Team:Kyoto/Notebook

From 2014.igem.org

(Difference between revisions)
Line 228: Line 228:
<tr><td>Annealing</td><td>55℃</td><td>30sec</td><td>30cycle</td></tr>
<tr><td>Annealing</td><td>55℃</td><td>30sec</td><td>30cycle</td></tr>
<tr><td>Extension</td><td>68℃</td><td>1min</td><td>30cycle</td></tr>
<tr><td>Extension</td><td>68℃</td><td>1min</td><td>30cycle</td></tr>
 +
</table>
</table>
Line 696: Line 697:
<tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>25cycle</td></tr>
<tr><td>Annealing</td><td>65℃</td><td>15sec</td><td>25cycle</td></tr>
<tr><td>Extension</td><td>72℃</td><td>5min</td><td>25cycle</td></tr>
<tr><td>Extension</td><td>72℃</td><td>5min</td><td>25cycle</td></tr>
 +
<tr><td>Final elongation</td><td>72℃</td><td>5min</td><td>1time</td></tr>
 +
<tr><td>Final hold</td><td>4℃</td><td>-</td><td>-</td></tr>
</table>
</table>
Line 775: Line 778:
<tr><td>6</td><td>9/8 Colony PCR Pcon+pSB3T5 Colony_5</td></tr>
<tr><td>6</td><td>9/8 Colony PCR Pcon+pSB3T5 Colony_5</td></tr>
<tr><td>7</td><td>9/8 Colony PCR pSB3T5(Nega) Colony_1</td></tr>
<tr><td>7</td><td>9/8 Colony PCR pSB3T5(Nega) Colony_1</td></tr>
-
<tr><td>8</td>9/8 Colony PCR Nega<td></td></tr>
+
<tr><td>8</td><td>9/8 Colony PCR Nega</td></tr>
</table>
</table>
Line 792: Line 795:
<h4>Gel Extractions</h4>
<h4>Gel Extractions</h4>
-
<span class="author">Matsumoto</span>
+
<span class="author">Tatsui and Yamaura</span>
<table class="note">
<table class="note">
<tr><th>Lane</th><th>Sample</th><th>Volume/(&micro;l)</th></tr>
<tr><th>Lane</th><th>Sample</th><th>Volume/(&micro;l)</th></tr>
-
<tr><td>1</td><td>9/1 pSB3T5 Colony_1 (Nega)</td><td>10</td></tr>
+
<tr><td>1</td><td>1kb ladder</td><td>-</td></tr>
-
<tr><td>2</td><td>9/1 pSB3T5 Colony_1 (E+P)(Confirmation)</td><td>5</td></tr>
+
<tr><td>2</td><td>-</td><td>-</td></tr>
-
<tr><td>3</td><td>9/1 pSB3T5 Colony_1 (E+P)(Extraction)</td><td>55</td></tr>
+
<tr><td>3</td><td>9/10 Digestion Pcon+pSB1C3(S+P)</td><td>-</td></tr>
-
<tr><td>4</td><td>1kb ladder</td><td>3</td></tr>
+
<tr><td>4</td><td>-</td><td>-</td></tr>
-
<tr><td>5</td><td>9/1 I732095 (Nega)</td><td>10</td></tr>
+
<tr><td>5</td><td>9/10 Digestion Pcon+pSB1C3(Nega)</td><td>-</td></tr>
-
<tr><td>6</td><td>9/1 I732095 (E+P)(Confirmation)</td><td>5</td></tr>
+
<tr><td>6</td><td>-</td><td>-</td></tr>
-
<tr><td>7</td><td>9/1 I732095 (E+P)(Extraction)</td><td>85</td></tr>
+
<tr><td>7</td><td>Pcon(X+P)</td><td>-</td></tr>
-
<tr><td>8</td><td>1kb ladder</td><td>3</td></tr>
+
<tr><td>8</td><td>-</td><td>-</td></tr>
-
<tr><td>9</td><td>9/1 pSB2K3 (Nega)</td><td>10</td></tr>
+
<tr><td>9</td><td>Pcon(Nega)</td><td>-</td></tr>
-
<tr><td>10</td><td>9/1 pSB2K3 (E+P)(Confirmation)</td><td>5</td></tr>
+
<tr><td>10</td><td>1kb ladder</td><td>-</td></tr>
-
<tr><td>11</td><td>9/1 pSB2K3 (E+P)(Extraction)</td><td>85</td></tr>
+
<tr><td>11</td><td>-</td><td>-</td></tr>
</table>
</table>
 +
<tr><th>DNA</th><th>Concentration/(&micro;g/ml)</th><th>260/280</th><th>260/230</th></tr>
 +
<tr><td>Pcon(X+P)</td><td>70</td><td>1.40</td><td>0.13</td></tr>
 +
</table>
 +
<h4>PCR</h4>
 +
<span class="author">No Name</span>
 +
<table class="note">
 +
<tr><th>Target</th><th>E+X+RBS+mamB+S</th><th>X+RBS+mamL+His+S+P</th><th>X+RBS+mamQ+His+S+P</th></tr>
 +
<tr><td>2×KAPA HiFi Hotstart Ready Mix/(&micro;l)</td><td>12.5</td><td>12.5</td><td>12.5</td></tr>
 +
<tr><td>Fw Primer/(&micro;l)</td><td>0.75(E+X+RBS+mamB)</td><td>0.75(X+RBS+mamL)</td><td>0.75(X+RBS+mamQ)</td></tr>
 +
<tr><td>Rv Primer/(&micro;l)</td><td>0.75(S+mamB)</td><td>0.75(P+S+His+mamL)</td><td>0.75(P+S+His+mamQ)</td></tr>
 +
<tr><td>9/9 DNA Purification Product/(&micro;l)</td><td>Trace</td><td>Trace</td><td>Trace</td></tr>
 +
<tr><td>MilliQ/(&micro;l)</td><td>11</td><td>11</td><td>11</td></tr>
 +
<tr><td>TOTAL/(&micro;l)</td><td>25</td><td>25</td><td>25</td></tr>
 +
</table>
 +
<table class="note">
 +
<tr><th>Steps</th><th>Temparature</th><th>Time</th><th>cycle</th></tr>
 +
<tr><td>Pre Denature</td><td>98℃</td><td>5min</td><td>1time</td></tr>
 +
<tr><td>Denature</td><td>98℃</td><td>20sec</td><td>25cycle</td></tr>
 +
<tr><td>Annealing</td><td>60℃</td><td>15sec</td><td>25cycle</td></tr>
 +
<tr><td>Extension</td><td>72℃</td><td>15sec</td><td>25cycle</td></tr>
 +
<tr><td>Final elongation</td><td>72℃</td><td>5min</td><td>1time</td></tr>
 +
<tr><td>Final hold</td><td>12℃</td><td>-</td><td>-</td></tr>
 +
</table>
 +
<h4>Electrophoresis</h4>
 +
<span class="author">Li</span>
 +
<table class="note">
 +
<tr><th>Lane</th><th>Cloning PCR Products</td></tr>
 +
<tr><td>1</td><td>1kb ladder</td></tr>
 +
<tr><td>2</td><td>9/10 PCR E+X+RBS+mamB+S(922bp)</td></tr>
 +
<tr><td>3</td><td>9/10 PCR X+RBS+mamL+His+S+P(340bp)</td></tr>
 +
<tr><td>4</td><td>-</td></tr>
 +
<tr><td>5</td><td>1kb ladder</td></tr>
 +
<tr><td>6</td><td>9/10 PCR X+RBS+mamQ+His+S+P(953bp)</td></tr>
 +
</table>
 +
<h4>DNA Purification</h4>
 +
<span class="author">Honda and Yamaura</span>
 +
<table class="note">
 +
<tr><th>DNA</th><th>Concentration/(&micro;g/ml)</th><th>260/280</th><th>260/230</th></tr>
 +
<tr><td>9/10 PCR E+X+RBS+mamB+S</td><td>200</td><td>1.84</td><td>2.15</td></tr>
 +
<tr><td>9/10 PCR X+RBS+mamL+His+S+P</td><td>120</td><td>1.69</td><td>1.84</td></tr>
 +
<tr><td>9/10 PCR X+RBS+mamQ+His+S+P</td><td>200</td><td>1.70</td><td>2.16</td></tr>
 +
</table>
 +
<h4>Restriction Enzyme Digestion</h4>
 +
<span class="author">Shimizu</span>
 +
<table class="note">
 +
<tr><th>Sample</th><th>DNA/(&micro;l)</th><th>Xba1/(&micro;l)</th><th>Pst1/(&micro;l)</th><th>Buffur/(&micro;l)</th><th>MilliQ/(&micro;l)</th><th>TOTAL/(&micro;l)</tr>
 +
<tr><td>9/10 PCR E+X+RBS+mamB+S</td><td>10</td><td>1</td><td>1</td><td>3(H)</td><td>15</td><td>30</td></tr>
 +
<tr><td>9/10 PCR X+RBS+mamL+His+S+P</td><td>16.7</td><td>1</td><td>1</td><td>3(H)</td><td>8.3</td><td>30</td></tr>
 +
<tr><td>9/10 PCR X+RBS+mamQ+His+S+P</td><td>10</td><td>1</td><td>1</td><td>3(H)</td><td>15</td><td>30</td></tr>
 +
</table>
<a name="0911" class="kyoto-jump"></a>
<a name="0911" class="kyoto-jump"></a>

Revision as of 08:44, 26 September 2014

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Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

Notebook

Calendar

July
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
Octorber
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31

アメフラシはこちら

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 Extractions

Matsumoto
LaneSampleVolume/(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 Extractions

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

Miniprep
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 Extractions

Tatsui and Yamaura
LaneSampleVolume/(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

9/11

9/12

9/13

9/14

9/15

9/16

9/17

9/18

9/19

9/20

9/22