240	KOD plus ver.2	Total
J23101-E0240₁	32	3	5	5	1.5	1.5	1	1	50
J23101-E0240₂	32	3	5	5	1.5	1.5	-	1	50

94℃	2min
98℃	10sec	30 cycles
55℃	30sec
68℃	4min
4℃	forever

PCR Purification

Name	Concentration(ng/µL)
J23101-E0240	40.6

Restriction Digestion

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
J23101-E0240(E-P)	45	6	0.6	EcoRI	0.2	PstI	0.2	8	60

PCR Purification

Name	Concentration(ng/µL)	Volume(µL)
J23101-E0240(E-P)	74.1	30

J23101-E0240(E-P) is concentrated 7µL

Ligation

	Vector	Insert		Ligation High		Total	Incubation
J23101-E0240[Low]	pSB4K5(E-P)	1	J23101-E0240(E-P)	1	2	4	20:00-20:30

Transformation

Name	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
R0011-E0240₁[Low]	-	1	20	21	LB kan	8/3~8/4
R0011-E0240₂[Low]	-	1	20	21
J23101-E0240[Low]	-	1	20	21

Thursday, August 5 By:

Result of Transformation

R0011-E0240₁[Low]	Many colonies
R0011-E0240₂[Low]
J23101-E0240[Low]

pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts. However, white colonies and green colonies are observed in R0011-E0240₁[Low] and R0011-E0240₂[Low] plate. We cultured both white and green colonies. In J23101-E0240[Low], Many of colonies are red, but green colonies are observed. We cultured green colonies.

Name	Color	Incubation
R0011-E0240₁[Low]-1	Green Colony	8/5-8/6
R0011-E0240₁[Low]-2	Green Colony
R0011-E0240₁[Low]-3	White Colony
R0011-E0240₁[Low]-4	White Colony
R0011-E0240₂[Low]-1	Green Colony
R0011-E0240₂[Low]-2	White Colony
R0011-E0240₂[Low]-3	White Colony
R0011-E0240₂[Low]-4	White Colony
J23101-E0240[Low]-1	Green Colony
J23101-E0240[Low]-2	Green Colony
J23101-E0240[Low]-3	Green Colony

Name	Concentration(ng/µL)
S_ΔTMD1-E0840₁-1-A	28.9
S_ΔTMD1-E0840₁-1-B	25.3
S_ΔTMD1-E0840₂-A	26.6
S_ΔTMD1-E0840₂-B	24.0

As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.

Friday, August 6

Miniprep

Name
R0011-E0240₁[Low]-1
R0011-E0240₁[Low]-2
R0011-E0240₁[Low]-3
R0011-E0240₁[Low]-4
R0011-E0240₂[Low]-1
R0011-E0240₂[Low]-2
R0011-E0240₂[Low]-3
R0011-E0240₂[Low]-4
J23101-E0240[Low]-1
J23101-E0240[Low]-2
J23101-E0240[Low]-3

Restriction Digestion

Name	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
R0011-E0240₁[Low]-1	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₁[Low]-2	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₁[Low]-3	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₁[Low]-4	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₂[Low]-1	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₂[Low]-2	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₂[Low]-3	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
R0011-E0240₂[Low]-4	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
J23101-E0240[Low]-1	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
J23101-E0240[Low]-2	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60
J23101-E0240[Low]-3	50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60

Electrophoresis

M	100bp	colspan="4"	colspan="4"\|
M	λ
M	λ
M	100bp
1	J23101-E0240[Low]-1
2	J23101-E0240[Low]-2
3	J23101-E0240[Low]-1
4	R0011-E0240₁[Low]-1		○
5	R0011-E0240₁[Low]-2		○
6	R0011-E0240₁[Low]-3		}
7	R0011-E0240₁[Low]-4		}
8	R0011-E0240₂[Low]-1		○
9	R0011-E0240₂[Low]-2		}
10	R0011-E0240₂[Low]-3		}
11	R0011-E0240₂[Low]-4		}
12	J23101-E0240[Low]-1		○
13	J23101-E0240[Low]-2		○

White colonies are not inserted <partinfo>R0011</partinfo> but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of lacI gene.

Error PCR (Retry)

Name	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template ΔTMD failed(50ng/µL)	KOD plus ver.2	Total
ΔTMD①	32	3	5	5	1.5	1.5	1	1	50
ΔTMD②	32	3	5	5	1.5	1.5	1	1	50

94℃	2min
98℃	10sec	25 cycles
68℃	4min	25 cycles
Add DpnI 2µl
Incubate	1h
4℃	forever

Transformation

Name	Conc(/µL)	Sample Volum(µL)	Competent Cell(µL)	Total	Plate	Incubation
ΔTMD①	-	4	50	54	LB kan	8/6~8/9
ΔTMD②	-	4	50	54
2-17-F	-	2	50	52
2-I-5		2	50	52	LB amp

Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep of MS and ML

Sample number	concentration(ng/µL)
MS	116.2
ML	146.6

Transfotrmation of MS and ML

Sample	conc(ng/µL)	Sample vol(µL)	Competent Cell	Competent cell vol(µL)	Total vol(µL)	Plate	Incuvation
MS	116.2	2	KRX	50	52	LB kanamycin	8/9 18:00‾8/10 12:00
ML	146.6	2	KRX	50	52	LB kanamycin	8/9 18:00‾8/10 12:00

Restriction enzyme digestion and ethanol precipitation

To use lac p for next ligation, we digested 1-6-G by EroRI and PstI

Sample	10x Buffer	BSA	Enzyme (EcoRI)	Enzyme (PstI)	MilliQ	Total
50	6	0.6	0.5	0.5	2.4	60

Incubate 37℃ 8/9 16:20‾18:20 After restriction enzyme digestion, we did ethanol precipitation.

Ligation and Transformation

Sample	Conc (nu/µL)	Sample vol (µL)	Competent cell	Competent cell vol (µL)	Total vol (µL)	Plate	Incuvation
Lac p (low)	-	2	KRX	50	52	LB kanamycin	8/9 20:00‾8/10 9:00
Lac p (low)	-	2	C2	50	52	LB kanamycin	8/9 20:00‾8/10 9:00

===Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka

Making culture plate on lac p (low), MS and ML

Lac p (low)	KRX	Many colonies
Lac p (low)	C2
MS	KRX
ML	KRX

Minprep of ΔTMD1+GFP

Sample number	Concentration (ng/µL)
1-1	9.9
1-2	27.3
2-1	43.2
2-2	34.7

37℃ 8/9 18:00‾8/10 9:00

Culture and Master Plate

===Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya

Sample	Medium	Cloud	Incubation
1	Kanamycin	o	37℃8/10 20:00‾8/11 9:00
1	Ampicillin	x
2	Kanamycin	o
2	Ampicillin	o
3	Kanamycin	o
3	Ampicillin	x
4	Kanamycin	o
4	Ampicillin	x
5	Kanamycin	o
5	Ampicillin	x
6	Kanamycin	o
6	Ampicillin	o
7	Kanamycin	o
7	Ampicillin	x

Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.

Miniprep of C2+lac(low), S-R-Rz 1', 3'

lac(low)1 : 31.2 (ng/µL) lac(low)2 : 29.9 (ng/µL)

Restriction Digestion and electrophoresis of lac (low) 1 and 3

Name	EcoRI	PstI
1	0.2	-
2	-	0.2
3	0.2	0.2
N	-	-

Sample: 1-1, 1-2, 1-3, 1-N, 3-1, 3-2, 3-3, 3-N M 1-1 1-2 1-3 1-N M M 3-1 3-2 3-3 3-N M Discussion: Each enzyme correctly cut samples.

Screening PCR of SRRz

Sample: 1-20 Control: P(1-23L) P'(2-8E) N Maker: lambda M N P P' P 1 2 3 4 5 6 M 7 8 9 10 11 12 13 M 14 15 16 18 19 20 M Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>

Sample name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Sample: 1-6, N Maker: lambda, 100 M 1 2 3 4 5 6 N M M M Discussion: Each enzyme correctly cut each sample and was active.

===Thursday, August 19 By: Wataru, Tomo, Ken

Miniprep of SΔTMD1GFP

29.6(ng/µg)

Point mutation PCR of ΔTMD1GFP

Sample number	Template	10xbuffer	dNTPs	MgSO4	Primer 1	Primer 2	Water	KOD-plus-	Total
1	1.5	5	5	3	1.5	1.5	31.5	1	50
2	1.5	5	5	3	1.5	1.5	31.5	1	50
control	1.5	5	5	3	1.5	1.5	32.5	-	50

94(℃)	2min
98	10sec	30cycles
55	30sec
68	3.5min
4.0	forever

Restriction Digestion(DpnI): 17:50-18:50

Electrophoresis

Sample: 1, 2, Control Marker: lambda, 100

Ligation and Transformation

We named point mutation PCR products rΔTMD1GFP.

Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku

Miniprep of ΔTMD1

Sample number	Concentration(ng/µg)
1-1	58.9
2-2	49.9

Sequencing of ΔTMD1 and MS

Sample: rδTMD1GFP1-1, 2-2, and MS Discussion: The sequencing was in success and the results were desirable. It meant point mutation of δTMD1GFP was succeeded and sequence of MS was confirmed. We decided to use rδTMD1GFP.

Screening PCR of SRRz-DT

Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N

90℃	10min
94℃	30sec	35cycles
50℃	30sec
72℃	1.5min
72℃	4min
4℃	hold

M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.

Deletion PCR of rΔTMD1GFP 2-2

Sample	10x	dNTPs	Primer1	Primer2	Template	Water	KOD-plus-	Total
1	5	5	1.5	1.5	1	35	1	50
2	5	5	1.5	1.5	1	35	1	50
Control	5	5	1.5	1.5	1	35	-	50

94℃	2min
94℃	10sec	35cycles
56℃	30sec
68℃	3.5min
4℃	hold

Restriction Digestion(DpnI)

Template	25(µL)
DpnI	1
Total	26

19:10-20:10

Ligation

Sample	Template	Water	Ligation high	T4 Kinase	total
1	3	6	5	1	15
2	3	6	5	1	15
Control	3	6	5	1	15

20:15-21:15

Transformation

We named sample 1, 2 and control rrδTMD1GFP1, 2 and control.

Tuesday, August 24 By:Ken, Tomo, Tasuku, Takuya

Retry of deletion PCR of rδTMD1 GFP

Sample	10x	dNTPs	MgSO4	Primer1	Primer2	Template	Water	KOD-plus-	Total
1	5	5	3	1.5	1.5	1	32	1	50
2	5	5	3	1.5	1.5	1	32	1	50
Control	5	5	3	1.5	1.5	1	32	1	50

94℃	2min
94℃	10sec	35cycles
58℃	30sec
68℃	3.5min
4℃	hold

Restriction Digestion (DpnI)

14:15-15:15

Electrophoreis

Sample: 1, 2, and control, Maker: 100 and lambda M 1 2 C M We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and RE were completed successfully.

Ligation

Point mutation of SRRz

Sample	10x	dNTPs	MgSO4	Primer1	Primer2	Template	Water	KOD-plus-	total
1	5	5	3	1.5	1.5	1	32	1	50
2	5	5	3	1.5	1.5	1	32	1	50
control	5	5	3	1.5	1.5	1	32	1	50

94℃	2min
98℃	10sec	30cycles
55℃	30sec
68℃	4min
4℃	hold

Restriction Digestion(DpnI), electrophoresis and ligation

We could find point mutation PCR and restriction enzyme of DpnI was done.

PCR of E0240

Sample	10}	dNTPs	MgSO4	VF2	VR	Template	Water	KOD-plus-	Total
1	5	5	3	1.5	1.5	1	31.5	1	50
2	5	5	3	1.5	1.5	1	31.5	1	50

PCR Purification

Sample1: 5.5*50(ng/µL) Sample2: 5.2*50(ng/µL)

Restriction Digestion(EcoRI, PstI) and Gel extraction

Sample1: 28.8 (ng/µL) Sample2: 26.4 (ng/µL)

Transformation

Sample: rrΔTMD1GFP1. 2. control, and rSRRz1. 2. control

Wednesday, August 25 By:Ken, Tomo, Kazuya, Tasuku, Takuya

Making culture and Master plate

rrΔTMD1-1	Many Colonies
rrΔTMD1-2	Many Colonies
rrΔTMD1-C-	zero
rSRRz-1	Many Colonies
rSRRz-2	Many Colonies
rSRRz-C-	zero

Miniprep of 1-5G

29.0 (ng/µL)

Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low

Sample name	Template	10xbuffer	100xbuffer	EcoRI	SpeI	PstI	Water	Total
1-5G	50	6	0.6	0.4	0.4	-	2.6	60
Lac low	10	4	0.4	-	0.3	0.3	25	40

Sample Name	Concentration(ng/µL)
1-5G	18.4
Lac low	8.6

Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation

===Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka

Miniprep

Sample name	Concentration(ng/µL)
constP(0.7)	44.5

Restriction Digestion of constP(0.7)

Template	10xbuffer	100xbuffer	SpeI	PstI	Water	Total
25	4	0.4	0.3	0.3	10	40

Purification of constP (0.7)

49.8 ng/µL

Friday, August 27 By:Ken, Tomo, Kazuya, Fumitaka

Making master plate of E0240 low

Sample Name	Concentration(ng/µL)
rrΔTMD1 1-2	20.9
rSRRz 1-1	16.4

Restriction Digestion of rrΔTMD1 and rSRRz

Sample name	Template	10xbuffer	100xbuffer	XbaI	PstI	Water	Total
rrΔTMD1 1-2	45	6	0.6	0.3	0.3	7.8	60
rSRRz 1-1	45	6	0.6	0.3	0.3	7.8	60

(13:20-14:20)

Purification

rrΔTMD1 1-2	44.7
rSRRz 1-1	56.1

Lagation and transformation

lacP + rrΔTMD1 1-2 constP (0.7) + rrΔTMD1 1-2 lac low + rSRRz 1-1

Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken

Making culture and Master plate

lacP rrΔTMD1GFP	Many colonies
lacP rrΔTMD1GFP(control)	Some colonies
constP rrΔTMD1GFP	Many colonies
constP rrΔTMD1GFP(control)	Many colonies
lacP rSRRz low	No colony
lacP rSRRz low(control)	No colony

Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate. On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.

===Tuesday, August 31 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken

Miniprep

constP (0.3)	48.5 (ng/µL)
lac rrΔTMD1	107.3

RE of constP (0.3) and lac rrΔTMD1

Gel Extraction of lac rrΔTMD1

File:KyotoExp100831-1.png

45min Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.

Purification of constP (0.3) and lac rrΔTMD1

constP (0.3)	5.8 (ng/µL)
lac rrΔTMD1	7.8 (ng/µL)

Ligation and transformation

Insert	Vector
lac rrΔTMD1	constP (0.3)

===Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken

Making culture and Master plate

lac rrΔTMD1 constP	many colonies
lac rrΔTMD1 const (control)	many colonies

Screenig PCR of lacP-rrΔTMD1GFP-constP Sample: 1-13 Control: Positive (1-23L) Maker: lambda, 100

   M  1  2  3  4   5  6  7     8  9  10 11 12  13  P M

File:KyotoExp100901.png

Discussion: All of the sample except sample 10 might be self-ligation products of constP.

Miniprep

rSRRz 1-1	33.8 (ng/µL)
low	56.0 (ng/µL)

Restriction Digestion of rSRRz and low

Sample name	Template	10xbuffer	100xbuffer	EcoRI	PstI	Water	Total
rSRRz	20	4	0.4	0.3	0.3	15	40
low	20	4	0.4	0.3	0.3	15	40

(13:25-14:30)

Purification

rSRRz	6.5 (ng/µL)
low	16.8

Ligation and transformation

Insert: rSRRz 1-1 Vector: low copy plasmid

Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken

Making culture and Master plate

rSRRz low	13 colonies
rSRRz low (Control)	13colonies

Screening PCR of rSRRz low

Sample: rSRRz (1-13) Maker: lambda, 100 Control: Positive (1-23L), Neganive M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M File:KyotoExp100902.png

Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, rSRRz might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.

Friday, September 3 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken

Making culture

lac rrΔTMD1 1, 3 rrΔTMD1 1-1, 1-2 rSRRz 1-1, 1-2 ML

Sample name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Sample name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10

Team:Kyoto/Notebook/Construction

From 2010.igem.org

Contents

Notebook: Construction for Lysisbox

Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.

Culture at 37℃ from 07/21 20:50 to 07/22 17:00 and Making Master Plate

PCR for SRRz and S

Thursday, July 22 By: Wataru

Electrophoresis (40min) of the PCR Products

Culture from 07/22 17:00 to 07/23 10:00 and Making Master Plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo>

Friday, July 23 By: Wataru, Tomo, Makoto

Standard PCR for SRRz

Restriction Digestion and Electrophoresis (35min) to check function of our Restriction Enzyme

Restriction Digestion and Ligation to insert S gene to <partinfo>E0840</partinfo>

Monday, July 26 By: Wataru, Tomonori, Makoto

Electrophoresis of PCR Products

PCR Purification

Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>

Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi

Colony PCR of SSam7-E0840 (Electrophoresis for 35min)

Wednesday, July 28 By:

Deletion PCR to delete a functional domain of S gene

Restriction Digestion to check the function of DpnI

Electrophoresis for 35min

Thursday, July 29 By:

Ligation and Phosphorylation

Monday, August 2 By: Wataru, Ken

Standard PCR of <partinfo>E0240</partinfo>

PCR Purification

Restriction Digestion for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly

PCR Purification

Error PCR

Tuesday, August 3 By:

Culture of each two colonies of SΔTMD1-E08401-1 and SΔTMD1-E08402 for 37℃ 08/03-08/04

Miniprep for Construction of Measure(lacP) and Measure(Standard)

PCR Purification

Ethanol Precipitation

Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ

Standard PCR of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU

Transformation

Thursday, August 5 By:

Result of Transformation

Friday, August 6

Miniprep

Restriction Digestion

Electrophoresis

Error PCR (Retry)

Transformation

Monday, August 9 By: Wataru, Tomonori, Ken, Takuya

Miniprep of MS and ML

Transfotrmation of MS and ML

Restriction enzyme digestion and ethanol precipitation

Ligation and Transformation

Making culture plate on lac p (low), MS and ML

Minprep of ΔTMD1+GFP

Culture and Master Plate

Miniprep of C2+lac(low), S-R-Rz 1', 3'

Restriction Digestion and electrophoresis of lac (low) 1 and 3

Screening PCR of SRRz

Thursday, August 12 By: Wataru, Ken

Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>

Miniprep of SΔTMD1GFP

Point mutation PCR of ΔTMD1GFP

Restriction Digestion(DpnI): 17:50-18:50

Electrophoresis

Ligation and Transformation

Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku

Miniprep of ΔTMD1

Sequencing of ΔTMD1 and MS

Screening PCR of SRRz-DT

Deletion PCR of rΔTMD1GFP 2-2

Restriction Digestion(DpnI)

Ligation

Transformation

Tuesday, August 24 By:Ken, Tomo, Tasuku, Takuya

Retry of deletion PCR of rδTMD1 GFP

Restriction Digestion (DpnI)

Electrophoreis

Ligation

Colony PCR of S_Sam7-E0840 (Electrophoresis for 35min)

Culture of each two colonies of S_ΔTMD1-E0840₁-1 and S_ΔTMD1-E0840₂ for 37℃ 08/03-08/04

Sample name	Template	10xbuffer	100xbuffer	EcoRI	XbaI 1	XbaI 2	SpeI	PstI 1	PstI 2	Water	Total
1	3	1	0.1	0.2	-	-	-	-	-	5.7	10
2	3	1	0.1	-	0.2	-	-	-	-	5.7	10
3	3	1	0.1	-	-	0.2	-	-	-	5.7	10
4	3	1	0.1	-	-	-	0.2	-	-	5.7	10
5	3	1	0.1	-	-	-	-	0.2	-	5.7	10
6	3	1	0.1	-	-	-	-	-	0.2	5.7	10
N	3	1	0.1	-	-	-	-	-	-	5.9	10