February 7

Preculture by_???
We started preculture at 12:10.

February 8

March 1

March 2

March 3

March 4

March 5

March 6

March 7

March 8

March 9

March 10

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5	Spe1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 29.0ng/μL

torA	Xba1	Pst1	BufferM	BSA	MilliQ	total
10	0.2	0.2	2	0.2	7.4	20

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torA	Lacp-pSB3C5	Ligation High Ver.2	total
4	2	3	9

torA : 929fmol
Lacp-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep
We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

Electrophoresis
1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

March 12

Transformation

DT(1ng/μL)	DT(0.1ng/μL)	Kil	lacP-torA	MilliQ	competent cell	total
1	0	0	0	0	20	21
0	1	0	0	0	20	21
0	0	5	0	0	50	51
0	0	0	5	0	50	51
0	0	0	0	1	20	21

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
10	0.5	0.5	3	0.3	15.7	30

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspA	pSB1C3	MilliQ	Ligation High Ver.2	total
4	2	0	3	9
2	2	0	2	6
0	2	2	2	6

March 13

Miniprep pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3	EcoR1	Spe1	BufferM	BSA	MilliQ	total
20	0.2	0.2	4	0.4	15.2	40

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9
MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for 1 hour

• torA : 0.707pmol
  
• pSB1C3 : 0.068pmol
  
  

Liquid culture
We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture
We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFP	EcoR1	Spe1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

DT	EcoR1	Xba1	Buffer2	BSA	MilliQ	total
10	0.5	0.5	3	0.5	15.5	30

for 2 hours at 37℃.

Miniprep
pspA (pSB1C3) 40.5ng/µL

Ligation

pspA	DT	Ligation High Ver.2	total
5	1.5	3	9.5

• pspA : 385fmol
  
• DT : 36fmol
  
  

Transformation

J23107-tatABCD	DT (0.1ng/µL)	DT (0.01ng/µL)	pspA-DT	competent cells on 3/15	total
2	0	0	0	20	22
0	2	0	0	20	22
0	0	2	0	20	22
0	0	0	2	20	22

Screening PCR
Kil, pspA and torA

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

March 16

Miniprep
torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick Taq	Primer-r	Primer-f	MilliQ	total
25	1	1	23	50

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

Checking Transformation Efficiency
competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	BufferH	MilliQ	total
20	0.2	0.2	0.3	3	0	6.3	30
5	0.2	0	0.2	0	2	12.6	20

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

for overnight at 37℃.
We did this to confirm.

Ligation

torA	pSB1C3	Ligation High Ver.2	total
4	2	3	9

MilliQ	pSB1C3	Ligation High Ver.2	total
4	2	3	9

at 16℃, for overnight

• torA→767fmol
  
• pSB1C3→68fmol
  

March 17

Miniprep J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCD	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Success.

pspA-DT	EcoR1	Pst1	BSA	BufferH	MilliQ	total
5	0.2	0.2	0.2	2	12.4	20
5	0.2	0	0.2	2	12.6	20

Failed.

March 19

Restriction

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

Kil	DT	Ligation High Ver.2	total
10	2	6	18

We did this for an hour at 16℃.

Restriction

GFP	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.5	0.5	0.5	3	15.5	30

We did this for 4 hours at 37℃

Ligation

pspA	DT	Ligation High Ver.2	total
5	5	5	15

pspA	pSB1C3	Ligation High Ver.2	total
4	1	3	8

We did these for an hour at 16℃.

• pspA (5µL)→377fmol
  
• DT→39fmol
  
• pspA (4µL)→339fmol
  
• pSB1C3→34fmol
  
  

Transformation
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick Taq	Primer-R	Primer-F	MilliQ	total
25	1	1	23	50

• pspA→○
  
• pspA-DT→○
  
• GFP-DT→○
  
• torA→×
  
• Kil-DT 6 of 8 sumples→○
  

Quick Taq	VR	VF	MilliQ	total
25	1	1	23	50

• pspA→○
  
• pspA-DT→×
  
  

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.3	0.3	0.3	3	16.1	30

DT	EcoR1	Xba1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep
GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30
5	0.2	0	0.2	2	12.6	20

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DT	EcoR1	Pst1	Xba1	BSA	BufferM	MilliQ	total
5	0.2	0.2	0	0.2	2	12.4	20
5	0.2	0	0	0.2	2	12.6	20
10	0.2	0	0.2	0.3	3	16.3	30

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

	torA	pSB1C3	pspA	DT	GFP-DT	Ligation High Ver.2	total
1	4	1	0	0	0	3	8
2	0	1	7	0	0	4	12
3	0	0	5	3	0	4	12
4	3	0	0	0	5	4	12

We did this for an hour at16℃.

March 22

PCR
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

Buffer	dNTPs	MgSO4	Primer-F	Primer-R	Template	MilliQ	KOD plus neo	total
5	5	3	1.5	1.5	0.5	32.5	1	50

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torA	EcoR1	Spe1	BSA	BufferM	MilliQ	total
10	0.2	0.2	0.3	3	16.3	30

Ligation

	torA	pSSB1C3	pspA	DT	GFP-DT	Ligation high	total
1	4	3	0	0	0	4	11
2	3	0	0	0	3	3	9
3	0	0	5	5	0	5	15

• torA (4µL)→512fmol
  
• pSB1C3→54fmol
  
• torA (3µL)→384fmol
  
• GFP-DT→36fmol
  
• pspA→377fmol
  
• DT→65fmol
  
  

March 23

Screening PCR
torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick Taq	VF	VR	MilliQ	total
25	1	1	23	50

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspA	EcoR1	Spe1	BufferM	BSA	MilliQ	total
5	0.3	0.3	3	0.3	21.1	30

And then we did ethanol precipitation

Ethanol precipitation
pspA 11.5ng/µL.

Miniprep
Lacp+pSB3C5-8 77.9µg/mL
Lacp+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

Lacp+pSB3C5-8	Spe1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

Kil+DT-4	Xba1	Pst1	Buffer2	BSA	MilliQ	total
20	0.5	0.5	3	0.5	5.5	30

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction
Lacp+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep
torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)	EcoR1	Pst1	BufferH	BSA	MilliQ	total
5	0.2	0.2	2	0.2	12.4	20
5	0.2	0	2	0.2	12.6	20

torA-GFP-DT	EcoR1	Xba1	Pst1	BufferH	BufferM	BSA	MilliQ	total
5	0.2	0	0.2	2	0	0.2	12.4	20
5	0.2	0	0	2	0	0.2	12.6	20
20	0	0.2	0.2	0	3	0.3	6.3	30

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

Lacp (pSB3C5)	torA-GFP-DT	Ligation High Ver.2	total
1	5	3	9

• Lacp : 22fmol
  
• torA-GFP-DT : 197fmol
  

pspA	pSB1C3	Ligation High Ver.2	total
10	1	5	16

pspA	DT	Ligation High Ver.2	total
10	1	5	16

• pspA : 180fmol
  
• pSB1C3 : 18fmol
  
• DT : 16fmol
  

LacP(pSB3C5)	Kil-DT	Ligation High Ver.2	total
1	5	3	9

for 2 hours at 16℃

Transformation

Lacp-Kil-DT	competent cell	total
1	10	11

March 27

Miniprep by We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

Name	Well	Sample	Competent Cells	Total	Plate	Colony
BBa_K117004	14J(2011 plate2)	5	20	?	?	?

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick Taq	VF2	VR	MilliQ	Total
25	1	1	23	50

Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture
Lacp-torA-GFP-DT

August 2

Mutation of FT by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.

Inverse PCR

10xBufer	2mM dNTPs	primer fwd	primer rev	template	polymerase	MilliQ	Total
5	5	1.5	1.5	0.5	1	35.5	50

94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold

Dpn1 Digestion

PCR product	Dpn1
50	2

37℃,1h incubate

Self-ligation

product	MilliQ	Ligase	T4 Kinase	Total
2	7	5	1	15

16℃, 1h incubate

Transformation

competent cell	DNA
20	2

Cells were stored on ice for 30min.
After 42℃ 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.

August 13

Liquid culture
FT at 37°C, for overnight.

August 14

Miniprep of FT : by Sato, Takeuchi
The concentration was 81.5ng/uL

Restriction digestion and Electrophoresis : by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.

DNA(FT,80ng/uL)	10xBuferH	EcoR1	Pst1	MilliQ	Total
5	2	1	-	12	20
5	2	-	1	12	20

37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)

Liquid culture
FT (4mL)

August 15

Miniprep of FT : by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.

Electrophoresis : by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.

August 16

Transformation : by Takeuchi, Ota

Name	Well	Sample	Competent Cells	Total	Plate	Colony
FT	-	1 µL	10	11	LB (Kan+)	×
pSB1C3	1-3-A	1	10	11	LB (CP+)	○
I719005	1-15-N	1	10	11	LB (Amp+)	○

August 17

Transformation : by Takeuchi

Name	Well	Sample	Competent Cells	MilliQ	Total	Plate	Colony
FT	-	2	2	18	22	LB (Kan+)	×

We found that mutation of FT was not successful.

August 20

We decided to do PCR using FT specific primers before mutation.
PCR of FT  : by Sato

10xBufer	dNTPs	MgSO4	primer fwd	primer rev	template	polymerase	MilliQ	Total
5	5	3	1	1	1(130ng/µL)	1(KOD plus neo)	33	50

94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.

Electrophoresis

Lane	Name	length(bp)
1	1kb ladder	-
2	FT	600

August 21

Restriction digestion  : by Sato

DNA(FT,203ng/µL)	10xBuferM	Xba11	Pst1	MilliQ	Total
10	4	1	1	24	40

37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

Ligation  : by Sato

Vector		Insert		Ligation High Ver.2
pSB1C3	1	FT	10	5.5

Liquid culture
T7 promoter, pSB1C3 (4mL)

August 22

Miniprep : by Sato

T7 promoter	pSB1C3
85.3ng/µL	82.93ng/µL

August 23

Ethanol Precipitation
diluted in 20µL 79.3ng/µL

mada dekite nai

August 24

Restriction enzyme processing

T7 promoter(85.3ng/µL)	Spel	Pstl	buffer M	MiliQ	Total
10	1	1	2	6	20

->purifying column 33.4ng/µL(dissolution 40µL)

pSB1C3(82.9ng/µL)	Xbal	Spel	buffer M	MiliQ	Total
20	1	1	4	14	40

->gene clean2 39.9ng/µL(dissolution 40µL)

<<picture1,2>>
Ligation

FT(600bp, 79.3ng/µL)	pSB1C3(2000bp,39.9ng/µL)	Ligation High Ver.2
3µL => 597fmol	2µL => 60fmol	2.5µL

FT(600bp, 79.3ng/µL)	T7(2100bp,33.4ng/µL)	Ligation High Ver.2
2.4µL => 478fmol	2µL => 48fmol	2.2µL

=> 16℃,1hr incubate

August 27

Colony PCR

2X Quick Tag	VF2	VR	MiliQ	Total
25	1	1	23	50

Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp

FT(TOPO) PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template(130ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	30

Liquid culture by Nobeyama
FT 4ml

August 28

Mutation of FT (re)

inverse PCR

MilliQ	buffer	dNTP	primer f	primer r	FT(130ng/µL)	KODplus	Total
35.5	5	5	1.5	1.5	0.5	1	50

94℃	98℃	68℃	cycles
2min	10sec	4min	18

Lane1: 1kb ladder
Lane2: FT

Miniprep FT(TOPO)
158ng/µL

Tranformation
competent cell: 20
BBa.I746902  : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)

August 29

Mutaion of FT(re;re)

Inverse PCR

first

MilliQ	buffer	dNTP	primer f	primer r	FT(130ng/µL)	KODplus	Total
35.5	5	5	1.5	1.5	0.5	1	50

second

MilliQ	buffer	dNTP	primer f	primer r	FT(52ng/µL)	KODplus	Total
35	5	5	1.5	1.5	1	1	50

94℃	98℃	68℃	cycles
2min	10sec	4min	30

Lane1: 1kb ladder
Lane2: first Template 130ng/µL
Lane3: second Template 53ng/µL

first sample	Dpnl	total
45µL	2µL	47µL

in 37℃, 1 hour

Self-Ligation

PCR products	MilliQ	Ligation High	T4 kinase	total
2µL	7µL	5µL	1µL	15µL

in 16℃, 1.5hour incubate
Transformation
competent cell: 20
DNA  : 2

Liquid culture(I746902): 3mL

August 30

Liquid culture(FT) 4mL x2

August 31

Miniprep(FT)

(1) 64.9ng/µL
(2) 52.6ng/µL

Restriction enzyme processing (Mutation check)

FT(52.6ng/µL)	bufferH	E.coli	Pst1	MilliQ	total
1µL	5µL	0.5µL	0.5µL	3.5µL	10µL

in 37℃,1.5hour
PCR(RBS primer)

buffer for KODplus neo	dNTPs	MgSO4	primer f	primer r	Template(52.6ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	30

Lane1: 1kb ladder
Lane2: FT
Lane3: FT(E.coli)
Lane4: FT(Pst1)
Lane5: FT PCR

PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template	KOD neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	15sec	35

Lane1: 1kb ladder
Lane2: FT

PCR(re)

buffer	dNTPs	MgSO4	primer f	primer r	Template	KOD neo	MilliQ	Total
5	5	2	1	1	1	1	34	50

94℃	98℃	68℃	cycles
2min	10sec	10sec	30

September 2

PCR(re;re)

first

buffer	dNTPs	MgSO4	primer f	primer r	Template(1ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

second

buffer	dNTPs	MgSO4	primer f	primer r	Template(10ng/µL)	KODplus neo	MilliQ	Total
5	5	3	1	1	1	1	33	50

94℃	98℃	68℃	cycles
2min	10sec	10sec	25

Lane1: first Template 1ng
Lane2: second Template 10ng
Lane3: 1kb ladder

refine first Template => 132ng/µL

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.