|
|
Line 1,971: |
Line 1,971: |
| </tr> | | </tr> |
| <tr> | | <tr> |
− | <td>Patp2-RBS-CI-TT | + | <td>Patp2-RBS-CI-TT 1 |
| <td>50uL</td> | | <td>50uL</td> |
| </tr> | | </tr> |
| + | <tr> |
| + | <td>Patp2-RBS-CI-TT 2 |
| + | <td>50uL</td> |
| + | </tr> |
| + | |
| </table> | | </table> |
| <br><img src="https://static.igem.org/mediawiki/2016/e/e2/T--HUST-China--notebook-patp2-R-CI-TT-fig01.png" alt=""> | | <br><img src="https://static.igem.org/mediawiki/2016/e/e2/T--HUST-China--notebook-patp2-R-CI-TT-fig01.png" alt=""> |
Line 2,159: |
Line 2,164: |
| </tr> | | </tr> |
| <tr> | | <tr> |
− | <td>Duet-pet | + | <td>pETDuet-1 |
| <td>EcoRI and PstI</td> | | <td>EcoRI and PstI</td> |
| </tr> | | </tr> |
Line 2,168: |
Line 2,173: |
| </table> | | </table> |
| </p> | | </p> |
− | <p>
| + | <p> |
− | <h6>9.7</h6>
| + | |
− | <h6>Transformation</h6>
| + | |
− | <h6>Zhenyi Guo</h6>
| + | |
− | <table border="1">
| + | |
− | <tr>
| + | |
− | <td>Name
| + | |
− | <td>Strain
| + | |
− | <td>Medium</td> | + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td>plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase
| + | |
− | <td>DH5α
| + | |
− | <td>LB(Chl added)</td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | </p>
| + | |
− | <p>
| + | |
| <h6>9.17</h6> | | <h6>9.17</h6> |
| <h6>DNA ligation&Transformation</h6> | | <h6>DNA ligation&Transformation</h6> |
Line 2,198: |
Line 2,186: |
| <tr> | | <tr> |
| <td>plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase | | <td>plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase |
− | <td>Duet-pet(T7)(cut by EcoRI and PstI) | + | <td>pETDuet-1(cut by EcoRI and PstI) |
| <td>DH5α | | <td>DH5α |
| <td>LB(Amp added) </td> | | <td>LB(Amp added) </td> |
Line 2,207: |
Line 2,195: |
| <h6>9.18</h6> | | <h6>9.18</h6> |
| <h6>Colony PCR</h6> | | <h6>Colony PCR</h6> |
− | <h6>Successfully align the sequencing results with the objective sequences:</h6>
| + | |
| <h6>Zhenyi Guo</h6> | | <h6>Zhenyi Guo</h6> |
| <table border="1"> | | <table border="1"> |
Revision as of 15:35, 18 October 2016
Notebook
Notebook
see how these parts were constructed?
CII
7.24
PCR
Yufeng Mo
Name
| Total |
CII 1
| 50 μl |
CII 2
| 50 μl |
CII control group
| 30 μl |
7.25
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
CII
| XbaI and PstI |
7.26
DNA ligation&transformation
Yufeng Mo
Name |
Backbone |
Strain |
Medium |
CII |
psb1c3(Xba I and Pst I cut) |
DH5α |
LB(chl added) |
7.27
Colony PCR
Yufeng Mo
CIII
7.14
PCR
Yufeng Mo
7.15
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
plasmid psb1c3 1
| XbaI and SpeI |
CIII
| XbaI and SpeI |
7.15
Dephosphorylation
Yufeng Mo
7.17
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
CIII
| psb1c3(XbaI and SpeI cut)
| DH5α
| LB(chl added) |
7.18
Colony PCR
Yufeng Mo
Number
| Name |
1
| CIII 1 |
2
| CIII 2 |
3
| CIII 3 |
4
| CIII 4 |
5
| CIII 5 |
6
| CIII 6 |
RBS-CII
PCR
Yufeng Mo
Name
| Total |
RBS-CII 2
| 40 μl |
RBS-CII 1
| 40 μl |
RBS-CII 0
| 20 μl |
9.4
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
RBS-CII
| XbaI and PstI |
9.4
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
RBS-CII
| psb1c3(Xba I and Pst I cut)
| DH5α
| LB(chl added) |
9.5
Colony PCR
Yufeng Mo
Number
| Name |
1
| RBS-CII 1 |
2
| RBS-CII 2 |
RBS-CIII
8.7
PCR
Ziyi Jiang
Name
| Total |
RBS-CIII
| 50 μl |
RBS-CIII
| 50 μl |
8.7
Restriction Enzyme Digestion
Ziyi Jiang
Name
| Enzyme |
psb1c3
| XbaI and PstI |
RBS-CIII
| XbaI and PstI |
8.10
DNA ligation&transformation
Ziyi Jiang
Name
| Backbone
| Strain
| Medium |
RBS-CIII
| psb1c3(XbaI and PstI cut)
| DH5α
| LB(chl added) |
8.11
Colony PCR
Ziyi Jiang
RBS-CIII-RBS-CIII
8.18
Restriction Enzyme Digestion
Ziyi Jiang
Name
| Enzyme |
RBS-CIII
| EcoRI and SpeI |
RBS-CIII
| EcoRI and XbaI |
8.18
DNA ligation&Transformation
Ziyi Jiang
Name
| Backbone
| Strain
| Medium |
CIII-RBS
| CIII-RBS-psb1c3(EcoRI and XbaI cut)
| DH5α
| LB(chl added) |
8.19
Colony PCR
Ziyi Jiang
Number
| Name |
13
| RBS-CIII-RBS-CIII-psb1c3 |
19
| RBS-CIII-RBS-CIII-psb1c3 |
RBS-CII-RBS-CII
9.12
PCR
Yufeng Mo
9.14
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
RBS-CII-psb1c3
| EcoRI and XbaI |
RBS-CII-psb1c3
| EcoRI and SpeI |
9.14
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
RBS-CII
| RBS-CII-psb1c3(EcoRI and XbaI cut)
| DH5α
| LB(chl added) |
9.15
Colony PCR
Zhi Zeng
Number
| Name |
1
| RBS-CII-RBS-CII-psb1c3 |
pRM-RBS-GFP-LAVtag
7.18
PCR
Xiaoyang Li
Name
| Total |
GFP-First
| 50 μl |
Name
| Total |
pRM-RBS-GFP-LAVtag
| 30 μl |
pRM-RBS-GFP-LAVtag
| 30 μl |
pRM-RBS-GFP-LAVtag
| 30 μl |
7.19
Restriction Enzyme Digestion
Xiaoyang Li
Name
| Enzyme |
GFP
| EcoRI and SpeI |
psb1c3
| EcoRI and SpeI |
7.20
DNA ligation&transformation
Xiaoyang Li
Name
| Backbone
| Strain
| Medium |
pRM-RBS-GFP-LAVtag
| psb1c3(EcoRI and SpeI cut)
| DH5α
| LB(chl added) |
7.24
Colony PCR
Xiaoyang Li
Number
| Name |
1
| pRM-RBS-GFP-LVAtag |
8.21
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
pRM-RBS-GFP-LVAtag
| EcoRI and PstI |
pETDuet-1
| EcoRI and PstI |
8.21
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
pRM-RBS-GFP-LVAtag
| pETDuet-1(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
8.22
Colony PCR
Yufeng Mo
Number
| Name |
1
| pRM-RBS-GFP-LVAtag-pETDuet 1 |
3
| pRM-RBS-GFP-LVAtag-pETDuet 3 |
4
| pRM-RBS-GFP-LVAtag-pETDuet 4 |
5
| pRM-RBS-GFP-LVAtag-pETDuet 5 |
6
| pRM-RBS-GFP-LVAtag-pETDuet 6 |
pRE-RBS-GFP-LVAtag
8.23
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
pRE-RBS-GFP-LVAtag
| XbaI and PstI |
8.23
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
pRE-RBS-GFP-LVAtag
| psb1c3(XbaI and PstI cut)
| DH5α
| LB(chl added) |
8.24
Colony PCR
Yufeng Mo
Number
| Name |
1
| pRE-RBS-GFP-LVAtag-psb1c3 |
8.29
Restriction Enzyme Digestion
Yufeng Mo,Zhenyi Guo
Name
| Enzyme |
pRE-RBS-GFP-LVAtag-psb1c3
| EcoRI and PstI |
8.29
DNA ligation&transformation
Xiao Deng,Zhenyi Guo
Name
| Backbone
| Strain
| Medium |
pRE-RBS-GFP-LVAtag
| petDuet-1(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
8.30
Colony PCR
Yufeng Mo
Number
| Name |
1
| pRE-RBS-GFP-LVAtag-petDuet-1 1 |
3
| pRE-RBS-GFP-LVAtag-petDuet-1 3 |
5
| pRE-RBS-GFP-LVAtag-petDuet-1 5 |
8
| pRE-RBS-GFP-LVAtag-petDuet-1 8 |
pR-RBS-GFP-LAVtag
8.2
PCR
Zhenyi Guo
Name
| Total |
pR-RBS-GFP-tag
| 50 μl |
pR-RBS-GFP-tag
| 50 μl |
pR-RBS-GFP-tag
| 50 μl |
8.2
Restriction Enzyme Digestion
Zhenyi Guo
Name
| Enzyme |
psb1c3
| EcoRI and PstI |
pR-RBS-GFP-tag
| EcoRI and PstI |
8.2
DNA ligation&Transformation
Zhenyi Guo
Name
| Backbone
| Strain
| Medium |
pR-RBS-GFP-tag
| psb1c3(EcoRI and PstI cut)
| DH5α
| LB(chl added) |
8.4
Colony PCR
Zhenyi Guo
Number
| Name |
2
| pR-RBS-GFP-tag |
8.24
Restriction Enzyme Digestion
Zhenyi Guo
Name
| Enzyme |
pR-RBS-GFP-tag
| EcoRI and PstI |
8.24
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
pR-RBS-GFP-tag
| pETDuet-1(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
8.26
Colony PCR
Yufeng Mo
Number
| Name |
4
| pR-RBS-GFP-tag-pETDuet-1 4 |
6
| pR-RBS-GFP-tag-pETDuet-1 6 |
7
| pR-RBS-GFP-tag-pETDuet-1 7 |
SnRK2.2
9.10
PCR
Yufeng Mo
Name
| Total |
SnRK2.2 1
| 40 μl |
SnRK2.2 2
| 40 μl |
SnRK2.2 0
| 20 μl |
9.10
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
SnRK2.2
| XbaI and PstI |
9.10
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
SnRK2.2
| psb1c3(XbaI and PstI cut)
| DH5α
| LB(chl added) |
9.12
Colony PCR
Yufeng Mo
Number
| Name |
1
| SnRK2.2-psb1c3 |
ABF2
9.10
PCR
Yufeng Mo
Name
| Total |
ABF2 1
| 40 μl |
ABF2 2
| 40 μl |
ABF2 0
| 20 μl |
9.10
Restriction Enzyme Digestion
Yufeng Mo
Name
| Enzyme |
ABF2
| XbaI and PstI |
9.10
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
ABF2
| psb1c3(XbaI and PstI cut)
| DH5α
| LB(chl added) |
9.11
Colony PCR
Yufeng Mo
Number
| Name |
2
| ABF2 2 |
3
| ABF2 3 |
4
| ABF2 4 |
PP2CA
7.25
PCR
Yufeng Mo
Name
| Total |
PP2CA 1
| 25 μl |
PP2CA 2
| 25 μl |
PP2CA control group
| 25 μl |
7.26
Restriction enzyme digestion
Yufeng Mo
Name
| Enzyme |
PP2CA
| XbaI and PstI |
7.26
DNA ligation&transformation
Yufeng Mo
Name
| Backbone
| Strain
| Medium |
PP2CA
| psb1c3(Xba I and Pst I cut)
| E.coli DH5α
| LB(chl added) |
7.27
Colony PCR
Yufeng Mo
prd29A
9.27
PCR
Zhi Zeng
Name
| Total |
prd29a 1
| 50 μl |
prd29a 2
| 50 μl |
prd29a 3
| 50 μl |
prd29a 4
| 50 μl |
9.27
Restriction Enzyme Digestion
Zhenyi Guo
Name
| Enzyme |
prd29a
| XbaI and PstI |
9.28
Colony PCR
Zhenyi Guo
Number
| Name |
1
| prd29a-psb1c3 |
2
| prd29a-psb1c3 |
CI-TT-pR-RBS-GFP-LVAtag&CII-TT-pRE-RBS-GFP-LVAtag
8.17
Restriction Enzyme Digestion
Lin Di
Name
| Enzyme |
Duet-pet
| EcoRI and PstI |
CI-TT-pR-RBS-GFP-LVAtag
| EcoRI and PstI |
CII-TT-pRE-RBS-GFP-LVAtag
| EcoRI and PstI |
![](https://static.igem.org/mediawiki/2016/d/df/T--HUST-China--notebook-CI-TT%26CII-TT-fig01.png)
8.17
DNA ligation&Transformation
Lin Di
Name
| Backbone
| Strain
| Medium |
CI-TTADH1-pR-RBS-GFP-LVAtag
| Duet-pet(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
CII-TTADH1-pRE-RBS-GFP-LVAtag
| Duet-pet(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
8.18
Colony PCR
Lin Di
Number
| Name |
6
| CI-TTADH1-pR-RBS-GFP-LVAtag-petDuet-1 6 |
5
| CII-TTADH1-pRE-RBS-GFP-LVAtag-petDuet-1 5 |
11
| CII-TTADH1-pRE-RBS-GFP-LVAtag -petDuet-1 11 |
cro-TT-pRM-RBS-GFP-LVAtag-PSB1C3
8.14
PCR
Zhi Zeng
8.15
Restriction Enzyme Digestion
Zhi Zeng
Name
| Enzyme |
pRM-RBS-GFP-LVAtag-psb1c3
| EcoRI and XbaI |
8.16
In-Fusion
Zhi Zeng
Insert
| Vector
| Total |
Cro&TT
| pRM-RBS-GFP-LVAtag-PSB1C3(Cut by EcoRⅠ&XbaⅠ)
| 5uL |
8.16
Transformation
Zhi Zeng
Name
| Strain
| Medium |
cro-TT-pRM-RBS-GFP-LVAtag-PSB1C3 (ligated by In-fusion)
| DH5α
| LB(Chl added) |
8.17
Colony PCR
Zhi Zeng
Name
| Total |
cro-TT-pRM-RBS-GFP-LVAtag-psb1c3
| 20uL |
8.18
Restriction Enzyme Digestion
Zhi Zeng
Name
| Enzyme |
PETDuet-1
| EcoRI and PstI |
cro-TT-pRM-RBS-GFP-LVAtag
| EcoRI and PstI |
8.18
DNA ligation&Transformation
Zhi Zeng
Name
| Backbone
| Strain
| Medium |
cro-TT-pRM-RBS-GFP-LVAtag
| pETDuet-1(EcoRI and PstI cut)
| DH5α
| LB(Amp added) |
PP2CA-TTADH1
8.14
Restriction Enzyme Digestion
Ziyi Jiang
Name
| Enzyme |
TTADH1-psb1c3
| EcoRI and XbaI |
PP2CA-psb1c3
| EcoRI and SpeI |
DNA ligation&Transformation
Ziyi Jiang
Name
| Backbone
| Strain
| Medium |
PP2CA
| TTADH1-psb1c3(EcoRI and XbaI cut)
| DH5α
| LB(Chl added) |
8.16
Colony PCR
Ziyi Jiang
Number
| Name |
5
| PP2CA-TTADH1-psb1c3 5 |
7
| PP2CA-TTADH1-psb1c3 7 |
12
| PP2CA-TTADH1-psb1c3 12 |
PP2CA-TTADH1-pCyc
8.22
Overlap PCR
Zhi Zeng
Name
| Total |
PP2CA-TTADH1-Pcyc
| 80uL |
8.22
Restriction Enzyme Digestion
Zhi Zeng
Backbone
| Enzyme |
psb1c3
| PstI and XbaI |
8.23
In-Fusion
Zhi Zeng
Insert
| Vector
| Total |
PP2CA-TTADH1-pCyc
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 5uL |
8.23
Transformation
Zhi Zeng
Name
| Strain
| Medium |
PP2CA-TTADH1-pCyc-psb1c3 (ligated by In-Fusion)
| DH5α
| LB(Chl added) |
8.24
Colony PCR
Zhi Zeng
Number
| Name |
3
| PP2CA-TTADH1-pCyc-psb1c3 |
4
| PP2CA-TTADH1-pCyc-psb1c3 |
7
| PP2CA-TTADH1-pCyc-psb1c3 |
13
| PP2CA-TTADH1-pCyc-psb1c3 |
14
| PP2CA-TTADH1-pCyc-psb1c3 |
Kozak-SnRK2.2-TTADH1-pCyc
8.30
Overlap PCR
Name
| Total |
Kozak-Snrk2.2-TTADH1-pCyc
| 80uL |
9.5
In-Fusion
Zhi Zeng
Insert
| Vector
| Total |
Kozak-Snrk2.2-TTADH1-pCyc
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 5uL |
9.5
Transformation
Zhi Zeng
Name
| Strain
| Medium |
Kozak-Snrk2.2-TTADH1-pCyc-psb1c3(ligated by In-Fusion)
| DH5α
| LB(Chl added) |
9.7
Colony PCR
Zhi Zeng
Name
| Total |
Kozak-Snrk2.2-TTADH1-pCyc-psb1c3
| 20uL |
placm-pRE-RBS-Cro-RBS-CII-TT
9.6
Overlap PCR
Jiyue Qin
Name
| Total |
Placm-pRE-RBS-Cro
| 50uL |
9.7
In-Fusion
Jiyue Qin
Insert
| Vector
| Total |
Placm-pRE-RBS-Cro&RBS-cII-TT
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 10uL |
9.6
Transformation
Jiyue Qin
Name
| Strain
| Medium |
Placm-pRE-RBS-Cro-RBS-CII-TT-psb1c3(ligated by In-Fusion)
| DH5α
| LB(Chl added) |
9.8
Colony PCR
Jiyue Qin
Number
| Name |
1
| Placm-pRE-RBS-Cro-RBS-CII-TT-psb1c3 |
patp2-RBS-CI-TTADH1-pR-RBS-CIII
9.2
Overlap PCR
Jiyue Qin
Name
| Total |
Patp2-RBS-CI-TT 1
| 50uL |
Patp2-RBS-CI-TT 2
| 50uL |
9.3
In-Fusion
Jiyue Qin
Insert
| Vector
| Total |
Patp2-RBS-CI-TT&pR-RBS-CIII
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 10uL |
9.3
Transformation
Jiyue Qin
Name
| Strain
| Medium |
Patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3
| DH5α
| LB(Chl added) |
9.4
Colony PCR
Jiyue Qin
Number
| Name |
1
| Patp2-RBS-CI-TT-pR-RBS-CIII-psb1c3 |
RBS-iLDH-TT-pRM-RBS-beta-galactosidase
9.1
Restriction Enzyme Digestion
Jiyue Qin
Name
| Enzyme |
GFP-LVAtag-psb1c3
| XbaI and PstI |
9.1
In-Fusion
Jiyue Qin
Insert
| Vector
| Total |
RBS-iLDH-TT&pRM-RBS-beta-galactosidase
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 10uL |
9.1
Transformation
Jiyue Qin
Name
| Strain
| Medium |
RBS-iLDH-TT-pRM-RBS-beta-galactosidase-psb1c3
| DH5α
| LB(Chl added) |
9.2
Colony PCR
Jiyue Qin
Number
| Name |
1
| RBS-iLDH-TT-pRM-RBS-beta-galactosidase-psb1c3 |
plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase
9.7
In-Fusion
Zhenyi Guo
Insert
| Vector
| Total |
plac-pRE-RBS-Cro-RBS-CII-TT&patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase
| psb1c3(Cut by PstⅠ&XbaⅠ)
| 10uL |
9.7
Transformation
Zhenyi Guo
Name
| Strain
| Medium |
plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase
| DH5α
| LB(Chl added) |
9.9
Colony PCR
Zhenyi Guo
Number
| Name |
1
| plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase |
9.11
Restriction Enzyme Digestion
Zhenyi Guo
Name
| Enzyme |
pETDuet-1
| EcoRI and PstI |
plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase -psb1c3
| EcoRI and PstI |
9.17
DNA ligation&Transformation
Zhenyi Guo
Name
| Backbone
| Strain
| Medium |
plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase
| pETDuet-1(cut by EcoRI and PstI)
| DH5α
| LB(Amp added) |
9.18
Colony PCR
Zhenyi Guo
Number
| Name |
1
| plac-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-galactosidase-Duet-pet |