Team:Kyoto/Notebook

Tuesday, July 20

By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto

1. Solubilization of antibiotics.

For Ampicillin(Amp), add 1.0g Amp to 20ml MilliQ (final concentration is 50mg/ml). For Kanamycin(Kan), add 0.5g Kan to 10ml MilliQ (final concentration is 50mg/ml). Dispense 1.1ml of the solution into 1.5ml tubes and store in the freezer (-20℃).

2. Make plates for LB (Amp+) and LB (Kan+).

3. Transformation of iGEM Parts.

Name	Well	Sample (µl)	Competent Cells (µl)	Total (µl)	Plate	Incubation	Result
<partinfo>J23100</partinfo>	1-18-C	1	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 "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".

Wednesday, July 21

By: Wataru, Ken, Makoto, Takuya Yamamoto

1. Culture plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.

2. Make a master plate of the above plates.

3. Retry Transformation of iGEM Parts.

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

4. PCR for S-R-Rz/Rz1 and S

Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.

No.	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	TemplateDNA (5ng/µl)	Primer Forward (10µM)	Primer S-R-Rz/Rz1 Reverse (10µM)	Primer S Reverse (10µM)	KOD Plus ver.2	Total
1	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
2	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
3	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
4	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
5	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
6	28µl	3µl	5µl	5µl	5µl	1.5µl	1.5µl	-	1µl	50µl
7	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl
8	28µl	3µl	5µl	5µl	5µl	1.5µl	-	1.5µl	1µl	50µl

Forward Primer of S-R-Rz/Rz1 and S is common. PCR condition: 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

Thursday, July 22

By: Wataru

1. Electrophoresis of the PCR products for 40min.

Length of S and S-R-Rz/Rz1 is 370bp, 1300bp, so PCR succeeded.

2. Miniprep of iGEM Parts.

Name	Concentration(ng/µl)
<partinfo>J23100</partinfo>	18.5
<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.

3. Culture plates and make master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 7/22 17:00 to 7/23 10:00.

Friday, July 23

By: Wataru, Tomo, Makoto

1. Miniprep of iGEM Parts.

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

2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.

Sample	Concentration (ng/µl)	New Name
1	18.6	-
3	77.6	S1
5	33.6	-
7	65.4	S2

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

3. Retry of PCR of S-R-Rz/Rz1.

Sample	Water	25mmol/l MgSO4	2mmol/l dNTPs	10×Buffer for KOD plus ver.2	Template DNA (5ng/µl)	Primer S-R-Rz/Rz1 Forward (10µmol/l)	Primer S-R-Rz/Rz1 Reverse (10µmol/l)	KOD plus ver.2	Total
1	28µl	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

PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.

4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.

Sample	10xBuffer	BSA	Enzyme	MilliQ	Total	Incubation
1	5µl	1	EcoRI 0.1	3.6	10	At 37℃ 7/23 18:00 - 7/23 18:30
2	5	1	XbaI 0.1	3.6	10
3	5	1	SpeI 0.1	3.6	10
4	5	1	PstI 0.1	3.6	10
5	5	1	-	3.7	10

5. Electrophoresis of above sample for 35min.

Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.

6. To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.

Sample	10×Buffer	Enzyme 1	Enzyme 2	MilliQ	Total	Incubation
S1	11µl	5	EcoRI 0.2	SpeI 0.2	33.6	50	At 37℃ for 2h
S2	11	5	EcoRI 0.2	SpeI 0.2	33.6	50
<partinfo>E0840</partinfo>(GFP)	45	5	EcoRI 0.2	XbaI 0.2	0	50

After PCR purification, evaporated them and diluted 3ul.

7. Ligated over night.

Sample	Vector	Insert	Ligation High	Total
S-E08401	<partinfo>E0840</partinfo> 0.5µl	S1 0.5	1	2
S-E08402	<partinfo>E0840</partinfo> 0.5	S2 0.5	1	2

Monday, July 26

By: Wataru, Tomonori, Makoto

1. Electrophoresis of PCR products

At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.

2. PCR purification

Sample	Concentration (ng/µl)	New Name
4	51.6	SRRz1
5	59.3
6	59.6	SRRz2

3. Transformation of iGEM Parts

Name	Well	Sample (µl)	Competent Cell (µl)	Total (µl)	Plate	Incubation	Result
	1-12-M	1	20	21	LB (Amplicillin+)	At 37℃ 7/26 - 7/27	×
	2-17-F	1	20	21	LB (Kanamycin+)		×
	1-5-E	1	20	21			×

4. Culture of 1-6-G, 1-12-O, and 1-23-L

Tuesday, July 27

By: Wataru, Tomo, Kazuya, Ken, Naoi

1. Colony PCR of S-E840

To check that S GFP is correctly inserted, we did colony PCR. Electrophoresis was done for 35min.

Marker	1	2	3	4	5	6	7	8	9	10	11	12	13	+	-	Marker
1kb	S-E08401						S-E08402							E0840	None	100bp

As a result, 1,3,5,6,11,12,13 are inserted S gene correctly. So, we decided to use 6 as S-E0840₁　and 11 as S-E0840₂.

2. Miniprep

Sample number	Concentration(ng/µL)
1-6-G	26.9
1-23-L	120.0
1-12-O	120.1

3. Restriction Digestion

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
1-23-L	30	5	0.5	EcoRI	0.4	XbaI	0.3	13.7	50
4.5②	40	5	0.5		0.4		0.4	3.8	50
6②	40	5	0.5		0.4		0.4	3.8	50

Incubate 37℃ 16:45~18:00

4. Ligation

5. Transformation

Wednesday, July 28

1. Result of Transformation

SRRz①-DT	Many colonies
SRRz②-DT

2. Deletion PCR

To delete functional domain of S gene, we did deletion PCR.

Miniprep

Sample number	Concentration(ng/µ)
S-E840①	95.5
S-E840②	98.6

Diluted S-GFP①　and S-GFP②　20 times with water, and used as template DNA.

Deletion PCR

To delete functional region of S gene, we did deletion PCR.

	Water	25mM MgSO4	2mM dNTPs	10x buffer for KOD Plus ver.2	Primer Deletion F(10µM)	Primer Deletion R(10µM)	Template S-E840①	Template S-E840②	KOD Plus ver.2	Total
Δ1-1	28	3	5	5	1.5	1.5	5	-	1	50
Δ1-2	28	3	5	5	1.5	1.5	5	-	1	50
Δ2-1	28	3	5	5	1.5	1.5	-	5	1	50

PCR program

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

3. RE

To check function of our Restriction enzymes, we digested S-E840① and S-E-840② by DpnI.

	Sample	fast digestion buffer	DpnI	MilliQ	Total
S-E840①	3	1	0.1	5.8	10
S-E840②	3	1	0.1	5.8	10

Electrophoresis

Gel: Agarose Time: 35min Voltage: 100V Maker: 1K 100

1 1k marker 2 not digested S-E840① 3 not digested S-E840② 4 digested S-E840① 5 digested S-E840② 6 100bp marker

1k 1 2 3 4 100

File:KyotoExp100728.png

Discussion

DpnI works correctly

Thursday, July 29

RE

	Sample volume	Fastdigestion buffer	Enzyme 1	MilliQ	Total
Δ1-1	50	6	DpnI 0.2	3.8	60
Δ2-1	50	6	DpnI 0.2	3.8	60

Incubate 7/29 9:40~7/29 11:00

Ligation and Pospholylation

	Sample	MilliQ	Ligation High	T4 Kinase	Total
Δ1-1	2	7	5	1	15
Δ2-1	2	7	5	1	15

Incubate 7/29 11:30~7/29 13:00

Transformation

Sample	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
Δ1-1	-	3	30	33	LB amp	7/29~7/30
Δ1-1	-	3	30	33

Friday, July 30

Result of transformation of Δ1 and Δ2 Many colonies are observed.

Monday, August 2

By: Wataru, Ken

1. Miniprep

Sample number	Concentration(ng/µL)
Δ1	52.7
Δ2	54.4
Δ3	89.5
pSB4K5	50.7
LacP	18.6

2. PCR and RE of E240

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.

	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template E240	KOD Pllus ver.2	Total
E240①	28	3	5	5	1.5	1.5	5	1	50
E240②	28	3	5	5	1.5	1.5	5	1	50

PCR program

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

Electrophoresis

PCR purification

Sample number	Concentration(ng/µL)
E240①	42.6
E240②	55.3

RE

To insert E240 to pSB4K5 by 3A assembly, we digested the PCR products of E240 by XbaI and PstI

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
E240①X-P	30	5	0.5	XbaI	0.2	PstI	0.2	14.1	50
E240②X-P	30	5	0.5	XbaI	0.2	PstI	0.2	14.1	50

PCR purification

Sample number	Concentration(ng/µL)	Volume(µL)
E240①X-P	21.8	40
E240②X-P	32.4	45

-20℃ freezer

3. Error PCR

	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
ΔTMD①	32	3	5	5	1.5	1.5	1	-	-	1	50
ΔTMD②	32	3	5	5	1.5	1.5	-	1	-	1	50
ΔTMD③	32	3	5	5	1.5	1.5	-	-	1	1	50

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

After the digestion by DpnI, we transfected 2µL of sample to 20µL of competent cell.

Tuesday, August 3

1. The result of transformation

ΔTMD①	Many colonies
ΔTMD②	No colony
ΔTMD③	Many colonies

We picked two colonies from ΔTMD① and ΔTMD③, and cultured 37℃ 8/3~8/4.

2. The construction of ML and MS

Miniprep

Sample number	Concentration(ng/µL)
pSB4K5	60.7
LacP	26.8

RE

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
LacP	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
E240①(X-P)	50	6	0.6	XbaI	0.2	PstI	0.2	3	60
E240②(X-P)	50	6	0.6	XbaI	0.2	PstI	0.2	3	60

Incubate

Purification

PCR purification

Sample number	Concentration(ng/µL)
pSB4K5 E-P	39.5
E240①X-P	21.8
E240②X-P	32.4

pSB4K5 E-P is concentrated 10µL and E240①X-P, E240②X-P are concentrated 1µL.

Ethanol precipitation

1-5-G dilute milliQ 2µL

Ligation

	Vector	Insert 1	Insert 2	Ligation High	Total
ML1	pSB4K5 E-P 1	LacI E-S 1	E240①X-P 1	3	15
ML2	pSB4K5 E-P 1	LacI E-S 1	E240②X-P 1	3	15

Incubation 17:30~20:20

PCR of J23101-E240

J23101-E240 is important in the measurement of RPU, so we amplified this parts by PCR.

	Water	25mM MgSO4	2mM dNTPs	10xBuffer for KOD Plus ver.2	Primer VF2(10µM)	Primer VR(10µM)	Template J23101-E240	KOD plus ver.2	Total
MS①	32	3	5	5	1.5	1.5	1	1	50
MS②	32	3	5	5	1.5	1.5	-	1	50

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

PCR purification

Sample number	Concentration(ng/µL)
J23101-E240	40.6

Discussion J23101-E240(MS) is amplified corrrecly.

RE

	Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
J23101-E240(E-P)	45	6	0.6	EcoRI	0.2	PstI	0.2	8	60

PCR purification

Sample number	Concentration(ng/µL)	Volume(µL)
J23101-E240 E-P	74.1	30

J23101-E240 is concentrated 7µL

Ligation

	Vector	Insert	Ligation High	Total
MS	pSB4K5 E-P 1	J23101-E240 E-P 1	2	4

Incubation 20:00~20:30

Transformation

Sample	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
ML1	-	1	20	21	LB kan	8/3~8/4
ML2	-	1	20	21
MS	-	1	20	21

Thursday, August 5

1. Result of transformation

ML1	Many colonies
ML2
MS

ML1 and ML2

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 ML1 and ML2 plate. We cultured both white and green colonies.

MS Self-ligated colony is red Many of colonies are red, however, green colonies are observed.

Culture and Master

Green colony ML1-1 ML1-2 ML2-1 MS1-1 MS1-2 MS1-3 White colony ML1-3 ML1-4 ML2-2 ML2-3 ML2-4

J23100 and LacP 8/5~8/6

2. Sequence

Sample number	Concentration(ng/µL)
ΔTMD①A	28.9
ΔTMD①B	25.3
ΔTMD③A	26.6
ΔTMD③B	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

1. Miniprep

ML1-1 ML1-2 ML1-3 ML1-4 ML2-1 ML2-2 ML2-3 ML2-4 MS1 MS2 MS3

2. RE

Sample volume	2 buffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total
50	6	0.6	EcoRI	0.3	PstI	0.3	2.8	60

Electrophoresis

1 100bp 2λ 3λ 4100bp 5MS1 6MS2 7MS3 8ML1-1 9ML1-2 10ML1-3 11ML1-4 12ML2-1 13ML2-2 14ML2-3 15ML2-4 16MS1 17MS2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Discussion MS1 and MS2 are inserted correctly. ML1-1 and ML1-2 are inserted correctly. ML2-1 are inserted correctly. White colonies are inserted not lacP but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of LacI.

Error PCR(Retry)

	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
4℃	forever

Add DpnI 2µL and incubate 1h.

Transformation

Sample	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

1. Miniprep of MS and ML

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

2. 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

3. 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.

4. 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
		2	C2	50	52

Tuesday, August 10

By: Wataru, Tomonori, Ken, Fumitaka

Making culture and Master plate

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

Lac p (low)	KRX	Many colonies
	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
	Ampicillin	x
2	Kanamycin	o
	Ampicillin	o
3	Kanamycin	o
	Ampicillin	x
4	Kanamycin	o
	Ampicillin	x
5	Kanamycin	o
	Ampicillin	x
6	Kanamycin	o
	Ampicillin	o
7	Kanamycin	o
	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)

RE and electrophoresis of lac (low) 1 and 3

Sample name	1	2	3	N
EcoRI	0.2	-	0.2	-
PstI	-	0.2	0.2	-

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

RE and electrophoresis of DT

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

1. Miniprep of ΔTMD1GFP

29.6(ng/µg)

2. 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

3. PCR condition

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

. RE(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, Humitaka, Tasuku

1. Miniprep of ΔTMD1

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

2. 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.

3. Screening PCR of SRRz-DT

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

PCR condition

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.

4. 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

PCR condition

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

RE (DpnI) and Ligation

Template	25(µL)
DpnI	1
Total	26

19:10‾20:10

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

1.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

PCR condition

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

RE (DpnI), electrophoresis and ligation RE: 14:15‾15:15 Electrophoresis: 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.

2.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

PCR condition

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

RE(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

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

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

Miniprep of 1-5G 29.0 (ng/µL)

RE 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 and transformation Ligation of E0240 and 1-5G

Thursday, August 26

By:Ken, Tomo, Kazuya, Tasuku, Takuya, Hashiya

Miniprep

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

RE 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, Hashiya

Making master plate of E0240 low

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

RE 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 Y, Takuya, 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, Humitaka, 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)

RE 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

9/2

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.

9/3

By: Tomonori, Tomo, Kazuya, Tasuku, Humitaka, Ken

Making culture

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

ML