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Team:OUC-China/Result/date
From 2011.igem.org
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7.21
I Prepare medium
1.soc medium
2.LB medium
3.LBliquid medium
II transform 2011 parts –OUC Order 1 to 3
III pick up a tested single colony
7.22
1．Extract plasmid of the practicing part
2. Measure density using distilled water as comparison
A260/A280 | A260/A230 | density（ng/ul） | |
1 | 1.892 | 2.115 | 0.035 |
2 | 1.841 | 1.227 | 0.0405 |
3 | 2.299 | 4.054 | 0.030 |
3. Digestion for 3 hours
1 | 2 | 3 | 4(Single enzyme cut) | |
DNA(ul) | 15 | 15 | 15 | 15 |
Buffer2(ul) | 5 | 5 | 5 | 5 |
Bsa(ul) | 0.5 | 0.5 | 0.5 | ----- |
Double-distilled water(ul) | 27.5 | 27.5 | 27.5 | 29 |
EcoR I(ul) | 1 | 1 | 1 | 1 |
Pst I(ul) | 1 | 1 | 1 | ------- |
Time(h) | 3 | 3 | 3 | 3 |
4.check by Electrophoresis
1、2、3：double enzyme，4、5、6：Single enzyme ，7：plasmid
5.transform 7new parts(number:4-10)
6.culture bacteria on LB medium
7.culture 3 new parts in Shaker
7.24
1. Presrvation species of parts No. ,4,5,6,7,8,9,10
2. Extract plasmid of 4,5,6,7,8,9,10
3.check through double enzyme Digestion
7.25
1. Measure density
5 | 0.020µg/µl |
4 | 0.012µg/µl |
6 | 0.020µg/µl |
2 | 0.015µg/µl |
2. Digestion
5 | 4 | 6 | 2 | |
DNA | 25 | 30 | 25 | 25 |
Buffer2 | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
H2O | 17.5 | 12.5 | 17.5 | 17.5 |
enzyme1 | EcoRⅠ1µl | EcoRⅠ1µl | SpeⅠ1µl | XbaⅠ1µl |
Enzyme2 | SpeⅠ1µl | XbaⅠ1µl | PstⅠ1µl | PstⅠ1µl |
3.check by Electrophoresis
4.gel extraction
5.measure density
OUC-order | 5 | 4 | 6 | 2 |
Density ug/µl | 0.016 | 0.021 | 0.027 | 0.028 |
6. Ligate overnight
1 | 2 | |
Parts | 2µl 5+3µl 4 | 3µl 2+3µl 6 |
Buffer | 2µl | 2µl |
enzyme | 1µl | 1µl |
DdH2O | 2µl | 1µl |
7.28
Repeat the ligation expriments of 7.25
7.29
Extract plasmids of parts No. 11,12,13,14
7.30
Medium has antibiotic resistance problem, re-transform 11\12\13\14
7.31
1.cultrue bacteria of 11,12,13,14 in LB liquid medium
2. Extract plasmid of 15,16,17,18,19,20,21,22
3. Optimization the Steps of Extracting plasmid
4.Chloramphericol preparing
5. NaAC’s DNA concentration
8.1
1. Extract plasmid of 11-14 and Measure density
ng/µl | A260∕A280 | |
11 | 12.7 | 1.917 |
12 | 10.3 | 1.981 |
13 | 35.5 | 1.919 |
14 | 15.7 | 1.975 |
2. Extract plasmid of No.5,4,6,2，15-20 parts
3.culture the colony of GFP on LB medium
8.2
Measure density of 15-22,2,4,5,6
Parts | µg/µl | A260∕A280 |
2 | 0.038 | 1.875 |
4 | 0.019 | 1.298 |
5 | 0.022 | 1.538 |
6 | 0.020 | 1.702 |
15 | 0.012 | 1.237 |
16 | 0.024 | 1.649 |
17 | 0.018 | 1.731 |
18 | 0.015 | 1.470 |
21 | 0.017 | 1.538 |
8.3
1. Concentrate plasmid using alcohol and NaAC
2. Measure density after concentrating
ng/µl | A260∕A280 | |
2 | 103 | 1.807 |
4 | 33 | 1.811 |
5 | 56 | 1.836 |
6 | 41 | 1.745 |
8.4
Digestion
Double enzymes | Single enzyme | |||
Parts | 5 | 2 | 4 | 6 |
DNA | 25 | 13 | 27 | 25 |
Buffer | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
ddH2O | 16.5 | 28.5 | 16.5 | 18.5 |
enzyme | EcoRⅠ 1.5 | XbaⅠ1.5 | XbaⅠ1 | SpeⅠ1 |
SpeⅠ1.5 | PstⅠ1.5 |
Parts | 4 | 6 |
DNA | 28.5 | 30 |
Buffer | EcoRⅠbuffer 5 | Buffer3 5 |
BSA | 0.5 | |
ddH2O | 15.5 | 13.5 |
enzyme | EcoRⅠ1 | PstⅠ1 |
8.5
1. Begin device 2
2. Design experimental procedures:
3. First construction with parts of device 2
8.6
2+6 successfully ligate
Measure density
Parts | µg/µl | A260∕A280 |
3 | 0.085 | 1.910 |
16 | 0.119 | 1.867 |
18 | 0.019 | 2.033 |
Measure density after concentrating
Parts | µg/µl | A260∕A280 |
18 | 0.0605 | 1.921 |
11 | 0.0335 | 2.107 |
Digestion
Double enzymes | Single enzyme | |||
Parts | 18 | 16 | 3 | 11 |
DNA | 20 | 10 | 15 | 29 |
Buffer | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
ddH2O | 21.5 | 31.5 | 28 | 14.5 |
enzyme | EcoRⅠ 1.5 | XbaⅠ1.5 | XbaⅠ1 | SpeⅠ1 |
SpeⅠ1.5 | PstⅠ1.5 |
After 8hours
Parts | 3 | 11 |
DNA | 20 | 20 |
Buffer | EcoRⅠbuffer 5 | Buffer3 5 |
BSA | 0.5 | |
ddH2O | 23.5 | 23 |
enzyme | EcoRⅠ1 | PstⅠ1 |
8.7
1. Checking 4+5, 2+6 ‘s ligation by Gel electrophoresis
Digestion
μL | 2+6 | 4+5 |
DNA | 2（53ng） | 3(28ng) |
Buffer2 | 1 | 1 |
BSA | 0.1 | 0.1 |
ddH2O | 6.7 | 5.9 |
EcoRⅠ | 0.1 | 0.1 |
PstⅠ | 0.1 | 0.1 |
check by small gel
8.8
1.transform the parts of 3-18,11-16,14-19
2. Active bacteria of 4.5.18.11 and culture in LB liquid medium
3. Presrvation species parts of 4-5
8.9
1.check 2+6,4+5 by small gel
2. Measure density
number | ng/µl | A260∕A280 | |
2+6 | 1 | 58 | 1.902 |
2 | 78.5 | 1.826 | |
3 | 62.5 | 1.953 | |
4 | 60.5 | 2.017 | |
4+5 | 1 | 31 | 2.081 |
2 | 33 | 2.089 | |
3 | 20.5 | 2.291 |
3. Digestion
parts | 2+6 | 5+4 | |||||
number | 1 | 2 | 3 | 4 | 1 | 2 | 3 |
DNA | 2 | 2 | 2 | 2 | 3 | 3 | 5 |
Buffer3 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
BSA | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
ddH2O | 6.5 | 6.5 | 6.5 | 6.5 | 5.5 | 5.5 | 5.5 |
EcoRⅠ | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
PstⅠ | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
4. Extract plasmid of 4,5,18,11
Measure density
number | ng/µl | A260∕A280 |
5 | 73 | 2.000 |
4 | 71.5 | 2.072 |
18 | 32.5 | 2.305 |
3 | 85 | 1.910 |
11 | 95.5 | 1.950 |
16 | 119 | 1.867 |
Digestion 50µl
5 | 4 | 18 | 3 | 11 | 16 | |
DNA | 13 | 13 | 30 | 11 | 10 | 8 |
Buffer3 | 5 | 5 | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
ddH2O | 29.5 | 29.5 | 19.5 | 31.5 | 30.5 | 34.5 |
enzyme | EcoRⅠ 1 | XbaⅠ1 | EcoRⅠ 1 | XbaⅠ1 | EcoRⅠ 1 | XbaⅠ1 |
SpeⅠ1 | PstⅠ1 | SpeⅠ1 | PstⅠ1 | SpeⅠ1 | PstⅠ1 |
8.11
1. Checking ligation correctness of 14&19 using small gel----fails
2.try to ligate using 3A
8.12
1. Extract plasmid of 5+4 and Measure density
number | ng/µl | A260∕A280 |
1 | 55 | 1.964 |
2 | 39 | 2.324 |
3 | 41 | 2.412 |
4 | 81 | 2.051 |
5 | 42 | 2.154 |
6 | 58 | 1.966 |
2.check by single enzyme digestion
number | 1 | 2 | 3 | 4 | 5 | 6 |
DNA | 3 | 4 | 4 | 2 | 4 | 3 |
Buffer EcoR I | 1 | 1 | 1 | 1 | 1 | 1 |
ddH2O | 5.5 | 4.5 | 4.5 | 6.5 | 4.5 | 5.5 |
EcoRⅠ | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
Check using small gel
3.cultrue bacteria of part 13
4. ligate 2+6+3,11+16,18+13
8.13
1.Extract plasmid
2.pick colony of 2+6+3,18+3,16+3 and culture in shaker
8.14
Checking whether colony of different size contain different plasmids, or whether could be used to pick colony raising transformation possibility. But it made no difference
8.15
1.measure density
number | µg/µl | A260∕A280 |
2+6+3-1 | 0.032 | 3.439 |
2+6+3-2 | 0.064 | 2.370 |
11+16 | 0.056 | 2.306 |
11+16 | 0.102 | 2.010 |
18+3 | 0.037 | 2.434 |
18+3 | 0.033 | 2.332 |
2. Digestion
Single enzyme 10µl
number | 2+6+3-1 | 2+6+3-2 | 3+18-1 | 3+18-2 | 11+16 | 11+16 |
DNA | 4 | 2 | 4 | 6 | 2.5 | 1.5 |
Buffer EcoR I | 1 | 1 | 1 | 1 | 1 | 1 |
ddH2O | 4.7 | 6.7 | 4.7 | 2.7 | 6.2 | 7.2 |
EcoRⅠ | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
Double enzyme 10µl
number | 18+3-1 | 18+3-2 | 18 | 2+6+3-1 | 2+6+3-2 | 2+6 | 11+16 | 11+16 |
DNA | 4 | 6 | 6 | 4 | 2 | 3 | 2.5 | 1.5 |
Buffer3 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
BSA | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
ddH2O | 4.3 | 2.3 | 2.4 | 4.3 | 6.3 | 5.3 | 5.8 | 6.8 |
EcoRⅠ | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
PstⅠ | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
8.16
Check yesterdays’ digestion by gel electrophoresis
Ladder | 18-3 | 18-3 | 18 | 6-2-3 | 6-2-3 | 6-2 | ||
100bp plus | single | double | single | double | double | double | double | double |
8.17
1. Presrvation species of pSB1AC3_BBa13017
2. Extract plasmid of 18，pSB1AC3_BBa13017，measure density
number | µg/µl | A260∕A280 |
pSB1AC3_BBa13017-1 | 0.199 | 2.036 |
pSB1AC3_BBa13017-2 | 0.168 | 2.036 |
pSB1AC3_BBa13017-3 | 0.113 | 2.083 |
pSB1AC3_BBa13017-4 | 0.103 | 2.102 |
pSB1AC3_BBa13017-5 | 0.174 | 2.129 |
pSB1AC3_BBa13017-6 | 0.147 | 2.115 |
pSB1AC3_BBa13017-7 | 0.064 | 2.345 |
pSB1AC3_BBa13017-8 | 0.106 | 2.186 |
pSB1AC3_BBa13017-9 | 0.163 | 2.138 |
pSB1AC3_BBa13017-10 | 0.170 | 2.092 |
18-1 | 0.110 | 2.095 |
18-2 | 0.124 | 2.102 |
18-3 | 0.140 | 2.121 |
3. Prepare medium of TY（culturing rhizobium）,CM（supplemented medium）
4. Digest pSB1AC3_BBa13017 as Carrier
DNA | 5 |
Buffer2 | 5 |
BSA | 0.5 |
ddH2O | 37.5 |
EcoRⅠ | 1 |
PstⅠ | 1 |
Extract 3055bp strip
8.18
1.Ligate 5,4
2.transform pSB1C3
8.19
1.culture rhizobium on TY medium
2.prepare CM,M9 medium
3.LeuB PCR
4. Presrvation species of Bean strain
5. Extract plasmid and Digest
number | 18 | 3 | 2+6 | 16 | 11 | 14+19 | 13 |
DNA | 12 | 10 | 18 | 9 | 7 | 7 | 6 |
Buffer2 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
ddH2O | 30.1 | 31.5 | 24.1 | 33.1 | 35.1 | 35.1 | 36.1 |
enzyme | EcoRI1.2 | XbaⅠ1.2 | EcoRI1.2 | XbaⅠ1.2 | EcoRI1.2 | EcoRI1.2 | XbaⅠ1.2 |
SpeⅠ1.2 | PstⅠ1.2 | SpeⅠ1.2 | PstⅠ1.2 | SpeⅠ1.2 | SpeⅠ1.2 | PstⅠ1.2 |
8.20
1. Presrvation species of 5+4 and extract plasmid,measure density
number | ng/µl | A260∕A280 |
1 | 99.5 | 2.098 |
2 | 140.5 | 2.162 |
3 | 99 | 2.152 |
4 | 123.5 | 2.093 |
5 | 137.5 | 2.083 |
2. Digestion
number | 1 | 2 | 3 | 4 | 5 |
DNA | 1.3 | 1 | 1.3 | 1 | 1 |
Buffer3 | 1 | 1 | 1 | 1 | 1 |
BSA | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
ddH2O | 7.1 | 7.4 | 7.1 | 7.4 | 7.4 |
EcoRⅠ | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
PstⅠ | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
8.22
1、Presrvation species of 5+4 and extract plasmid,measure density, digest
2、pick single colonies of 19-14&13；2-6&3 , 11&16 and culture in shaker
3、PCR Leu（50ul），recover and check
10×Taq Buffer(with MgSO4) 5μL
MgCl2 4μL
Primer-F 1.5μL
Primer-R 1.5μL
H2O 33.5μL
dNTP 2μL
DNA 2μL
Taq 0.25μL
Pfu 0.25μL
Recommended By Advicer Tan
Result: success
8.23
1、Presrvation species of 2 6&3，11&16，14 19&13 and extract plasmid, measure density，digest
density
number | 2 6&3 | 11&16 | 1419&13 | | | | |
density （ng/ul） | 30 | 49 | 45 | 132 | 62 | 42 | 64 |
Digestion
Single enzyme
2 6&3 | 11&16 | 1419&13 | | | | | |
DNA | 3.3 | 2 | 2.2 | 1 | 1.5 | 2.3 | 1.5 |
Buffer4 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
DdH2O | 5.4 | 6.6 | 6.4 | 7.6 | 7.1 | 6.3 | 7.1 |
XbaI | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
BSA | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
Double enzymes
2 6&3 | 11&16 | 1419&13 | | | | | |
DNA | 3.3 | 2 | 2.2 | 1 | 1.5 | 2.3 | 1.5 |
Buffer4 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
BSA | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
H2O | 5.1 | 6.4 | 6.2 | 7.4 | 6.9 | 6.1 | 6.9 |
EcoRI-HF | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
SpeI | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
result：success
2、Preservation species of CFN42 WT and BL21
3、check 18&3 be small gel ——succeed
4、Prepare medium：LB liquid medium 300ml，
LB medium 150ml
TY medium 150ml
5、Prepare SMM medium，+ pro+ Vb1
8.24
1、Prepare SMM medium :MM+pro+Vb1 MM+pro+Vb1+leu
2、check by small gel
8.25
1、ligate 11-16&18-13 and 5&4 using 3A
Measure density：
11-16 | 18-13 | 5 | 4 | |
density（ng/ul） | 46.5 | 136.5 | 85.5 | 71 |
Double enzymes
5 | 4 | 18-3 | 11-16 | |
DNA | 12 | 10 | 10 | 25 |
Buffer4 | 5 | 5 | 5 | 5 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
H2O | 29.5 | 32.5 | 31.5 | 16.5 |
enzyme1 | EcoRI-HF 1.5 | XbaI 1 | EcoRI-HF 1.5 | XbaI 1.5 |
enzyme 2 | SpeI 1.5 | PstI 1 | SpeI 1.5 | PstI 1.5 |
Check by gel
Ligation of 5&4
5 | 4 | AC | T4 | Buffer | H2O |
6 | 4 | 4 | 1 | 2 | 3 |
Ligation of 11-16&18-13：（ul）
K381001 | 18-3 | 11-16 | Buffer | T4 |
5 | 6 | 6 | 2 | 1 |
2、inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu)，culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30℃
8.27
1、transform 18-3&11
2、pick 3 colonies of 5&4 and culture in l LB liquid medium
3、culture LeuB and K115001 in LB liquid medium
4、recover 22,21,3 and ligate 21&3,22&3
Ligation of 21&3：(ul)
22 | 3 | T4 | Buffer | ddH2O |
7 | 4 | 1 | 2 | 6 |
Ligation of 22&3：(ul)
21 | 3 | T4 | Buffer | ddH2O |
8.5 | 4 | 1 | 2 | 4.5 |
8.28
1、Prepare medium：LB liquid medium 500ml
2、transform 5&4 and 18-3&11-16 again，and transform leu 21&3 and 22&3
3、 culture deficient cells
4、culture 18-3&11 in LB liquid medium
8.29
1、PCR :5&4 and leu
PCR（ul）
ddH2O | dNTP | Buffer | Primer-F | Primer-R | dna | Taq |
39.5 | 2 | 5 | 1 | 1 | 1 | 0.5 |
2、Presrvation species of 18-3&11 and extract plasmid,measure density, digest
density（1）19.5（2）130.5
digestion
single enzyme
（1） | （2） | |
DNA | 6 | 1 |
Buffer | 1 | 1 |
BSA | 0.1 | 0.1 |
ddH2O | 2.65 | 7.65 |
XbaI | 0.35 | 0.35 |
Double enzymes:
（1） | （2） | |
DNA | 6 | 1 |
Buffer | 1 | 1 |
BSA | 0.1 | 0.1 |
ddH2O | 2.4 | 7.4 |
EcoRI | 0.25 | 0.25 |
PstI | 0.25 | 0.25 |
Check by small gel
3、pick single colony of 5&4，leu,21&3,22&3，18-3&11-16 and culture in LB liquid medium。
4、prepare TY medium 100ml
8.30
1、Presrvation species of 5&4,leuB89,leuB13 and extract plasmid,measure density, digest
density：
leuB13（1） | 5&4（1） | 5&4（2） | 5&4（3） | leuB89（1） | leuB89（2） | leuB89(3) |
24.5 | 188.0 | 62.0 | 132.0 | 6.8 | 3.1 | 10.8 |
Digestion of 5&4
（1） | （2） | （3） | |
DNA | 1 | 2 | 1 |
Buffer3 | 1 | 1 | 1 |
ddH2O | 7.5 | 6.5 | 7.5 |
BSA | 0.1 | 0.1 | 0.1 |
EcoRI | 0.2 | 0.2 | 0.2 |
PstI | 0.2 | 0.2 | 0.2 |
Check by small gel
Result:5&4 and leuB successfully ligate
8.31
1、did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.
density：
2 part 37.5
K115001 part 120
digestion(ul)
K115001 | 2 | |
DNA | 9 | 9 |
10*H Buffer | 2 | 2 |
EcoRI | 1 | 1 |
PstI | 1 | 1 |
ddH2O | 7 | 7 |
9.1
1、prepare 0147TY medium
Tryptone 5.0g Yeast extract 3.0g CaCl.6H2O 1.3g Agar 15.0g Distilled water 1.0L
2、ligate 18-3&11-16 and 18-3&11
density：
18-3（1） | 18-3（2） | 11-16（1） | 11-16（2） | K carrier（1） | K carrier（2） | AC（1） | AC(2) |
140 | 98 | 278.5 | 203．0 | 17.5 | 17.5 | 7.9 | 13.1 |
Digestion:
18-3 | 11 | 11-16 | |
DNA | 7 | 10 | 4 |
Buffer | 2 | 2 | 2 |
enzyme1 | E 1 | X 1 | X 1 |
enzyme 2 | S 1 | P 1 | P 1 |
DdH2O | 9 | 6 | 12 |
Ligation:
18-3：3ul 16:1ul 2：6ul T4：1ul Buffer：2ul H2O：7ul
18-3：3ul 11-16：4ul 2：6ul T4：1ul Buffer：2ul ddH2O：4ul
9.2
1、Presrvation species of leuB and extract plasmid,measure density, digest
density：
1 | 2 | 3 | 4 |
292.5 | 21.5 | 223.5 | 32.0 |
Digestion:
leuB | K(pSB1C3) | |
EcoRI | 1 | 1 |
PstI | 1 | 1 |
Buffer | 2 | 2 |
DNA | 14 | 8.5 |
ddH2O | 2 | 7.5 |
Check by small gel:
recover：
2、pick single colony of 18-3&11-16 and 18-3&11 and culture in shaker
3、transform BBa_I751250,as LuxI_AHL producer。Transform BBa_J04450(placI+mRFP)
9.3
1、ligate leuB, K(pSB1C3) carrier and transform
density：
Before Concentration | After Concentration | |
LeuB | 15 | 36 |
pSB1C3 | 8 | 25 |
ligation
LeuB | Psb1c3 | T4 | Buffer | DdH2O |
6 | 4 | 1 | 2 | 7 |
2、extract plasmid of 8-3&11-16 and 18-3&11, digest，and check by small gel
Density:
18-3&11-161 | 18-3&11-162 | 18-3&11 1 | 18-3&11 2 | 5-4（AC）1 | 5-4（AC） 2 | 2-6-3 |
265 | 242 | 192 | 217.5 | 193 | 72 | 63.5 |
digestion：
18-3&11-16(1) | 18-3&11-16(2) | 18-3&11(1) | 18-3&11(2) | |
DNA | 3.5 | 4 | 5 | 4.5 |
Buffer | 2 | 2 | 2 | 2 |
EcoRI | 1 | 1 | 1 | 1 |
PstI | 1 | 1 | 1 | 1 |
ddH2O | 12.5 | 12 | 11 | 11.5 |
Check by gel：
2、ligate 5-4&2-6-3
ligation
K | 5-4 | 2-6-3 | T4连接酶 | Buffer | DdH2O |
6 | 4 | 2 | 1 | 2 | 5 |
9．4
1、transform 5-4&2-6-3，extract plasmid of 2-6-3与2
2、transform Pea Rhizobium
9.5
1、Presrvation species of leuB and extract plasmid,measure density, digest, PCR ,check by gel
PCR
1 | 2 | 3 | |||
ddH2O | 37.5 | 37 | 36.5 | 36.5 | 36.5 |
dNTPmix | 4 | 4 | 4 | 4 | 4 |
Buffer | 5 | 5 | 5 | 5 | 5 |
VF2 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
VR | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
tamplate | 0 | 0.5 | 1 | 1 | 1 |
Taq | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
Check by gel
digestion
LeuB 1 | LeuB 3 | |
DNA | 3 | 3 |
BufferM | 0.5 | 0.5 |
ddH2O | 1.1 | 1.1 |
SpeI | 0.2 | 0.2 |
XbaI | 0.2 | 0.2 |
Check by gel
9.6
1、Presrvation species of 54263 and extract plasmid, digest, PCR ,check by gel
digestion
1 | 2 | 3 | |
DNA | 1.4 | 1.4 | 1.7 |
Buffer | 0.5 | 0.5 | 0.5 |
DdH2O | 2.5 | 2.5 | 2.2 |
EcoRI | 0.3 | 0.3 | 0.3 |
PstI | 0.3 | 0.3 | 0.3 |
9.7
1、measure OD of Deficient cell
MM（leuB-） | 1 leuB+ | 2 leuB+ | 3 leuB+ | 4 leuB+ | |
OD（A340） | 1.611-1 | 1.698-1 | 1.818-1 | 1.548-1 | 1.881-1 |
2、digest leuB、③、K115001
digestion：
8 | 6 | LeuB | 3 | T1 | T2 | T3 | |
DNA | 9 | 9 | 10 | 10 | 10 | 13 | 12 |
Buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
DdH2O | 7 | 6.6 | 5.6 | 5.6 | 5.6 | 2.6 | 3.6 |
EcoRI | 1 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 |
SpeI | 1 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 |
density：
LeuB 98.0
T 1:93.5 2:73.5 3:87
3 1:97 2:73 3:82
9.8
1、extract plasmid of 6，and digest with LeuB
Density of 6：
1 | 2 | 3 |
57 | 68 | 50 |
digestion
6 | 6 | LeuB | |
DNA | 6 | 6 | 10 |
Buffer | 2 | 2 | 2 |
DdH2O | 9.6 | 9.6 | 5.6 |
EcoRI | 1.2 | 1.2 | 1.2 |
SpeI | 1.2 | 1.2 | 1.2 |
2、ligate leuB&3
ligation：
T4 | Buffer | leuB | 3 | T carrier | DdH2O |
1 | 2 | 3 | 0.7 | 10 | 3.3 |
9.9
1、check 6, leuB, 8
Gel：
Ligate 8&6、6&leuB
Ligation of 8&6：
T carrier | 8 | 6 | T4 | Buffer | DdH2O |
10 | 1 | 1 | 1 | 2 | 5 |
Ligation of 6&leuB
T carrier | 6 | LeuB | T4 | Buffer | DdH2O |
10 | 1 | 3 | 1 | 2 | 3 |
9.10
1、transform 8&6 and leuB
2、culture leuB&3 in LB medium
3、device checking：
Device1 has strong Fluorescent and device 2 hasslight one
Picture:
9.11
1、PCR：sinR、pro-RBS-sinR、raiR、raiR-pro
density
sinI | 12.8 |
SinI pro | 9.6 |
Pro-RBS-sinI | 15.1 |
RaiI | 14.2 |
RaiI pro | 9.4 |
Pro-RBS-raiI | 11.4 |
SinR pro | 11.5 |
Pro-RBS-raiR | 15.0 |
2、pick single colony of 6&leuB
9.12
1、digest 21、22 and ligate
digestion
21 | 22 | |
Buffer | 2 | 2 |
NDA | 13.6 | 15 |
DdH2O | 2 | 0.6 |
EcoRI | 1.2 | 1.2 |
SpeI | 1.2 | 1.2 |
ligation
21&3
21 | 3 | Buffer | T4 | DdH2O | T |
3 | 1 | 2 | 1 | 3 | 10 |
22&3
22 | 3 | Buffer | T4 | DdH2O | T |
5 | 1 | 2 | 1 | 1 | 10 |
2、extract plasmid of leuB-6 and measure density
density：16.5
9．13
1、transform and culture 21&3,22&3,8&6，recover PSB1C3 carrier
2.Pcr
ddH2O | Pfu with MgSO4 buffer | dNTP | PF | PR | Template | Pfu |
38µl | 5µl | 4µl | 1µl | 1µl | 0.5µl | 0.5µl |
Results：failed
3.digest sinR, sinI,pro- sinI
Digestion:20µl
sinR | sinI | pro- sinI | |
DNA | 10 | 11 | 16 |
Buffer 10×H | 2 | 2 | 2 |
ddH2O | 6 | 5 | 0 |
EcoRI | 1 | 1 | 1 |
PstI | 1 | 1 | 1 |
9.15
Digestion of Leu&3 :20µl
number | 1 | 2 | 3 |
DNA(µl) | 10 | 10 | 9 |
Buffer 10×H(µl) | 2 | 2 | 2 |
ddH2O(µl) | 6 | 6 | 7 |
XbaⅠ(µl) | 1 | 1 | 1 |
PstⅠ(µl) | 1 | 1 | 1 |
9.16
Digest 6 and leub
number | 6 | Leu |
DNA(µl) | 15 | 9 |
Buffer 10×M(µl) | 2 | 2 |
ddH2O(µl) | 2 | 7 |
enzymew | SpeⅠ(µl) 1 | XbaⅠ(µl) 1 |
PstⅠ(µl) 1 |
9.18
Ligate Leu and 6 using 3A
Leu | 6 | buffer | T4 | |
10µl | 2µl | 6µl | 2µl | 1µl |
Gel picture：
Digest 6
number | DNA(µl) | Buffer 10× (µl) | PstⅠ(µl) | ddH2O(µl) |
6 | 9 | 2 | 1 | 8 |
9.20
Measure density
number | ng/µl | A260∕A280 |
6 | 179.5 | 1.851 |
pSB1C3-1 | 6.3 | |
pSB1C3-2 | 8.6 | 3.510 |
pSB1C3-3 | 5.1 |
Digest Leu
DNA(µl) | Buffer 10×M(µl) | ddH2O(µl) | PstⅠ(µl) | XbaⅠ(µl) |
6 | 2 | 2 | 2 | 8 |
Ligate Leu& 6
Leu | 6 | buffer | T4 | ddH2O |
6 | 4 | 2 | 1 | 7 |
9.21
Measure density
number | ng/µl | A260∕A280 |
raiⅠpro-3 | 29.5 | 1.666 |
raiⅠpro-2 | 28.5 | 1.885 |
raiⅠpro-1 | 23.0 | 1.818 |
C0076 | 161.0 | 1.793 |
E0020e0fp-1 | 28.5 | 1.676 |
E0020e0fp-2 | 19.5 | 2.19 |
