Template:Kyoto/Notebook/Magnetosome

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Revision as of 09:44, 12 October 2014 by Hideaki TSUJII (Talk | contribs)

7/15

Transformation

Tatsui, Matsumoto and Sukegawa

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_J23101(Pcon)220(2/23)22LB(CP)
BBa_B0032(RBS)220(2/23)22LB(CP)

7/16

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_B0030(1)220(2/23)22LB(CP)
BBa_B0030(2)220(bought)22LB(CP)
BBa_B0032(1)220(2/23)22LB(CP)
BBa_B0032(2)220(bought)22LB(CP)

7/17

Colony PCR

Tatsui, Matsumoto, Li, and Yamauchi

NameⅠNameⅡ
7/16 BBa_B0030(1) Colony_17/16 BBa_B0032(1) Colony_1
7/16 BBa_B0030(1) Colony_27/16 BBa_B0032(1) Colony_2
7/16 BBa_B0030(1) Colony_37/16 BBa_B0032(1) Colony_3
7/16 BBa_B0030(2) Colony_17/16 BBa_B0032(2) Colony_1
7/16 BBa_B0030(2) Colony_27/16 BBa_B0032(2) Colony_2
7/16 BBa_B0030(2) Colony_37/16 BBa_B0032(2) Colony_3
Negative Control-
--

Liquid Culture

Tatsui, Matsumoto, Li, and Yamauchi

Namemedium
7/16 BBa_B0030(1) Colony_1Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_2Plusgrow medium(CP)
7/16 BBa_B0030(1) Colony_3Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_1Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_2Plusgrow medium(CP)
7/16 BBa_B0030(2) Colony_3Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_1Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_2Plusgrow medium(CP)
7/16 BBa_B0032(1) Colony_3Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_1Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_2Plusgrow medium(CP)
7/16 BBa_B0032(2) Colony_3Plusgrow medium(CP)

7/18

Miniprep

Tatsui, Matsumoto, Tsujii, Stephane, and Yamauchi

DNAConcentration/(µg/ml)260/280260/230
7/16 BBa_B0030(1) Colony_12291.961.40
7/16 BBa_B0030(1) Colony_22091.951.50
7/16 BBa_B0030(1) Colony_31082.122.12
7/16 BBa_B0030(2) Colony_1---
7/16 BBa_B0030(2) Colony_21742.021.56
7/16 BBa_B0030(2) Colony_31582.011.68
7/16 BBa_B0032(1) Colony_11792.002.06
7/16 BBa_B0032(1) Colony_2---
7/16 BBa_B0032(1) Colony_32211.901.37
7/16 BBa_B0032(2) Colony_12251.891.37
7/16 BBa_B0032(2) Colony_22701.981.63
7/16 BBa_B0032(2) Colony_31941.891.33

7/30

Restriction Enzyme Digestion

No name

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)Buffur/(µl)BSA/(µl)MilliQ/(µl)TOTAL/(µl)
7/16 BBa_B0030(2) Colony_110.30001(H)07.710
7/16 BBa_B0030(2) Colony_1100.3001(M)16.710
7/16 BBa_B0030(2) Colony_11000.301(M)07.710
7/16 BBa_B0030(2) Colony_110000.31(H)07.710
7/16 BBa_B0030(2) Colony_1(Nega)100001(M)0810

Electrophoresis

No name

LaneRestriction Enzyme Digestion Products
17/30 BBa_B0030(2) Colony_1(E CUT)
27/30 BBa_B0030(2) Colony_1(X CUT)
37/30 BBa_B0030(2) Colony_1(S CUT)
47/30 BBa_B0030(2) Colony_1(P CUT)
57/30 BBa_B0030(2) Colony_1(Nega)
61kbp ladder

7/31

Transformation

Hashitani, Tatsui, Li and Yamauchi

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_E0240(RBS-GFP-DT)22022LB(CP)
J23100(Pcon)22022LB(AMP)

Liquid Culture

No name

Namemedium
7/31 J23100(Pcon) Colony_1Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_2Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_3Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_4Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_5Plusgrow medium(AMP)
7/31 J23100(Pcon) Colony_6Plusgrow medium(AMP)

Restriction Enzyme Digestion

Hashitani, Tatsui, Li and Yamauchi

7/18 RBS SampleDNA/(µl)EcoR1/(µl)Spe1/(µl)Pst1/(µl)Xba1/(µl)Buffur/(µl)BSA/(µl)MilliQ/(µl)TOTAL/(µl)
BBa_B003250.30001(H)03.710
BBa_B0032500.3001(M)03.710
BBa_B00325000.301(H)03.710
BBa_B003250000.31(M)12.710
BBa_B0032(Nega)500001(M)0410

8/1

Electrophoresis

Honda, Li and Yamauchi

LaneRestriction Enzyme Digestion Products
11kb ladder
27/31 BBa_B0032(E CUT)
37/31 BBa_B0032(S CUT)Error
4-
5-
67/31 BBa_B0032(P CUT)
77/31 BBa_B0032(X CUT)
87/31 BBa_B0032(Nega)

PCR

Tatsui

BBa_E0240(RBS-GFP-DT)Volume
10×buffur/(µl)5
2mMdNTPs/(µl)5
25mM MgSO4/(µl)2
VF Primer/(µl)1.5
VR Primer/(µl)1.5
template/(µl)0.5
KOD-plus/(µl)1
MilliQ/(µl)33.5
TOTAL/(µl)50
StepsTemparatureTimecycle
Pre Denature94℃2min1time
Denature94℃15sec30cycle
Annealing55℃30sec30cycle
Extension68℃1min30cycle
DNAConcentration/(µg/ml)260/280260/230
Product801.911.78

8/2

Miniprep

Tatsui, Honda, Matsumoto, Yamauchi, Li, Tsujii, and Nakamura

DNAConcentration/(µg/ml)260/280260/230
7/31 J23100(Pcon) Colony_11452.113.09
7/31 J23100(Pcon) Colony_2---
7/31 J23100(Pcon) Colony_31602.013.13
7/31 J23100(Pcon) Colony_41401.872.70
7/31 J23100(Pcon) Colony_51052.395.90
7/31 J23100(Pcon) Colony_61801.901.37

8/20

Transformation

Honda

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
7/31 BBa_E0240(RBS-GFD-DT)22022LB(CP)
BBa_I73209522022LB(CP)

8/21

Colony PCR

Shimazaki

NameⅠ
8/20 BBa_I732095 Colony_1
8/20 BBa_I732095 Colony_2
8/20 BBa_I732095 Colony_3
Negative control

Electrophoresis

Honda

LanePCR Product
11kb ladder
28/21 BBa_I732095 Colony_1
38/21 BBa_I732095 Colony_2
48/21 BBa_I732095 Colony_3
5Negative Control

Liquid Culture

Honda and Tatsui

Namemedium
8/20 BBa_I732095 Colony_1Plusgrow medium(CP)
8/20 BBa_I732095 Colony_2Plusgrow medium(CP)
8/20 BBa_I732095 Colony_3Plusgrow medium(CP)

8/22

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
8/20 BBa_I732095 Colony_14301.682.13
8/20 BBa_I732095 Colony_22551.871.81
8/20 BBa_I732095 Colony_32151.781.35

Restriction Enzyme Digestion

Nakamura

SampleDNA/(µl)EcoR1/(µl)Spe1/(µl)Buffur/(µl)MilliQ/(µl)TOTAL/(µl)
8/22 BBa_I732095 Colony_14.7113(H)20.330
8/22 BBa_I732095 Colony_27.8113(H)17.230
8/22 BBa_I732095 Colony_39.3113(H)15.730

Transformation

Tatsui and Yamauchi

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_J04450(pSB3C5)22022LB(CP)

8/25

Transformation

Tatsui, Kita and Li

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_J04450(pSB2K3)22022LB(KAN)
BBa_C0051(pSB1A2)22022LB(AMP)

Liquid Culture

Tatsui

Namemedium
8/22 BBa_J04450(pSB3C5)Plusgrow medium(CP)*5

8/26

Miniprep

Tsujii, Honda and Matsumoto

DNAConcentration/(µg/ml)260/280260/230
8/25 BBa_J04450(pSB3C5)5751.371.37

Liquid Culture

Tatsui, Matsumoto and Nakamura

Namemedium
8/25 BBa_J04450(pSB2K3)Plusgrow medium(KAN)*5
8/25 BBa_J0051(pSB1A2)Plusgrow medium(AMP)*2

8/27

Miniprep

No Name

DNAConcentration/(µg/ml)260/280260/230
8/26 BBa_J04450(pSB2K3)10451.431.11
8/26 BBa_J0051(pSB1A2)7901.390.90

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_J04450(pSB3T5)22022LB(TET)

8/28

Liquid Culture

Yamauchi

Namemedium
8/27 BBa_J04450(pSB3T5)Plusgrow medium(TET)

8/29

Miniprep

Matsumoto

DNAConcentration/(µg/ml)260/280260/230
8/28 BBa_J04450(pSB3T5)1801.601.67

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
BBa_J04450(pSB4A5)22022LB(AMP)

9/1

Colony PCR

NameⅠ
-
-
-
9/1 pSB4A5 Colony_1
9/1 pSB4A5 Colony_2
Negative Control

Restriction Enzyme Digestion

No name

SampleDNA/(µl)EcoR1/(µl)Pst1/(µl)Buffur/(µl)MilliQ/(µl)TOTAL/(µl)
8/28 Miniprep pSB3T525226(H)2560
Negative control_13.7001(H)5.310
8/22 Miniprep I73209514339(H)6190
Negative control_21.5001(H)7.510
8/27 Miniprep pSB2K36339(H)6990
Negative control_31001(H)810
8/2 Miniprep Pcon Colony_3201.51.55(H)1745
Negative control_44001(H)510
8/2 Miniprep Pcon Colony_1201.51.55(H)1745
Negative control_54001(H)510

Liquid Culture

No name

Namemedium
8/29 BBa_J04450(pSB4A5) Colony_1Plusgrow medium(AMP)*2
8/1 Pcon Colony_1Plusgrow medium(AMP)*2
8/27 pSB3T5 Colony_1Plusgrow medium(TET)*2

9/2

Miniprep

Honda, Yamauchi and Li

DNAConcentration/(µg/ml)260/280260/230
8/29 BBa_J04450(pSB4A5) Colony_12101.611.18
8/1 Pcon Colony_12151.791.87
8/27 pSB3T5 Colony_12151.580.92

Gel Extraction

Matsumoto

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
19/1 pSB3T5 Colony_1 (Nega)10
29/1 pSB3T5 Colony_1 (E+P)(Confirmation)5
39/1 pSB3T5 Colony_1 (E+P)(Extraction)55
41kb ladder3
59/1 I732095 (Nega)10
69/1 I732095 (E+P)(Confirmation)5
79/1 I732095 (E+P)(Extraction)85
81kb ladder3
99/1 pSB2K3 (Nega)10
109/1 pSB2K3 (E+P)(Confirmation)5
119/1 pSB2K3 (E+P)(Extraction)85
LaneSampleVolume/(µl)
19/1 Pcon Colony_3 (E+P)(Confirmation)5
29/1 Pcon Colony_3 (E+P)(Extraction)20
39/1 Pcon Colony_3 (E+P)(Extraction)20
49/1 Pcon Colony_3 (Nega)10
51kb ladder3
69/1 Pcon Colony_1 (E+P)(Confirmation)5
79/1 Pcon Colony_1 (E+P)(Extraction)20
89/1 Pcon Colony_1 (E+P)(Extraction)20
99/1 Pcon Colony_1 (Nega)10

9/3

Concentration Measurement

Matsumoto, Yamauchi and Li

DNAConcentration/(µg/ml)260/280260/230
pSB2K3401.480.10
Pcon Colony_1301.760.62
pSB3T5502.350.50
pSB1C3652.140.10
Pcon Colony_3451.560.58

Ligation

No name

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
Pcon2.1pSB1C31.5150.410
-(Negative Control)0pSB1C31.5152.510
Pcon1.3pSB2K32.5150.210
-(Negative Control)0pSB2K32.5151.510
Pcon10pSB3T52.3150.710
-(Negative Control)0pSB3T52.3151.710

Transformation

Matsumoto

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/3 Ligation Pcon+pSB1C331518LB(CP)
9/3 Ligation pSB1C3(Negative Control)81018LB(CP)
9/3 Ligation Pcon+pSB2K331518LB(KAN)
9/3 Ligation pSB2K3(Negative Control)81018LB(KAN)
9/3 Ligation Pcon+pSB3T531518LB(TET)
9/3 Ligation pSB3T5(Negative Control)21012LB(TET)

9/4

Colony PCR

Honda and Yamauchi

NameⅠNameⅡNameⅢNameⅣ
-9/3 pSB1C3(Nega) Colony_79/3 Pcon+pSB3T5 Colony_59/3 Pcon+pSB2K3 Colony_1
-9/3 pSB1C3(Nega) Colony_89/3 Pcon+pSB3T5 Colony_69/3 Pcon+pSB2K3 Colony_2
9/3 pSB1C3(Nega) Colony_19/3 Pcon+pSB3T5 Colony_19/3 Pcon+pSB3T5 Colony_79/3 Pcon+pSB2K3 Colony_3
9/3 pSB1C3(Nega) Colony_29/3 Pcon+pSB3T5 Colony_29/3 Pcon+pSB3T5 Colony_89/3 Pcon+pSB2K3 Colony_4
9/3 pSB1C3(Nega) Colony_39/3 Pcon+pSB3T5 Colony_39/3 Pcon+pSB3T5 Colony_99/3 Pcon+pSB2K3 Colony_5
9/3 pSB1C3(Nega) Colony_49/3 Pcon+pSB3T5 Colony_49/3 Pcon+pSB3T5 Colony_109/3 Pcon+pSB2K3 Colony_6
9/3 pSB1C3(Nega) Colony_59/3 pSB1C3(Nega) Colony_99/3 pSB3T5(Nega) Colony_19/3 Pcon+pSB2K3 Colony_7
9/3 pSB1C3(Nega) Colony_69/3 pSB1C3(Nega) Colony_109/3 pSB3T5(Nega) Colony_29/3 Pcon+pSB2K3 Colony_8
NameⅤNameⅥNameⅦNameⅧ
9/3 Pcon+pSB2K3 Colony_99/3 Pcon+pSB1C3 Colony_29/3 Pcon+pSB1C3 Colony_109/3 pSB1C3(Nega) Colony_8
9/3 Pcon+pSB2K3 Colony_109/3 Pcon+pSB1C3 Colony_39/3 pSB1C3(Nega) Colony_19/3 pSB1C3(Nega) Colony_9
9/3 pSB2K3(Nega) Colony_19/3 Pcon+pSB1C3 Colony_49/3 pSB1C3(Nega) Colony_29/3 pSB1C3(Nega) Colony_10
9/3 pSB2K3(Nega) Colony_29/3 Pcon+pSB1C3 Colony_59/3 pSB1C3(Nega) Colony_39/3 pSB1C3(Nega) Colony_11
9/3 pSB2K3(Nega) Colony_39/3 Pcon+pSB1C3 Colony_69/3 pSB1C3(Nega) Colony_4-
9/3 pSB2K3(Nega) Colony_49/3 Pcon+pSB1C3 Colony_79/3 pSB1C3(Nega) Colony_5-
9/3 pSB2K3(Nega) Colony_59/3 Pcon+pSB1C3 Colony_89/3 pSB1C3(Nega) Colony_6-
9/3 Pcon+pSB1C3 Colony_19/3 Pcon+pSB1C3 Colony_99/3 pSB1C3(Nega) Colony_7-
NameⅨNameⅩ
9/3 Pcon+pSB1C3 Colony_118/22 Miniprep I732095 Colony_3
Negative Control-

Restriction Enzyme Digestion

No name

SampleDNA/(µl)EcoR1/(µl)Pst1/(µl)Buffur/(µl)MilliQ/(µl)TOTAL/(µl)
8/27 Miniprep pSB2K32113(H)2330
8/27 Miniprep pSB2K3(Nega)1001(H)810
9/2 Miniprep pSB3T510113(H)1530
9/2 Miniprep pSB3T5(Nega)3001(H)610
9/2 Miniprep pSB4A510113(H)1530
9/2 Miniprep pSB4A5(Nega)3001(H)610
8/22 Miniprep pSB1C3 Colony_22111(H)510

9/5

Electrophoresis

Honda and Matsumoto

LaneColony PCR ProductsⅠLaneColony PCR ProductsⅠ
11kb ladder109/4 pSB1C3(Nega) Colony_7
2-119/4 pSB1C3(Nega) Colony_8
3-129/4 pSB1C3(Nega) Colony_9
49/4 pSB1C3(Nega) Colony_1139/4 pSB1C3(Nega) Colony_10
59/4 pSB1C3(Nega) Colony_2149/4 Pcon+pSB2K3 Colony_1
69/4 pSB1C3(Nega) Colony_3159/4 Pcon+pSB2K3 Colony_2
79/4 pSB1C3(Nega) Colony_4169/4 Pcon+pSB2K3 Colony_3
89/4 pSB1C3(Nega) Colony_5171kb ladder
99/4 pSB1C3(Nega) Colony_6
LaneColony PCR ProductsⅡLaneColony PCR ProductsⅡ
11kb ladder109/4 pSB3T5(Nega) Colony_2
29/4 Pcon+pSB2K3 Colony_4119/4 Pcon+pSB2K3 Colony_1
39/4 Pcon+pSB2K3 Colony_5129/4 Pcon+pSB2K3 Colony_2
49/4 Pcon+pSB2K3 Colony_6139/4 Pcon+pSB2K3 Colony_3
59/4 Pcon+pSB2K3 Colony_7149/4 Pcon+pSB2K3 Colony_4
69/4 Pcon+pSB2K3 Colony_8159/4 Pcon+pSB2K3 Colony_5
79/4 Pcon+pSB2K3 Colony_9169/4 Pcon+pSB2K3 Colony_6
89/4 Pcon+pSB3T5 Colony_10171kb ladder
99/4 pSB3T5(Nega) Colony_1
LaneColony PCR ProductsⅢLaneColony PCR ProductsⅢ
11kb ladder109/4 pSB2K3(Nega) Colony_5
29/4 Pcon+pSB2K3 Colony_7119/4 pSB1C3(Nega) Colony_1
39/4 Pcon+pSB2K3 Colony_8129/4 pSB1C3(Nega) Colony_2
49/4 Pcon+pSB2K3 Colony_9139/4 pSB1C3(Nega) Colony_3
59/4 Pcon+pSB2K3 Colony_10149/4 pSB1C3(Nega) Colony_4
69/4 pSB2K3(Nega) Colony_1159/4 pSB1C3(Nega) Colony_5
79/4 pSB2K3(Nega) Colony_2169/4 pSB1C3(Nega) Colony_6
89/4 pSB2K3(Nega) Colony_3171kb ladder
99/4 pSB2K3(Nega) Colony_4
LaneColony PCR ProductsⅣLaneColony PCR ProductsⅣ
11kb ladder109/4 Pcon+pSB1C3 Colony_5
29/4 pSB1C3(Nega) Colony_7119/4 Pcon+pSB1C3 Colony_6
39/4 pSB1C3(Nega) Colony_8129/4 Pcon+pSB1C3 Colony_7
49/4 pSB1C3(Nega) Colony_9139/4 Pcon+pSB1C3 Colony_8
59/4 pSB1C3(Nega) Colony_10149/4 Pcon+pSB1C3 Colony_9
69/4 Pcon+pSB1C3 Colony_1159/4 Pcon+pSB1C3 Colony_10
79/4 Pcon+pSB1C3 Colony_2168/22 Miniprep BBa_I732095 Colony_3
89/4 Pcon+pSB1C3 Colony_3171kb ladder
99/4 Pcon+pSB1C3 Colony_4
LaneColony PCR ProductsⅤ
11kb ladder
29/4 Pcon+pSB1C3 Colony_11
39/4 Negative Control
48/22 Miniprep BBa_I732095 Colony_3
5-
6-
7-
8-

Gel Extraction

No Name

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
11kb ladder5
29/4 pSB2K3(E+P)5
3--
49/4 pSB2K3(E+P)25
5--
69/4 pSB2K3(Nega)10
LaneRestriction Enzyme Digestion ProductⅡVolume/(µl)
11kb ladder5
29/4 pSB3T5(E+P)5
3--
49/4 pSB3T5(E+P)25
5--
69/4 pSB3T5(Nega)10
LaneRestriction Enzyme Digestion ProductⅢVolume/(µl)
11kb ladder5
29/4 pSB4A5(E+P)5
3--
49/4 pSB4A5(E+P)25
5--
69/4 pSB4A5(Nega)10
LaneRestriction Enzyme Digestion ProductⅣVolume/(µl)
11kb ladder5
2--
3--
49/4 pSB1C3(E+P)10
5--
6--
DNAConcentration/(µg/ml)260/280260/230
9/4 pSB2K3(E+P)361.250.24
9/4 pSB3T5(E+P)101.00-/td>
9/4 pSB4A5(E+P)40-0.01
9/4 pSB1C3(E+P)751.350.14

9/8

Cloning PCR

Honda and Li

TargetmamBmamQmamL
2×KAPA HiFi Hotstart Ready Mix/(µl)12.512.512.5
Fw Primer/(µl)1.5(mamB)1.5(mamQ)1.5(mamL)
Rv Primer/(µl)1.5(mamB)1.5(mamQ)1.5(mamL)
AMB-1 Genome/(µl)111
MilliQ/(µl)101010
TOTAL/(µl)252525
StepsTemparatureTimecycle
Pre Denature98℃5min1time
Denature98℃20sec25cycle
Annealing65℃15sec25cycle
Extension72℃5min25cycle
Final elongation72℃5min1time
Final hold4℃--

Ligation

Honda

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
Pcon1pSB3T510111022
-(Negative Control)0pSB1C330306

Electrophoresis

Li

LaneCloning PCR Products
11kb ladder
29/8 Cloning PCR (AMB-1 mamB)
39/8 Cloning PCR (AMB-1 mamQ)
49/8 Cloning PCR (AMB-1 mamL)
51kb ladder

Transformation

Honda

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/8 Ligation Pcon+pSB3T533033LB(TET)
9/8 Ligation pSB3T5(Negative Control)33033LB(TET)

Liquid Culture

No name

Namemedium
9/3 Transformation Pcon+pSB1C3 Colony_8Plusgrow medium(CP)*2

9/9

DNA Purification

Li

DNAConcentration/(µg/ml)260/280260/230
9/8 Cloning PCR (AMB-1 mamB)1651.541.57
9/8 Cloning PCR (AMB-1 mamQ)2501.691.92
9/8 Cloning PCR (AMB-1 mamL)1901.561.79

Miniprep

Li

DNAConcentration/(µg/ml)260/280260/230
9/8 Liquid Culture Pcon+pSB1C3901.651.17

Colony PCR

Honda and Li

NameⅠ
-
9/8 Transformation Pcon+pSB3T5 Colony_1
9/8 Transformation Pcon+pSB3T5 Colony_2
9/8 Transformation Pcon+pSB3T5 Colony_3
9/8 Transformation Pcon+pSB3T5 Colony_4
9/8 Transformation Pcon+pSB3T5 Colony_5
9/8 Transformation pSB3T5(Nega) Colony_1
Negative control

Electrophoresis

Honda and Li

LaneCloning PCR Products
11kb ladder
29/8 Colony PCR Pcon+pSB3T5 Colony_1
39/8 Colony PCR Pcon+pSB3T5 Colony_2
49/8 Colony PCR Pcon+pSB3T5 Colony_3
59/8 Colony PCR Pcon+pSB3T5 Colony_4
69/8 Colony PCR Pcon+pSB3T5 Colony_5
79/8 Colony PCR pSB3T5(Nega) Colony_1
89/8 Colony PCR Nega

Restriction Enzyme Digestion

No name

SampleDNA/(µl)Pst1/(µl)Spe1/(µl)Buffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/9 Miniprep Pcon+pSB1C322113(H)330
9/9 Miniprep Pcon+pSB1C3(Nega)1001(H)810
8/2 Miniprep Pcon Colony_414113(H)1130
8/2 Miniprep Pcon Colony_4(Nega)1001(H)810

9/10

Gel Extraction

Tatsui and Yamaura

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
11kb ladder-
2--
39/10 Digestion Pcon+pSB1C3(S+P)-
4--
59/10 Digestion Pcon+pSB1C3(Nega)-
6--
7Pcon(X+P)-
8--
9Pcon(Nega)-
101kb ladder-
11--
DNAConcentration/(µg/ml)260/280260/230
Pcon(X+P)701.400.13

PCR

No Name

TargetE+X+RBS+mamB+SX+RBS+mamL+His+S+PX+RBS+mamQ+His+S+P
2×KAPA HiFi Hotstart Ready Mix/(µl)12.512.512.5
Fw Primer/(µl)0.75(E+X+RBS+mamB)0.75(X+RBS+mamL)0.75(X+RBS+mamQ)
Rv Primer/(µl)0.75(S+mamB)0.75(P+S+His+mamL)0.75(P+S+His+mamQ)
9/9 DNA Purification Product/(µl)TraceTraceTrace
MilliQ/(µl)111111
TOTAL/(µl)252525
StepsTemparatureTimecycle
Pre Denature98℃5min1time
Denature98℃20sec25cycle
Annealing60℃15sec25cycle
Extension72℃15sec25cycle
Final elongation72℃5min1time
Final hold12℃--

Electrophoresis

Li

LaneCloning PCR Products
11kb ladder
29/10 PCR E+X+RBS+mamB+S(922bp)
39/10 PCR X+RBS+mamL+His+S+P(340bp)
4-
51kb ladder
69/10 PCR X+RBS+mamQ+His+S+P(953bp)

DNA Purification

Honda and Yamaura

DNAConcentration/(µg/ml)260/280260/230
9/10 PCR E+X+RBS+mamB+S2001.842.15
9/10 PCR X+RBS+mamL+His+S+P1201.691.84
9/10 PCR X+RBS+mamQ+His+S+P2001.702.16

Restriction Enzyme Digestion

Shimizu

SampleDNA/(µl)Xba1/(µl)Pst1/(µl)Buffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/10 PCR E+X+RBS+mamB+S10113(H)1530
9/10 PCR X+RBS+mamL+His+S+P16.7113(H)8.330
9/10 PCR X+RBS+mamQ+His+S+P10113(H)1530

Ligation

Tatsui and Murada

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
9/10 DNA Purification mamQ1YCplac111115210
9/10 DNA Purification mamL1YCplac111115210
9/10 DNA Purification mamB1YCplac111115210
Control0YCplac11111529

Transformation

Tatsui and Murada

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/10 Ligation mamQ+YCplac111----
9/10 Ligation mamL+YCplac111----
9/10 Ligation mamB+YCplac111----

9/11

DNA Purification

No Name

DNAConcentration/(µg/ml)260/280260/230
9/10 Digestion mamB(X+P)651.680.51
9/10 Digestion mamL(X+P)301.950.81
9/10 Digestion mamQ(X+P)601.651.22

Electrophoresis

Murada

LaneColony PCR Products
LaneColony PCR Products
LaneColony PCR Products
LaneColony PCR Products
LaneColony PCR Products

Ligation

No Name

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
--------

Transformation

Yamauchi

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/11 Ligation mamL21012-
9/11 Ligation mamQ156-

Liquid Culture

Honda

Namemedium
9/8 Master Pcon+pSB1C3 Colony_3Plusgrow medium(CP)
9/10 Transformation mamL+YCplac111Plusgrow medium(AMP)
9/10 Transformation mamQ+YCplac111Plusgrow medium(AMP)

9/12

Colony PCR

Yamaura

NameⅠ

Electrophoresis

Yamaura

LaneColony PCR Products

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
9/11 Liquid Culture Pcon+pSB1C31101.832.63
9/11 Liquid Culture mamL+YCplac1113001.752.38
9/11 Liquid Culture mamQ+YCplac1111601.812.37

Restriction Enzyme Digestion

Yasuda

SampleDNA/(µl)Xba1/(µl)Pst1/(µl)Spe1/(µl)Buffur/(µl)BSAMilliQ/(µl)TOTAL/(µl)
9/12 Miniprep mamL+YCplac1116.71103(H)315.330
9/12 Miniprep mamQ+YCplac11112.51103(H)39.530
9/12 Miniprep Pcon+pSB1C318.10113(H)06.930

Ligation

Honda

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
9/12 Digestion mamB(E+S)39/8 Digestion pSB1C3(E+S)115010
9/10 mamB0.5YCplac1111152.510

DNA Purification

Tatsui

DNAConcentration/(µg/ml)260/280260/230
mamB(E+S)501.752.04

Transformation

Honda

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/12 Ligation mamB+pSB1C311011LB(CP)
9/12 Ligation mamB+YCplac11111011LB(AMP)

Colony PCR

Li

NameⅠNameⅡ
9/11 Transformation mamL+pSB1A2 Colony_19/11 Transformation mamL+pSB1A2 Colony_9
9/11 Transformation mamL+pSB1A2 Colony_29/11 Transformation mamL+pSB1A2 Colony_10
9/11 Transformation mamL+pSB1A2 Colony_39/11 Transformation mamQ+pSB1A2 Colony_1
9/11 Transformation mamL+pSB1A2 Colony_49/11 Transformation mamQ+pSB1A2 Colony_2
9/11 Transformation mamL+pSB1A2 Colony_59/11 Transformation mamQ+pSB1A2 Colony_3
9/11 Transformation mamL+pSB1A2 Colony_69/11 Transformation mamQ+pSB1A2 Colony_4
9/11 Transformation mamL+pSB1A2 Colony_79/11 Transformation mamQ+pSB1A2 Colony_5
9/11 Transformation mamL+pSB1A2 Colony_89/11 Transformation mamQ+pSB1A2 Colony_6

Electrophoresis

Li

LaneColony PCR ProductsⅠLaneColony PCR ProductsⅠ
19/12 mamL+pSB1A2 Colony_1109/12 mamL+pSB1A2 Colony_9
29/12 mamL+pSB1A2 Colony_2119/12 mamL+pSB1A2 Colony_10
39/12 mamL+pSB1A2 Colony_3129/12 mamQ+pSB1A2 Colony_1
49/12 mamL+pSB1A2 Colony_4139/12 mamQ+pSB1A2 Colony_2
59/12 mamL+pSB1A2 Colony_5149/12 mamQ+pSB1A2 Colony_3
69/12 mamL+pSB1A2 Colony_6159/12 mamQ+pSB1A2 Colony_4
79/12 mamL+pSB1A2 Colony_7169/12 mamQ+pSB1A2 Colony_5
89/12 mamL+pSB1A2 Colony_8179/12 mamQ+pSB1A2 Colony_6
91kb ladder

Colony PCR

Honda

NameⅠ
9/11 Transformation mamL+pSB1A2 Green_1
9/11 Transformation mamL+pSB1A2 Green_2
9/11 Transformation mamL+pSB1A2 Green_3
9/11 Transformation mamL+pSB1A2 Green_4
9/11 Transformation mamL+pSB1A2 Green_5
9/11 Transformation mamL+pSB1A2 Black_7
9/11 Transformation mamL+pSB1A2 Black_8
9/11 Transformation mamQ+pSB1A2 Black_1

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠ
19/12 mamL+pSB1A2 Green_1
29/12 mamL+pSB1A2 Green_2
39/12 mamL+pSB1A2 Green_3
49/12 mamL+pSB1A2 Green_4
59/12 mamL+pSB1A2 Green_5
69/12 mamL+pSB1A2 Black_7
79/12 mamL+pSB1A2 Black_8
89/12 mamQ+pSB1A2 Black_1
91kb ladder

Liquid Culture

Yamauchi

Namemedium
9/11 Transformation mamQ+pSB1A2 Colony_1Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_2Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_3Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_4Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_5Plusgrow medium(AMP)
9/11 Transformation mamQ+pSB1A2 Colony_6Plusgrow medium(AMP)

9/13

Miniprep

Tatsui

DNAConcentration/(µg/ml)260/280260/230
9/12 Liquid Culture mamQ+pSB1A2 Colony_1 Error---
9/12 Liquid Culture mamQ+pSB1A2 Colony_2 Error---
9/12 Liquid Culture mamQ+pSB1A2 Colony_3 Error---
9/12 Liquid Culture mamQ+pSB1A2 Colony_4802.022.76
9/12 Liquid Culture mamQ+pSB1A2 Colony_5702.133.41
9/12 Liquid Culture mamQ+pSB1A2 Colony_61101.961.70
9/12 Liquid Culture Pcon+pSB1C31101.961.70

PCR Labeling and Sequence

No Name

Target9/13 mamQ+pSB1A2 Colony_49/13 mamQ+pSB1A2 Colony_59/13 mamQ+pSB1A2 Colony_6Pcon+pSB1A29/13 mamQ+pSB1A2 Colony_49/13 mamQ+pSB1A2 Colony_59/13 mamQ+pSB1A2 Colony_6
Template/(µl)5464564
Big dye/(µl)0.50.50.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.751.751.75
Primer/(µl)0.5(VF)0.5(VF)0.5(VF)0.5(VF)0.5(VR)0.5(VR)0.5(VR)
MilliQ/(µl)2.751.753.753.752.751.753.75
TOTAL/(µl)10.510.510.510.510.510.510.5
Target9/12 mamQ+YCplac1119/12 mamL+YCplac1119/12 mamQ+YCplac1119/12 mamL+YCplac1119/12 mamQ+YCplac111
Template/(µl)31.531.53
Big dye/(µl)0.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.75
Primer/(µl)0.5(M13F)0.5(M13F)0.5(M13R)0.5(M13R)0.5(seqpLQB-2)
MilliQ/(µl)4.756.254.756.254.75
TOTAL/(µl)10.510.510.510.510.5

Gel Extraction

No Name

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
11kb ladder-
2--
39/12 Digestion mamQ+YCpla111(X+P)(992bp)-
4--
5--
6--
79/12 Digestion mamL+YCpla111(X+P)(340bp)-
8--
99/12 Digestion Pcon+pSB1C3(Nega)(5600bp)-
10--
119/12 Digestion Pcon+pSB1C3(S+P)(2070bp)-
DNAConcentration/(µg/ml)260/280260/230
9/12 Digestion mamL+YCpla111(X+P)101.000.70
9/12 Digestion Pcon+pSB1C3(S+P)1501.250.71

Restriction Enzyme Digestion

Li

SampleDNA/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)EcoR1/(µl)Buffur/(µl)BSAMilliQ/(µl)TOTAL/(µl)
9/13 Miniprep mamQ+pSB1A2 Colony_412.50.500.501.7(M)1.6016.8
9/13 Miniprep mamQ+pSB1A2 Colony_514.30.500.501.9(M)1.86019.06
9/13 Miniprep mamQ+pSB1A2 Colony_69.10.500.501.5(M)1.51.915
9/13 Miniprep mamQ+pSB1A2 Colony_6(Nega)300000.5(M)0.515

Ligation

Yasuda

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
9/13 Gel Extraction mamL(X+P)3Pcon+pSB1C3(S+P)115010

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
9/12 Liquid Culture Pcon+pSB1C32401.781.59

9/14

Gel Extraction

Honda

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
11kb ladder3
2--
39/13 mamQ Colony_4(X+P)16.8
4--
51kb ladder3
6--
79/13 mamQ Colony_5(X+P)19.06
8--
99/13 mamQ Colony_6(Nega)5
10--
119/13 mamQ Colony_6(X+P)15

Ligation

Tatsui

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
mamB(E+S)4pSB1C3(E+S)318016
mamL(X+P)3.3pSB1C3(E+S)0.715010

PCR

Tatsui

TargetE+X+RBS+mamB+S
2×KAPA HiFi Hotstart Ready Mix/(µl)12.5
Fw Primer/(µl)0.75(E+X+RBS+mamB)
Rv Primer/(µl)0.75(S+mamB)
9/9 DNA Purification Product/(µl)Trace
MilliQ/(µl)11
TOTAL/(µl)25
StepsTemparatureTimecycle
Pre Denature98℃5min1time
Denature98℃20sec25cycle
Annealing60℃15sec25cycle
Extension72℃15sec25cycle
Final elongation72℃5min1time
Final hold12℃--

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/14 Ligation Pcon+mamL+pSB1C311011LB(CP)
9/14 Ligation Pcon+mamL+pSB1C3(Nega)11011LB(CP)
9/14 Ligation mamB+pSB1C311011LB(CP)

9/15

Colony PCR

Tatsui

NameⅠNameⅡ
9/14 Transformation mamB+pSB1C3 Colony_19/14 Transformation mamB+pSB1C3 Colony_9
9/14 Transformation mamB+pSB1C3 Colony_29/14 Transformation mamB+pSB1C3 Colony_10
9/14 Transformation mamB+pSB1C3 Colony_39/14 Transformation mamB+pSB1C3 Colony_11
9/14 Transformation mamB+pSB1C3 Colony_49/14 Transformation mamB+pSB1C3 Colony_12
9/14 Transformation mamB+pSB1C3 Colony_59/14 Transformation mamB+pSB1C3 Colony_13
9/14 Transformation mamB+pSB1C3 Colony_69/14 Transformation mamB+pSB1C3 Colony_14
9/14 Transformation mamB+pSB1C3 Colony_79/14 Transformation mamB+pSB1C3 Colony_15
9/14 Transformation mamB+pSB1C3 Colony_8-

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠ
19/14 mamB+pSB1C3 Colony_1
29/14 mamB+pSB1C3 Colony_2
39/14 mamB+pSB1C3 Colony_3
49/14 mamB+pSB1C3 Colony_4
59/14 mamB+pSB1C3 Colony_5
69/14 mamB+pSB1C3 Colony_6
79/14 mamB+pSB1C3 Colony_7
89/14 mamB+pSB1C3 Colony_8
91kb ladder
109/14 mamB+pSB1C3 Colony_9
119/14 mamB+pSB1C3 Colony_10
129/14 mamB+pSB1C3 Colony_11
139/14 mamB+pSB1C3 Colony_12
149/14 mamB+pSB1C3 Colony_13
159/14 mamB+pSB1C3 Colony_14
169/14 mamB+pSB1C3 Colony_15

Colony PCR

No Name

NameⅠ
9/14 Transformation Pcon+mamL+SB1C3 Colony_1
9/14 Transformation mamB+pSB1C3 Colony_9

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
mamB+YCplac11111011LB(AMP)
Pcon+mamL+pSB1C311011LB(CP)

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
mamB+pSB1C3 Colony_9---
mamB+pSB1C3 Colony_10---
mamB+pSB1C3 Colony_11---
mamL+pSB1A2 Colony_11701.741.41
mamL+pSB1A2 Colony_21401.951.43
mamL+pSB1A2 Colony_31351.951.60
mamL+pSB1A2 Colony_4---
mamL+pSB1A2 Colony_5---
mamL+pSB1A2 Colony_6---

Liquid Culture

Honda

Namemedium
9/14 Transformation Pcon+mamL+pSB1C3 Colony_1Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_9Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_10Plusgrow medium(CP)
9/14 Transformation mamB+pSB1C3 Colony_11Plusgrow medium(CP)

Restriction Enzyme Digestion

Li

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSAMilliQ/(µl)Buffur/(µl)TOTAL/(µl)
9/15 DNA Purification mamB251010003(H)30
9/15 Mniprep mamL+pSB1A2 Colony_111.80101310.23(M)30
9/15 Mniprep mamL+pSB1A2 Colony_1(Nega)0.6000018.41(M)10
9/15 Mniprep mamL+pSB1A2 Colony_214.3010137.73(M)30
9/15 Mniprep mamL+pSB1A2 Colony_2(Nega)0.7000018.31(M)10
9/15 Mniprep mamL+pSB1A2 Colony_314.8010137.23(M)30
9/15 Mniprep mamL+pSB1A2 Colony_3(Nega)0.7000018.31(M)10
9/15 Mniprep mamL+pSB1A2 Colony_413.3010138.73(M)30
9/15 Mniprep mamL+pSB1A2 Colony_4(Nega)0.7000018.31(M)10
9/13 Pcon+pSB1C3200011053(H)30
9/13 Pcon+pSB1C3(Nega)10000063(H)10

PCR

Honda

TargetmamB Colony_10mamB Colony_11
2×PrimeSTAR Max Premix/(µl)2525
Fw mamB Mutation Primer/(µl)11
Rv mamB Mutation Primer/(µl)11
9/15 Miniprep mamB+pSB1C3/(µl)Colony_10(Trace)Colony_11(Trace)
MilliQ/(µl)2323
TOTAL/(µl)5050
StepsTemparatureTimecycle
Pre Denature98℃2min1time
Denature98℃10sec30cycle
Annealing55℃15sec30cycle
Extension72℃15sec30cycle
Final elongation72℃5min1time
Final hold12℃--

9/16

PCR Labeling and Sequence

No Name

Target9/15 Pcon+mamL+pSB1C39/15 mamL+pSB1A2 Colony_19/15 mamL+pSB1A2 Colony_29/15 mamL+pSB1A2 Colony_39/15 mamL+pSB1A2 Colony_49/15 mamB+pSB1C3 Colony_99/15 mamB+pSB1C3 Colony_109/15 mamB+pSB1C3 Colony_11
Template/(µl)1.82.352.93.03.01.141.21.2
Big dye/(µl)0.50.50.50.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.751.751.751.75
Primer(VF)/(µl)0.50.50.50.50.50.50.50.5
MilliQ/(µl)5.955.44.854.754.756.616.556.55
TOTAL/(µl)10.510.510.510.510.510.510.510.5
Target9/15 Pcon+mamL+pSB1C39/15 mamL+pSB1A2 Colony_19/15 mamL+pSB1A2 Colony_29/15 mamL+pSB1A2 Colony_39/15 mamB+pSB1C3 Colony_99/15 mamB+pSB1C3 Colony_109/15 mamB+pSB1C3 Colony_11
Template/(µl)1.82.352.93.01.141.21.2
Big dye/(µl)0.50.50.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.751.751.75
Primer(VR)/(µl)0.50.50.50.50.50.50.5
MilliQ/(µl)5.955.44.854.756.616.556.55
TOTAL/(µl)10.510.510.510.510.510.510.5
StepsTemparatureTimecycle
Pre Denature96℃2min1time
Denature96℃10sec30cycle
Annealing55℃5sec30cycle
Extension72℃4min30cycle
Final elongation72℃1min1time
Final hold4℃--

Gel Extraction

Honda

LaneRestriction Enzyme Digestion ProductⅠVolume/(µl)
11kb ladder-
21kb ladder-
39/15 mamL Colony_1(X+P)-
4--
59/15 mamL Colony_2(Nega)-
6--
79/15 mamL Colony_2(X+P)-
81kb ladder-
91kb ladder-
109/15 mamL Colony_3(Nega)-
119/15 mamL Colony_3(X+P)-
LaneRestriction Enzyme Digestion ProductⅡVolume/(µl)
11kb ladder-
2--
3--
49/15 mamQ(X+P)-
5--
69/15 mamQ(Nega)-
DNAConcentration/(µg/ml)260/280260/230
mamL Colony_1451.690.60
mamL Colony_2601.330.70
mamL Colony_3401.760.08
mamQ651.350.83

Dephosphorylation

Honda

Sample/(µL)
9/15 Digestion Pcon+pSB1C3(S+P)-
9/15 Gel Extraction Pcon+pSB1C3(S+P)-

DNA Purification

Honda

DNAConcentration/(µg/ml)260/280260/230
9/15 Mutation PCR mamB+pSB1C3 Colony_102251.641.74
9/15 Mutation PCR mamB+pSB1C3 Colony_112051.591.76

Gel Extraction

Yamauchi

LaneSampleⅠVolume/(µl)
11kb ladder-
2--
39/15 Digestion Pcon+pSB1C3(S+P)-
4--
51kb ladder-
6--
79/15 Gel Extraction Pcon+pSB1C3(S+P)-
8--
91kb ladder-
10--
11--

Transformation

Honda and Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/16 DNA Purification mamB+pSB1C3 Colony_1011011LB(CP)
9/16 DNA Purification mamB+pSB1C3 Colony_1111011LB(CP)

Restriction Enzyme Digestion

No Name

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSAMilliQ/(µl)Buffur/(µl)TOTAL/(µl)
9/15 mamQ+pSB1A2 Colony_426.70202617.36(M)60
9/15 mamQ+pSB1A2 Colony_4(Nega)0.7000017.31(M)10
9/15 Pcon+mamL+pSB1C31500220356(H)60
9/15 Pcon+mamL+pSB1C3(Nega)0.5000008.51(H)10

PCR

Tatsui

TargetE+Pcon+X+RBS+mamB+His+S(*2)
2×KAPA HiFi Hotstart Ready Mix/(µl)12.5
Fw E+Pcon+X+RBS+mamB/(µl)0.75
Rv S+His+mamB/(µl)0.75
9/9 DNA Purification mamB/(µl)Trace
MilliQ/(µl)11
TOTAL/(µl)25

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
9/15 mamB+pSB1C3 Colony_93501.681.81
9/15 mamB+pSB1C3 Colony_103251.651.50
9/15 mamB+pSB1C3 Colony_113401.671.68
Pcon+mamL+pSB1C32201.752.09

9/17

Colony PCR

Tatsui

NameⅠ
9/16 Transformation Mutation mamB Colony_11_1
9/16 Transformation Mutation mamB Colony_11_2
9/16 Transformation Mutation mamB Colony_11_3
9/16 Transformation Mutation mamB Colony_11_4
9/16 Transformation Mutation mamB Colony_10_1
9/16 Transformation Mutation mamB Colony_10_2
9/16 Transformation Mutation mamB Colony_10_3
9/16 Transformation Mutation mamB Colony_10_4

Miniprep

Tatsui and Yamauchi

DNAConcentration/(µg/ml)260/280260/230
9/15 Pcon+pSB1C3 Colony_31101.661.48

Gel Extraction

Yamauchi

LaneSampleⅠVolume/(µl)
19/16 Mutation PCR Pcon+mamB+His-
2Pcon+mamL(Nega)-
3Pcon+mamL(S+P)-
41kb ladder-
59/16 Mutation PCR Pcon+mamB+His-
6--
7mamQ+pSB1A2 Colony_4(X+P)-
81kb ladder-
9mamQ(Nega)-
10--
11mamQ+pSB1A2 Colony_4(X+P)-
DNAConcentration/(µg/ml)260/280260/230
Pcon+mamB+His2451.872.10
mamQ+pSB1A2 Colony_4(X+P)351.870.580
Pcon+mamL(S+P)285 Error1.140.85

Ligation

Tatsui

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
mamQ(X+P)3Pcon+mamL(S+P)115010

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/17 Ligation Pcon+mamL+mamQ+pSB1C311011LB(CP)

Restriction Enzyme Digestion

Tatsui

SampleDNA/(µl)EcoR1/(µl)Spe1/(µl)Xba1/(µl)Pst1/(µl)BSABuffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/17 Gel Extraction Pcon+mamB+His16.3220006(H)33.760
9/17 Gel Extraction Pcon+mamB+His(Nega)1000001(H)810
9/17 Miniprep Pcon+pSB1C336.4020206(H)13.660
9/17 Miniprep Pcon+pSB1C3(Nega)1000001(H)810

Liquid Culture

Tatsui

Namemedium
Mutation mamB Colony_10_3Plusgrow medium(CP)
Mutation mamB Colony_10_4 Plusgrow medium(CP)
Mutation mamB Colony_11_3Plusgrow medium(CP)
Mutation mamB Colony_11_4Plusgrow medium(CP)
mamQ+pSB1A2 Colony_4Plusgrow medium(AMP)*2
Pcon+mamL Colony_1Plusgrow medium(CP)*3.5

9/18

Miniprep

Tatsui and Honda

DNAConcentration/(µg/ml)260/280260/230
9/17 Liquid Culture Mutation mamB Colony_10_33551.692.02
9/17 Liquid Culture Mutation mamB Colony_10_42651.521.55
9/17 Liquid Culture Mutation mamB Colony_11_33551.621.77
9/17 Liquid Culture Mutation mamB Colony_11_42501.621.51
9/17 Liquid Culture mamQ+pSB1A2 Colony_42901.411.10
9/17 Liquid Culture Pcon+mamL+pSB1C3 Colony_1851.471.56

PCR Labeling and Sequence

No Name

Target9/18 Miniprep Mutation mamB Colony_10_39/18 Miniprep Mutation mamB Colony_10_49/18 Miniprep Mutation mamB Colony_11_39/18 Miniprep Mutation mamB Colony_11_49/18 Miniprep Mutation mamB Colony_10_39/18 Miniprep Mutation mamB Colony_10_49/18 Miniprep Mutation mamB Colony_11_39/18 Miniprep Mutation mamB Colony_11_4
Template/(µl)1.11.51.11.61.11.51.11.6
Big dye/(µl)22222222
5×Buffer/(µl)11111111
Primer/(µl)0.16(VF)0.16(VF)0.16(VF)0.16(VF)0.16(VR)0.16(VR)0.16(VR)0.16(VR)
MilliQ/(µl)5.745.345.745.245.745.345.745.24
TOTAL/(µl)1010101010101010
Target9/18 Miniprep Mutation mamB Colony_10_39/18 Miniprep Mutation mamB Colony_10_49/18 Miniprep Mutation mamB Colony_11_39/18 Miniprep Mutation mamB Colony_11_4
Template/(µl)1.11.51.11.6
Big dye/(µl)2222
5×Buffer/(µl)1111
Primer/(µl)0.16(pQLB seq_2)0.16(pQLB seq_2)0.16(pQLB seq_2)0.16(pQLB seq_2)
MilliQ/(µl)5.745.345.745.24
TOTAL/(µl)10101010
StepsTemparatureTimecycle
Pre Denature96℃2min1time
Denature96℃10sec30cycle
Annealing50℃5sec30cycle
Extension60℃4min30cycle
Final elongation4℃1min1time
Final hold4℃--

Colony PCR

Tatsui

NameⅠNameⅡ
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_19/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_9
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_29/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_10
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_39/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_11
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_49/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_12
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_59/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_13
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_69/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_14
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_79/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_15
9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_89/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony_16

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠ
19/18 Pcon+mamL+mamQ+pSB1C3 Colony_1
29/18 Pcon+mamL+mamQ+pSB1C3 Colony_2
39/18 Pcon+mamL+mamQ+pSB1C3 Colony_3
49/18 Pcon+mamL+mamQ+pSB1C3 Colony_4
59/18 Pcon+mamL+mamQ+pSB1C3 Colony_5
69/18 Pcon+mamL+mamQ+pSB1C3 Colony_6
79/18 Pcon+mamL+mamQ+pSB1C3 Colony_7
89/18 Pcon+mamL+mamQ+pSB1C3 Colony_8
91kb ladder
109/18 Pcon+mamL+mamQ+pSB1C3 Colony_9
119/18 Pcon+mamL+mamQ+pSB1C3 Colony_10
129/18 Pcon+mamL+mamQ+pSB1C3 Colony_11
139/18 Pcon+mamL+mamQ+pSB1C3 Colony_12
149/18 Pcon+mamL+mamQ+pSB1C3 Colony_13
159/18 Pcon+mamL+mamQ+pSB1C3 Colony_14
169/18 Pcon+mamL+mamQ+pSB1C3 Colony_15
179/18 Pcon+mamL+mamQ+pSB1C3 Colony_16

DNA Purification

Tatsui

DNAConcentration/(µg/ml)260/280260/230
9/17 Digestion Pcon+mamB+His(E+S)165--

Gel Extraction

Tatsui

LaneSampleⅠVolume/(µl)
1--
21kb ladder-
39/17 Digestion Pcon+pSB1C3(Nega)-
49/17 Digestion Pcon+pSB1C3(S+P)-
5--
6--
LaneSampleⅡVolume/(µl)
1--
2--
31kb ladder-
49/17 Digestion Pcon+pSB1C3(S+P)-
5--
6--
DNAConcentration/(µg/ml)260/280260/230
9/17 Digestion Pcon+pSB1C3---

Ligation

Tatsui

Insert/(µL)Vector/(µL)T4 Ligase/(µl)10*Buffer/(µl)MilliQ/(µl)Total/(µl)
mamQ(X+P)69/18 Gel Extraction Pcon+pSB1C3(S+P)Trace0.512.510
9/18 DNA Purification Pcon+mamB+His(E+S)59/14 pSB3T5(E+S)20.511.510

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/18 Ligation Pcon+mamQ+pSB1C311011LB(CP)
9/18 Ligation Pcon+mamB+His+pSB3T511011LB(TET)

Liquid Culture

Tatsui

Namemedium
pSB3T5Plusgrow medium(TET)*2
9/17 Transformation Pcon+mamL+mamQ+pSB1C3Plusgrow medium(CP)

Restriction Enzyme Digestion

No Name

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/18 Miniprep Mutation mamB Colony_10_311.202020.88(D)5680
9/18 Miniprep Mutation mamB Colony_10_3(Nega)1000001(D)810
9/18 Miniprep Mutation mamB Colony_10_415.102020.88(D)52.180
9/18 Miniprep Mutation mamB Colony_10_4(Nega)1000001(D)810
9/18 Miniprep Mutation mamB Colony_11_311.202020.88(D)5680
9/18 Miniprep Mutation mamB Colony_11_3(Nega)1000001(D)810
9/18 Miniprep Mutation mamB Colony_11_41602020.88(D)51.280
9/18 Miniprep Mutation mamB Colony_11_4(Nega)1000001(D)810
9/18 Miniprep mamQ+pSB1A234.50505220(D)133.5200
9/18 Miniprep mamQ+pSB1A2(Nega)1000001(D)810
9/17 Miniprep Pcon+pSB1C34000220.88(B)27.280
9/17 Miniprep Pcon+pSB1C3(Nega)1000001(B)810
9/18 Miniprep Pcon+mamL+pSB1C34700220.88(B)20.280
9/18 Miniprep Pcon+mamL+pSB1C3(Nega)1000001(B)810

9/19

Miniprep

Tatsui

DNAConcentration/(µg/ml)260/280260/230
9/18 Liquid Culture pSB3T51151.521.22
9/18 Liquid Culture Pcon+mamL+mamQ+pSB1C3751.591.20

PCR Labeling and Sequence

No Name

Target9/19 Miniprep Pcon+mamL+mamQ+pSB1C39/19 Miniprep Pcon+mamL+mamQ+pSB1C39/19 Miniprep Pcon+mamL+mamQ+pSB1C39/19 Miniprep Pcon+mamL+mamQ+pSB1C3
Template/(µl)3.53.53.53.5
Big dye/(µl)0.50.50.50.5
5×Buffer/(µl)1.751.751.751.75
Primer/(µl)0.5(VF)0.5(VR)0.5(seq_1)0.5(seq_2)
MilliQ/(µl)4.254.254.254.25
TOTAL/(µl)10.510.510.510.5
StepsTemparatureTimecycle
Pre Denature96℃2min1time
Denature96℃10sec30cycle
Annealing50℃5sec30cycle
Extension60℃4min30cycle
Final elongation4℃1min1time
Final hold4℃--

Gel Extraction

Tatsui

LaneSampleⅠVolume/(µl)
1--
2--
39/18 Digestion Mutation mamB Colony_10_3(X+P)40
49/18 Digestion Mutation mamB Colony_10_3(Nega)10
51kb ladder-
6--
79/18 Digestion Mutation mamB Colony_10_3(X+P)40
8--
91kb ladder-
109/18 Digestion Mutation mamB Colony_10_4(Nega)10
119/18 Digestion Mutation mamB Colony_10_4(X+P)40
LaneSampleⅡVolume/(µl)
19/18 Digestion mamQ+pSB1A2(X+P)200
2--
3--
4--
5--
6--
LaneSampleⅢVolume/(µl)
1100b ladder-
29/18 Digestion Pcon+pSB1C3(Nega)10
39/18 Digestion Pcon+pSB1C3(S+P)80
DNAConcentration/(µg/ml)260/280260/230
mamB Colony_10_3(X+P)---
mamB Colony_10_4(X+P)---
mamQ+pSB1A2(X+P)---
Pcon+pSB1C3(S+P)---

Colony PCR

Tatsui

NameⅠ
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_1
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_2
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_3
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_4
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_5

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠ
1-
2-
3Pcon+mamQ+pSB1C3 Colony_1
4Pcon+mamQ+pSB1C3 Colony_2
5Pcon+mamQ+pSB1C3 Colony_3
6Pcon+mamQ+pSB1C3 Colony_4
7Pcon+mamQ+pSB1C3 Colony_5
8-

Restriction Enzyme Digestion

Tatsui

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/19 Miniprep Pcon+mamL+mamQ+pSB1C334.700220.88(B)32.580
9/19 Miniprep pSB3T553.320200.88(B)1480
9/19 Miniprep pSB3T5(Nega)1000001(B)810

PCR

Tatsui

TargetPcon+mamB+His
KOD Plus/(µl)1
10×Buffer/(µl)5
2mM dNTPS/(µl)5
25mM MgSO4/(µl)3
Fw Primer(E+Pcon+X+RBS+mamB)/(µl)2
Rv Primer(S+His+mamB)/(µl)2
Template/(µl)0.5
MilliQ/(µl)31.5
Total/(µl)50
StepsTemparatureTimecycle
Pre Denature98℃2min1time
Denature98℃10sec25cycle
Annealing55℃15sec25cycle
Extension72℃1min25cycle
Final elongation72℃5min1time
Final hold12℃--

DNA Purification

Honda

DNAConcentration/(µg/ml)260/280260/230
9/18 Digestion mamL+pSB1C3(S+P)551.661.66
9/19 PCR Pcon+mamB+His1501.631.52

Restriction Enzyme Digestion

Honda

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/19 DNA Purification Pcon+mamB+His2010100.88(B)49.280

Liquid Culture

Tatsui

Namemedium
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_1Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_2Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_3Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_4Plusgrow medium(TET)
9/18 Transformation Pcon+mamB+His+pSB3T5 Colony_5Plusgrow medium(TET)

Colony PCR

Tatsui

NameⅠ
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_1
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_2
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_3
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_4
9/18 Transformation Pcon+mamQ+pSB1C3 Colony_5

9/20

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠ
1-
2100b ladder
3Pcon+mamQ+pSB1C3 Colony_1
4Pcon+mamQ+pSB1C3 Colony_2
5Pcon+mamQ+pSB1C3 Colony_3
6Pcon+mamQ+pSB1C3 Colony_4
7Pcon+mamQ+pSB1C3 Colony_5

Miniprep

Tatsui

DNAConcentration/(µg/ml)260/280260/230
9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony_3---
9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony_5---

Colony PCR

Tatsui

NameⅠ
Pcon+mamB+His+pSB3T5 Colony_1(VF+VR)
Pcon+mamB+His+pSB3T5 Colony_1(seq_4+VR)

Electrophoresis

Yasuda

LaneColony PCR ProductsⅠ
1-
2100b ladder
3100b ladder
4Pcon+mamB+His+pSB3T5 Colony_1(VF+VR)
5Pcon+mamB+His+pSB3T5 Colony_1(seq_4+VR)
6100b ladder

Restriction Enzyme Digestion

Honda

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffur/(µl)MilliQ/(µl)TOTAL/(µl)
9/18 Miniprep Mutation mamB Colony_10_311.202020.88(D)5680
9/18 Miniprep Mutation mamB Colony_10_3(Nega)1000001(D)810
9/18 Miniprep Mutation mamB Colony_10_415.102020.88(D)5683.9
9/18 Miniprep Mutation mamB Colony_10_4(Nega)1101001(D)810

Gel Extraction

Tatsui

LaneSampleⅠVolume/(µl)
1--
2100b ladder-
3Digestion Mutation mamB Colony_10_3(X+P)-
4--
5--
6--
7Digestion Mutation mamB Colony_11_3(X+P)-
8Digestion Mutation mamB Colony_11_3(Nega)-
91kb ladder-
10--
11Digestion Mutation mamB Colony_11_3(X+P)-
LaneSampleⅡVolume/(µl)
1--
2100b ladder-
39/19 Digestion pSB3T5(Nega)-
49/19 Digestion pSB3T5(E+S)-
LaneSampleⅢVolume/(µl)
1--
2100b ladder-
3Digestion Mutation mamB Colony_11_4(X+P)-
4Digestion Mutation mamB Colony_11_4(Nega)-
DNAConcentration/(µg/ml)260/280260/230
mamB Colony_10_3(X+P)60--
mamB Colony_11_3(X+P)851.382.04
mamB Colony_11_4(X+P)600.582.32
pSB3T5(E+S)700.442.04

DNA Purification

Tatsui

DNAConcentration/(µg/ml)260/280260/230
Pcon+mamL+mamQ(S+P)---

Ligation

Tatsui

Insert/(µL)Vector/(µL)T4 Ligase/(µl)2*Buffer/(µl)MilliQ/(µl)Total/(µl)
mamB Colony_10_3(X+P)1.7Pcon+mamL+mamQ(S+P)2.315010
mamB Colony_10_4(X+P)1.7Pcon+mamL+mamQ(S+P)2.315010
mamB Colony_11_3(X+P)1.7Pcon+mamL+mamQ(S+P)2.315010
mamB Colony_11_4(X+P)1.7Pcon+mamL+mamQ(S+P)2.315010
pSB3T5(E+S)1.4Pcon+mamB+His(E+S)2.615010

Transformation

Tatsui

Sample/(µL)CompetentCells/(µl)Total/(µl)Medium
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_10_311011LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_10_411011LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_11_311011LB(CP)
9/20 Ligation Pcon+mamL+mamQ+mamB Colony_11_411011LB(CP)
9/20 Ligation Pcon+mamB+His11011LB(TET)

9/22

Colony PCR

Tatsui

NameⅠNameⅡNameⅢNameⅣ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_1
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_2
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_3
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_4
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_5
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_6
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_7
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_8
NameⅤNameⅥ
9/20 Transformation Pcon+mamB+His Colony_19/19 Transformation Pcon+mamB+His Colony_1
9/20 Transformation Pcon+mamB+His Colony_29/19 Transformation Pcon+mamB+His Colony_2
9/20 Transformation Pcon+mamB+His Colony_39/19 Transformation Pcon+mamB+His Colony_3
9/20 Transformation Pcon+mamB+His Colony_4-
9/20 Transformation Pcon+mamB+His Colony_5-

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠColony PCR ProductsⅡ
1Pcon+mamL+mamQ+mamB Colony_10_3_1Pcon+mamL+mamQ+mamB Colony_11_3_1
2Pcon+mamL+mamQ+mamB Colony_10_3_2Pcon+mamL+mamQ+mamB Colony_11_3_2
3Pcon+mamL+mamQ+mamB Colony_10_3_3Pcon+mamL+mamQ+mamB Colony_11_3_3
4Pcon+mamL+mamQ+mamB Colony_10_3_4Pcon+mamL+mamQ+mamB Colony_11_3_4
5Pcon+mamL+mamQ+mamB Colony_10_3_5Pcon+mamL+mamQ+mamB Colony_11_3_5
6Pcon+mamL+mamQ+mamB Colony_10_3_6Pcon+mamL+mamQ+mamB Colony_11_3_6
7Pcon+mamL+mamQ+mamB Colony_10_3_7Pcon+mamL+mamQ+mamB Colony_11_3_7
8Pcon+mamL+mamQ+mamB Colony_10_3_8Pcon+mamL+mamQ+mamB Colony_11_3_8
9100b ladder100b ladder
10Pcon+mamL+mamQ+mamB Colony_10_4_1Pcon+mamL+mamQ+mamB Colony_11_4_1
11Pcon+mamL+mamQ+mamB Colony_10_4_2Pcon+mamL+mamQ+mamB Colony_11_4_2
12Pcon+mamL+mamQ+mamB Colony_10_4_3Pcon+mamL+mamQ+mamB Colony_11_4_3
13Pcon+mamL+mamQ+mamB Colony_10_4_4Pcon+mamL+mamQ+mamB Colony_11_4_4
14Pcon+mamL+mamQ+mamB Colony_10_4_5Pcon+mamL+mamQ+mamB Colony_11_4_5
15Pcon+mamL+mamQ+mamB Colony_10_4_6Pcon+mamL+mamQ+mamB Colony_11_4_6
16Pcon+mamL+mamQ+mamB Colony_10_4_7Pcon+mamL+mamQ+mamB Colony_11_4_7
17Pcon+mamL+mamQ+mamB Colony_10_4_8Pcon+mamL+mamQ+mamB Colony_11_4_8

Ligation

No Name

Insert/(µL)Vector/(µL)T4 Ligase/(µl)10*Buffer/(µl)MilliQ/(µl)Total/(µl)
mamB Colony_10_3(X+P)1Pcon+mamL+mamQ(S+P)7.50.51010

Restriction Enzyme Digestion

Tatsui

SampleDNA/(µl)EcoR1/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffur/(µl)CLAP/(µl)MilliQ/(µl)TOTAL/(µl)
9/19 Miniprep Pcon+mamL+mamQ+pSB1C334.800220.88(B)131.480

Colony PCR

Honda

NameⅠNameⅡNameⅢNameⅣ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_19/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_1
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_29/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_2
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_39/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_3
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_49/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_4
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_59/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_5
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_69/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_6
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_79/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_7
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_3_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_10_4_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_89/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_4_8
NameⅤ
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(VF+VR)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(seq_4+VR)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(L_F+B_R)
9/20 Transformation Pcon+mamL+mamQ+mamB Colony_11_3_2(Q_F+B_R)

Electrophoresis

Tatsui

LaneColony PCR ProductsⅠColony PCR ProductsⅡColony PCR ProductsⅢ
1Pcon+mamL+mamQ+mamB Colony_10_3_1Pcon+mamL+mamQ+mamB Colony_11_3_1-
2Pcon+mamL+mamQ+mamB Colony_10_3_2Pcon+mamL+mamQ+mamB Colony_11_3_2-
3Pcon+mamL+mamQ+mamB Colony_10_3_3Pcon+mamL+mamQ+mamB Colony_11_3_3-
4Pcon+mamL+mamQ+mamB Colony_10_3_4Pcon+mamL+mamQ+mamB Colony_11_3_4-
5Pcon+mamL+mamQ+mamB Colony_10_3_5Pcon+mamL+mamQ+mamB Colony_11_3_5-
6Pcon+mamL+mamQ+mamB Colony_10_3_6Pcon+mamL+mamQ+mamB Colony_11_3_6-
7Pcon+mamL+mamQ+mamB Colony_10_3_7Pcon+mamL+mamQ+mamB Colony_11_3_7-
8Pcon+mamL+mamQ+mamB Colony_10_3_8Pcon+mamL+mamQ+mamB Colony_11_3_8-
9100b ladder100b ladder100b ladder
10Pcon+mamL+mamQ+mamB Colony_10_4_1Pcon+mamL+mamQ+mamB Colony_11_4_19/19 Transformation Pcon+mamB+His Colony_1
11Pcon+mamL+mamQ+mamB Colony_10_4_2Pcon+mamL+mamQ+mamB Colony_11_4_29/19 Transformation Pcon+mamB+His Colony_2
12Pcon+mamL+mamQ+mamB Colony_10_4_3Pcon+mamL+mamQ+mamB Colony_11_4_39/19 Transformation Pcon+mamB+His Colony_3
13Pcon+mamL+mamQ+mamB Colony_10_4_4Pcon+mamL+mamQ+mamB Colony_11_4_4Pcon+mamL+mamQ+mamB Colony_11_3_2(VF+VR)
14Pcon+mamL+mamQ+mamB Colony_10_4_5Pcon+mamL+mamQ+mamB Colony_11_4_5Pcon+mamL+mamQ+mamB Colony_11_3_2(seq_4+VR)
15Pcon+mamL+mamQ+mamB Colony_10_4_6Pcon+mamL+mamQ+mamB Colony_11_4_6Pcon+mamL+mamQ+mamB Colony_11_3_2(L_F+B_R)
16Pcon+mamL+mamQ+mamB Colony_10_4_7Pcon+mamL+mamQ+mamB Colony_11_4_7Pcon+mamL+mamQ+mamB Colony_11_3_2(Q_F+B_R)
17Pcon+mamL+mamQ+mamB Colony_10_4_8Pcon+mamL+mamQ+mamB Colony_11_4_8-

Miniprep

Tatsui

DNAConcentration/(µg/ml)260/280260/230
Pcon+mamQ+pSB1C3 Colony_3851.510.79
Pcon+mamQ+pSB1C3 Colony_5601.941.12

9/27

Western Blotting

Honda

Electrophoresis:200V 25mA 30min

Transcription:50V 200mA 73min

Make the 20% acrilamid gel apray the following

LaneSample
1-5 9-12Sample buffer
6T7-RBS-1C3(control)
7pL(1C3)
8protein marker

Liquid Culture

Sample
pLQB(9/22 master)
pLQ(9/22 master)

9/28

Fixation

Honda

25% Glyceraldehyde/(ml)Phosphate buffer/(ml)MilliQ/(ml)Total/(ml)
1.255.03.7510.0

9/29

Liquid Culture

Honda

Sample
9/22 master pLQB
9/22 master pLQ
9/22 master pL
9/22 master pQ-3
9/22 master pQ-5
9/20 master pB-22
9/20 master pB-23

Fixation

Honda

Osmic acid/(ml)Phosphate buffer/(ml)Total/(ml)
448

9/30

Miniprep

Tatsui

DNAConcentration/(µg/ml)260/280260/230
9/29 Liquid Culture pLQB---
9/29 Liquid Culture pLQ---

Western Blotting

Honda

Electrophoresis:200V 25mA 1h30min

Transcription:50V 200mA 73min

Make the 20% acrilamid gel apray the following

LaneSample
2Marker
3pLQB
4pLQ
5pL
6pQ-3
7pQ-5
8pB
9GTP-His
10T7-RBS-1C3(control)
11Sample buffer

Liquid Culture

Honda

Sample
9/22 master pLQB
9/22 master pQ(3)

10/1

Ethanol Precipitation

Tatsui

Sample
9/28 M pL(1C3)(VF)
9/28 M pL(1C3)(VR)
9/22 M pB(22)(VF)
9/22 M pB(22)(VR)
9/22 pB(23)(VF)
9/22 pB(23)(VR)
10/1 M pQ(3)(VF)
10/1 M pQ(3)(VR)
10/1 M pLQB(VF)
10/1 M pLQB(seq-1)
10/1 M pLQB(seq-2)
10/1 M pLQB(seq-3)
10/1 M pLQB(seq-4)
10/1 M pLQB(VR)

Liquid Culture

Honda

Sample
9/22 master Plate pLQB
9/22 master Plate pQ(3)

Restriction Enzyme Digestion

SampleDNA/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)BSABuffer/(µl)MilliQ/(µl)TOTAL/(µl)
9/23 pQ30303194490
pQ(control)100001810
9/2 Pcon(1C3)30033194490
9/2 Pcon(1C3)(control)100001810

Miniprep

Honda

DNAConcentration/(µg/ml)260/280260/230
9/29 Liquid Culture pLQB---
9/29 Liquid Culture pQ(3)---
9/29 Liquid Culture pQ(22)---
9/29 Liquid Culture pQ(23)---

Sequence PCR

Target9/28 M pL(1C3)(VF)9/28 M pL(1C3)(VR)9/22 M pB(22)(VF)9/22 M pB(22)(VR)9/22 pB(23)(VF)9/22 pB(23)(VR)10/1 M pQ(3)(VF)10/1 M pQ(3)(VR)10/1 M pLQB(VF)10/1 M pLQB(seq-1)10/1 M pLQB(seq-2)10/1 M pLQB(seq-3)10/1 M pLQB(seq-4)10/1 M pLQB(VR)
Template/(µl)4.74.71.61.63.23.255555555
Big dye/(µl)0.50.50.50.50.50.50.50.50.50.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.751.751.751.751.751.751.751.751.751.75
Primer(VF)/(µl)0.50.50.50.50.50.50.50.50.50.50.50.50.50.5
MilliQ/(µl)3.053.056.156.154.554.552.752.752.752.752.752.752.752.75
TOTAL/(µl)10.510.510.510.510.510.510.510.510.510.510.510.510.510.5
StepsTemparatureTimecycle
Pre Denature96℃2min1time
Denature96℃10sec30cycle
Annealing55℃5sec30cycle
Extension60℃4min30cycle
Final elongation4℃1min1time
Final hold4℃infinity-

Gel Extraction

(1)

LaneSampleⅠVolume/(µl)
1blank-
21400b ladder-
3His-OE-1-
41kb ladder-
5750b ladder-
6His-OE-2-

(2)

LaneSampleⅡVolume/(µl)
3,6pQ(X,P)-
4100b ladder-

(3)

LaneSampleⅢVolume/(µl)
3,6Pcon 1C3(S,P)-
41kb ladder-

(4)

LaneSampleⅣVolume/(µl)
3DiDECARB-4-
6DiDECARB-5-
2DiDECARB-4(control)-
5DiDECARB-5(control)-
41kb ladder-

10/2

Miniprep

DNAConcentration/(µg/ml)260/280260/230
pLQB3901.701.20
pLQ3191.640.92
j044502551.591.04

10/3

PCR

Honda

TargetVolume
10×buffur for KOD Plus Ver.2/(µl)5
2mMdNTPs/(µl)5
25mM MgSO4/(µl)3
VF Primer/(µl)1.5
VR Primer/(µl)1.5
template/(µl)1
KOD-plus/(µl)1
MilliQ/(µl)33
TOTAL/(µl)50

DNA Purification

DNAConcentration/(µg/ml)260/280260/230
10/3 PB39.81.750.92

Digestion

SampleDNA/(µl)EcoRI/(µl)Spe1/(µl)BSABuffer/(µl)MilliQ/(µl)TOTAL/(µl)
10/3 Column pB15.8110.22-20

Restriction Enzyme Digestion

Honda

SampleDNA/(µl)Xba1/(µl)Pst1/(µl)BSABuffer/(µl)MilliQ/(µl)TOTAL/(µl)
DiDECARB-410220.4421.640
DiDECARB-4(control)10001810
DiDECARB-510220.4421.640
DiDECARB-5(control)10001810

Gel Extraction

Tatsui

DNAConcentration/(µg/ml)260/280260/230
pSB1C3(X,P)-49421.891.26
pSB1C3(X,P)-55181.871.26

10/4

Digestion

Tatsui

SampleDNA/(µl)Xba1/(µl)Pst1/(µl)BSABuffer/(µl)MilliQ/(µl)TOTAL/(µl)
9/18 MP pL-1C3(85)24110.449.640
9/18 MP pL-1C3(85)(control)10001810
9/18 MP Bµ-1C3-11-3(355)6110.4427.640
9/18 MP Bµ-1C3-11-3(355)(control)10001810

Gel Extraction

(1)

LaneSampleⅠVolume/(µl)
110/4 Bµ 1C3-11-3(control)-
3,410/4 Bµ 1C3-11-3(XP)-
6ladder-

(2)

LaneSampleⅡVolume/(µl)
1ladder-
3,410/4 pL-1C3(XP)-
610/4 pL-1C3(control)-

Ligation

No name

SampleVector/(µL)Insert/(µL)Ligase/(µl)Buffer/(µl)MilliQ/(µl)Total/(µl)
9/20 Q-1C3(B)1315010
9/16 Q-1C3(B)1315010
9/20 Q-1C3(B)(control)1015310
9/16Q-1C3(B)(control)1015310

Transformation

SamplePlasmid/(µl)CompetentCells(DH5α)/(µl)
E1210
E2210
E3210
E4210

10/5

Ethanol Precipitation

Honda

10/3 Gel Extraction Pcon1C3(SP)

Ligation

Honda

Vector/(µL)Insert/(µL)Toyobo Ligation high/(µl)
10/5 Pcon1C3(SP)+9/20pQ(3)(XP)112
10/5 Pcon1C3(SP)(control)112

Transformation

Honda

SamplePlasmid/(µl)CompetentCells(DH5α)/(µl)
F1210
F2210

Colony PCR

LaneColony PCR ProductsⅠ
9-14E1 1-6
15-20E2 1-6

Liquid Culture

Sample
E1 1-6
E2 1-6

DNA Purification

Honda

DNAConcentration/(µg/ml)260/280260/230
pB PCR572.062.39

Digestion

SampleDNA/(µl)EcoRI/(µl)Xba1/(µl)Spe1/(µl)Pst1/(µl)Buffer/(µl)BSAMilliQ/(µl)TOTAL/(µl)
9/18 MP Bµ(1C3)11-4(ES)10101040.423.640
9/18 MP Bµ(1C3)11-4(control)1000010810
9/22 MP pQ-3(XP)400404161.694.4160
9/22 MP pQ-3(control)1000010810
9/22 Pcon Master(SP)10001140.423.640
9/22 Pcon Master(control)1000010810
9/22 pB(PCR Product)(ES)20101040.413.640
9/22 pB(PCR Product)(control)100010/td>10810

PCR

No Name

TargetVolume
2×KAPA HiFi Hotstart Ready Mix/(µl)12.5
Fw Primer/(µl)0.75
Rv Primer/(µl)0.75
Template(9/22 Mini Pcon-mamQ-3)pick up
MilliQ/(µl)11
TOTAL/(µl)25
StepsTemparatureTimecycle
Pre Denature98℃5min1time
Denature98℃20sec30cycle
Annealing60℃15sec30cycle
Extension72℃30sec30cycle
Final elongation72℃5min1time
Final hold12℃--

10/6

Sequence PCR

Target10/6 M T7-RBS-his(1C3)-1-VF10/6 M T7-RBS-his(1C3)-1-VR10/6 M T7-RBS-his(1C3)-2-VF10/6 M T7-RBS-his(1C3)-2-VR10/6 M T7-RBS-his(1C3)-3-VF10/6 M T7-RBS-his(1C3)-3-VR10/6 M Q(1C3)E1-1 VF10/6 M Q(1C3)E1-1 VR10/6 M Q(1C3)E1-2-VF10/6 M Q(1C3)E1-2-VR10/6 M Q(1C3)E1-3-VF10/6 M Q(1C3)E1-3-VR10/6 M Q(1C3)E2-1-VF10/6 M Q(1C3)E2-1-VR10/6 M Q(1C3)E2-2-VF10/6 M Q(1C3)E2-2-VR
Template/(µl)2.22.22.52.52.52.51.851.851.11.11.71.71.81.81.71.7
Big dye/(µl)0.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.5
5×Buffer/(µl)1.751.751.751.751.751.751.751.751.751.751.751.751.751.751.751.75
Primer/(µl)0.50.50.50.50.50.50.50.50.50.50.50.50.50.50.50.5
MilliQ/(µl)5.555.555.255.255.255.255.95.96.656.656.056.055.955.956.056.05
TOTAL/(µl)10.510.510.510.510.510.510.510.510.510.510.510.510.510.510.510.5
StepsTemparatureTimecycle
Pre Denature96℃2min1time
Denature96℃10sec30cycle
Annealing50℃5sec30cycle
Extension60℃4min30cycle
Final elongation4℃1min1time
Final hold4℃infinity-

Miniprep

DNAConcentration/(µg/ml)260/280260/230
T7-RBS-his(1C3)-11801.841.76
T7-RBS-his(1C3)-21601.921.92
T7-RBS-his(1C3)-31561.691.64
E1-12161.842.04
E1-23551.822.31
E1-32381.812.11
E2-12251.832.00
E2-22301.761.84

Gel Extraction

(1)

LaneSampleⅠVolume/(µl)
110/5 B&micro(1C3)(control)-
310/5 B&micro(1C3)(ES)-
610/5 pB(ES)-

(2)

LaneSampleⅡVolume/(µl)
1pQ-3(control)-
3pQ-3(XP)-
4Pcon(control)-
6Pcon(SP)-