Team:HUST-China/Notebook

Notebook

「Notebook」

Sensing part

PmrC-GFPPmrC-GFP-pBAD30

7.10

PCR

Yiyan Yu

Name Total
PmrC-GFP 50μl
demo1

7.19

Restriction Enzyme Digestion

Yiyan Yu

Name Enzyme
PmrC-GFP EcoRI and Hind III
pBAD30 EcoRI and Hind III

7.20

DNA ligation & transformation

Yiyan Yu & Qingyang Yu

Name Backbone Strain Medium
PmrC-GFP pBAD30(EcoR I and Hind III cut) Top10 LB(amp added)

7.21

Colony PCR

Yiyan Yu

Number Name
1 PmrC-GFP-pBAD30
5 PmrC-GFP-pBAD30
6 PmrC-GFP-pBAD30

PmrA-PmrB(Fe)PmrA-PmrB(Fe)-pBAD30

7.8

PCR

Long Cheng

Name Total
PmrA-PmrB(Fe) 50 μl
demo1

7.19

Restriction Enzyme Digestion

Yiyan Yu

Name Enzyme
PmrA-PmrB(Fe) EcoRI and KpnI
pBAD30 EcoRI and KpnI

7.20

Restriction Enzyme Digestion

Yiyan Yu & Qingyang Yu

Name Backbone Strain Medium
PmrA-PmrB(Fe) pBAD30(EcoR I and Hind III cut) Top10 LB(amp added)

7.21

Colony PCR

Yiyan Yu

Number Name
1 PmrA-PmrB(Fe)-pBAD30
5 PmrA-PmrB(Fe)-pBAD30
6 PmrA-PmrB(Fe)-pBAD30
demo1

PmrA-PmrB(Fe)-pBAD30→PmrA-PmrB(LBTX)-pBAD30

7.25

PCR

Yiyan Yu

Name Total
LBT 1 to 12 50 μl each
demo1

8.10

Point mutation

Xiaoyang Li

Name Total
PmrA-PmrB(Fe)-pBAD30 50 μl

8.20

Restriction Enzyme Digestion

Xiaoyang Li

Name Enzyme
PmrA-PmrB(Fe)-pBAD30 AhdI and SacI
LBT 1 AhdI and SacI
LBT 11 AhdI and SacI
demo1

8.20

Transformation

Xiaoyang Li & Yating Hu

Name Backbone Strain Medium
LBT 1 PmrA-PmrB(Fe)-pBAD30(AhdI and SacI cut) Top10 LB(amp added)
LBT 11 PmrA-PmrB(Fe)-pBAD30(AhdI and SacI cut) Top10 LB(amp added)

8.21

Colony PCR

Xiaoyang Li

Number Name
1 PmrA-PmrB(LBT 1)-pBAD30
1 PmrA-PmrB(LBT 11)-pBAD30
demo1

8.31

Restriction Enzyme Digestion

Xiaoyang Li

Name Enzyme
PmrA-PmrB(LBT2) EcoRI and KpnI
PmrA-PmrB(LBT3) EcoRI and KpnI
PmrA-PmrB(LBT4) EcoRI and KpnI
PmrA-PmrB(LBT5) EcoRI and KpnI
PmrA-PmrB(LBT6) EcoRI and KpnI
PmrA-PmrB(LBT7) EcoRI and KpnI
PmrA-PmrB(LBT8) EcoRI and KpnI
PmrA-PmrB(LBT9) EcoRI and KpnI
PmrA-PmrB(LBT10) EcoRI and KpnI
PmrA-PmrB(LBT12) EcoRI and KpnI
demo1

8.31

Transformation

Xiaoyang Li & Yating Hu

Name Backbone Strain Medium
PmrA-PmrB(LBT 2,3,4,5,6,7,8,9,10,12) pBAD30(EcoR I and Kpn I cut) Top10 LB(amp added)

9.3

Colony PCR

Xiaoyang Li

Number Name
1 PmrA-PmrB(LBT2)
2 PmrA-PmrB(LBT3)
3 PmrA-PmrB(LBT4)
4 PmrA-PmrB(LBT5)
5 PmrA-PmrB(LBT6)
6 PmrA-PmrB(LBT7)
7 PmrA-PmrB(LBT8)
8 PmrA-PmrB(LBT9)
9 PmrA-PmrB(LBT10)
10 PmrA-PmrB(LBT12)
demo1

PmrA-PmrB(LBT X)-pBAD30→PmrA-PmrB(LBT X)-PmrC-GFP-pBAD30

10.18

PCR

Long Cheng

Name Total
PmrA-PmrB(LBT 5) 30 μl
PmrA-PmrB(LBT 9) 30 μl
PmrA-PmrB(LBT 11) 30 μl
PmrC-GFP 50 μl

10.19

SOE PCR

Xiaoyang Li & Qingyang Yu

Name Total
PmrA-PmrB(LBT 5) & PmrC-GFP 50 μl
PmrA-PmrB(LBT 9) & PmrC-GFP 50 μl
PmrA-PmrB(LBT 11) & PmrC-GFP 50 μl
demo1

10.19

Restriction Enzyme Digestion

Xiaoyang Li

Name Enzyme
PmrA-PmrB(LBT 5) & PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 9) & PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 11) & PmrC-GFP EcoRI and Hind III
pBAD30 EcoRI and Hind III

10.19

DNA ligation & transformation

Xiaoyang Li & Yating Hu

Name Backbone Strain Medium
PmrA-PmrB(LBT 5)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 9)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 11)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)

10.20

Colony PCR

Xiaoyang Li

Number Name
1 PmrA-PmrB(LBT 5)-PmrC-GFP
1 PmrA-PmrB(LBT 9)-PmrC-GFP
1 PmrA-PmrB(LBT 11)-PmrC-GFP

10.19

SOE PCR

Qingyang Yu

Name Total
PmrA-PmrB(LBT 3) 50 μl
PmrA-PmrB(LBT 6) 50 μl
PmrA-PmrB(LBT 7) 50 μl
PmrA-PmrB(LBT 10) 50 μl
PmrA-PmrB(LBT 11) 50 μl
PmrC-GFP 50 μl

10.24

SOE PCR

Qingyang Yu

Name Total
PmrA-PmrB(LBT3)-PmrC-GFP 50 μl
PmrA-PmrB(LBT6)-PmrC-GFP 50 μl
PmrA-PmrB(LBT 7)-PmrC-GFP 50 μl
PmrA-PmrB(LBT 9)-PmrC-GFP 50 μl
PmrA-PmrB(LBT 10)-PmrC-GFP 50 μl

10.19

Restriction Enzyme Digestion

Xiaoyang Li

Name Enzyme
PmrA-PmrB(LBT 3)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 5)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 6)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 7)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 9)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 10)-PmrC-GFP EcoRI and Hind III
PmrA-PmrB(LBT 11)-PmrC-GFP EcoRI and Hind III
pBAD30 EcoRI and Hind III

10.26

DNA ligation & transformation

Yiyan Yu

Name Backbone Strain Medium
PmrA-PmrB(LBT 3)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 5)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 6)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 7)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 9)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 10)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)
PmrA-PmrB(LBT 11)-PmrC-GFP pBAD30(EcoRI and Hind III cut) Top10 LB(amp added)

10.28

Colony PCR

Xiaoyang Li

Number Name
1 PmrA-PmrB(LBT 5)-PmrC-GFP
2 PmrA-PmrB(LBT 5)-PmrC-GFP
3 PmrA-PmrB(LBT 5)-PmrC-GFP
4 PmrA-PmrB(LBT 5)-PmrC-GFP
5 PmrA-PmrB(LBT 5)-PmrC-GFP
6 PmrA-PmrB(LBT 5)-PmrC-GFP
3 PmrA-PmrB(LBT 6)-PmrC-GFP
4 PmrA-PmrB(LBT 6)-PmrC-GFP
2 PmrA-PmrB(LBT 7)-PmrC-GFP
3 PmrA-PmrB(LBT 7)-PmrC-GFP
1 PmrA-PmrB(LBT 9)-PmrC-GFP
2 PmrA-PmrB(LBT 9)-PmrC-GFP
3 PmrA-PmrB(LBT 9)-PmrC-GFP
4 PmrA-PmrB(LBT 9)-PmrC-GFP
5 PmrA-PmrB(LBT 9)-PmrC-GFP
6 PmrA-PmrB(LBT 9)-PmrC-GFP
3 PmrA-PmrB(LBT 10)-PmrC-GFP
4 PmrA-PmrB(LBT 10)-PmrC-GFP
1 PmrA-PmrB(LBT 11)-PmrC-GFP
2 PmrA-PmrB(LBT 11)-PmrC-GFP
3 PmrA-PmrB(LBT 11)-PmrC-GFP
4 PmrA-PmrB(LBT 11)-PmrC-GFP
5 PmrA-PmrB(LBT 11)-PmrC-GFP
6 PmrA-PmrB(LBT 11)-PmrC-GFP
7 PmrA-PmrB(LBT 11)-PmrC-GFP
8 PmrA-PmrB(LBT 11)-PmrC-GFP
9 PmrA-PmrB(LBT 11)-PmrC-GFP

Capture part

oprFoprF-pET28a

7.2

PCR

Efan Wang

Name Total
oprF 1 50 μl
oprF 2 50 μl
oprF 3 50 μl
oprF 4 50 μl
demo1

7.17

Restriction Enzyme Digestion

Ziyang Xiao & Zichun Weng & Yuqi Wu

Name Enzyme
pET28a NheI and BamHI
oprF 1 NheI and BamHI
oprF 2 NheI and BamHI

7.27

DNA ligation & transformation

Ziyang Xiao

Name Backbone Strain Medium
oprF pET28a(BamH I and Nhe I cut) Top10 LB(kan added)

7.28

Colony PCR

Xiao Deng

Number Name
1 oprF-pET28a
2 oprF-pET28a
3 oprF-pET28a
4 oprF-pET28a
5 oprF-pET28a
demo1

3×LBT1-12 & SitagoprF-3×LBT X(or Sitag)-pET28a

7.22

PCR

Ziyang Xiao & Zichun Weng

Name Total
3 x LBT1 50μl
3 x LBT2 50μl
3 x LBT3 50μl
3 x LBT4 50μl
3 x LBT9 50μl
3 x LBT10 50μl
3 x LBT11 50μl
3 x LBT12 50μl

7.27

PCR

Efan Wang & Ziyang Xiao

Name Total
3 x LBT5_1 50μl
3 x LBT5_2 50μl
3 x LBT6_1 50μl
3 x LBT6_2 50μl
3 x LBT7_1 50μl
3 x LBT7_2 50μl
3 x LBT8_1 50μl
3 x LBT8_2 50μl
Sitag 1 50μl
Sitag 2 50μl
demo1

7.27

Restriction Enzyme Digestion

Ziyang Xiao & Ting Peng

Name Enzyme
oprF-pET28a HindIII and BamHI
3 x LBT2 HindIII and BamHI
3 x LBT3 HindIII and BamHI
3 x LBT4 HindIII and BamHI
3 x LBT8 HindIII and BamHI
Sitag HindIII and BamHI

8.4

Restriction Enzyme Digestion

Xiao Deng

Name Enzyme
3 x LBT1 HindIII and BamHI
3 x LBT5 HindIII and BamHI
3 x LBT6 HindIII and BamHI
3 x LBT7 HindIII and BamHI
3 x LBT9 HindIII and BamHI
3 x LBT10 HindIII and BamHI
3 x LBT11 HindIII and BamHI
3 x LBT12 HindIII and BamHI

7.28

DNA ligation & transformation

Xiao Deng & Ting Peng

Name Backbone Stain Medium
3 x LBT2 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT3 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT4 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT8 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
Sitag oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)

7.29

Colony PCR

Xiao Deng

Number Name
1 oprF-3×LBT2-pET28a
2 oprF-3×LBT3-pET28a
3 oprF-3×LBT4-pET28a
4 oprF-3×LBT8-pET28a
5 oprF-Sitag-pET28a
demo1

8.12

DNA ligation & transformation

Xiao Deng & Nan Hu & Shaofeng Liao

Name Backbone Stain Medium
3 x LBT1 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT5 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT6 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT7 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT9 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT10 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT11 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)
3 x LBT12 oprF-pET28a(BamHI and HindIII cut) Top10 LB(kan added)

8.13

Colony PCR

Xiao Deng

Number Name
1 oprF-3×LBT2-pET28a
2 oprF-3×LBT3-pET28a
3 oprF-3×LBT4-pET28a
4 oprF-3×LBT8-pET28a
5 oprF-Sitag-pET28a
6 oprF-3×LBT4-pET28a
7 oprF-3×LBT8-pET28a
8 oprF-Sitag-pET28a
demo1

Standardization Part

8.20

PCR

Yubei Sun & Huiping Shi

Name Total
oprF-3 X LBT1 50 μl
oprF-3 X LBT4 50 μl
oprF-3 X LBT8 50 μl
oprF-3 X LBT10 50 μl
oprF-3 X LBT11 50 μl
oprF-3 X LBT12 50 μl
demo1

9.20

SOE PCR

Efan Wang

Name Total
oprF-3 X LBT3 50 μl
oprF-3 X LBT6 50 μl
oprF-3 X LBT7 50 μl
oprF-Sitag 50 μl
demo1

9.5

Restriction Enzyme Digestion

Yubei Sun & Ziyi Li

Name Enzyme
oprF-3 X LBT 1 EcoRI and Spel I
oprF-3 X LBT 4 EcoRI and Spel I
oprF-3 X LBT 10 EcoRI and Spel I
oprF-3 X LBT 11 EcoRI and Spel I
pSB1C3 EcoRI and Spel I
pSB1C3 Xbal I and Pst I

9.20

Restriction Enzyme Digestion

Huiping Shi

Name Enzyme
oprF-3 X LBT 3 EcoRI and Spel I
oprF-3 X LBT 6 EcoRI and Spel I
oprF-3 X LBT 7 EcoRI and Spel I
oprF-3 X LBT 8 EcoRI and Spel I
oprF-3 X LBT 11 EcoRI and Spel I
oprF-3 X LBT 12 EcoRI and Spel I

9.27

DNA ligation & transformation

Yubei Sun & Huiping Shi

Name Backbone Strain Medium
oprF-3 X LBT 1 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 3 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 4 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 8 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-Sitag pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)

9.27

DNA ligation & transformation

Ziyang Xiao

Name Backbone Strain Medium
oprF-3 X LBT 6 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 7 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 10 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 11 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)
oprF-3 X LBT 12 pSB1C3(EcoRI and Spel I cut) DH5a LB(chl added)

9.28

Colony PCR

Yubei Sun

Number Name
1 oprF-3×LBT1-pSB1C3
2 oprF-3×LBT3-pSB1C3
3 oprF-3×LBT4-pSB1C3
4 oprF-3×LBT8-pSB1C3
5 oprF-Sitag-pSB1C3

9.30

Colony PCR

Ziyang Xiao

Number Name
1 oprF-3×LBT6-pSB1C3
2 oprF-3×LBT7-pSB1C3
3 oprF-3×LBT10-pSB1C3
4 oprF-3×LBT11-pSB1C3
5 oprF-3×LBT12-pSB1C3

PmrC-GFP-pBAD30→PmrC-GFP-pSB1C3

10.2

PCR

Ziyang Xiao

Name Total
PmrC-GFP 30 μl

10.4

Restriction Enzyme Digestion

Yubei Sun

Name Enzyme
PmrC-GFP Pstl I and XbaI

10.5

DNA ligation & transformation

Ziyang Xiao

Name Backbone Strain Medium
PmrC-GFP pSB1C3(Pstl I and XbaI cut) DH5a LB(chl added)

10.6

Colony PCR

Ziyang Xiao

Number Name
1 PmrC-GFP-pSB1C3
2 PmrC-GFP-pSB1C3
3 PmrC-GFP-pSB1C3
4 PmrC-GFP-pSB1C3
5 PmrC-GFP-pSB1C3

PmrA-PmrB(LBT X)-PmrC-GFP-pBAD30→PmrA-PmrB(LBT X)-PmrC-GFP-pSB1C3

10.26

PCR

Ziyang Xiao

Name Total
PmrA-PmrB(LBT 5)PmrC-GFP 30 μl
PmrA-PmrB(LBT 9)-PmrC-GFP 30 μl
PmrA-PmrB(LBT 11)-PmrC-GFP 30 μl

10.4

Restriction Enzyme Digestion

Huiping Shi

Name Enzyme
PmrA-PmrB(LBT 5)-PmrC-GFP Pst I and XbaI
PmrA-PmrB(LBT 9)-PmrC-GFP Pst I and XbaI
PmrA-PmrB(LBT 11)-PmrC-GFP Pst I and XbaI

10.5

DNA ligation & transformation

Huiping Shi

Name Backbone Strain Medium
PmrA-PmrB(LBT 5)-PmrC-GFP pSB1C3(Pst I and Spel I cut) DH5a LB(chl added)
PmrA-PmrB(LBT 9)-PmrC-GFP pSB1C3(Pst I and Spel I cut) DH5a LB(chl added)
PmrA-PmrB(LBT 11)-PmrC-GFP pSB1C3(Pst I and Spel I cut) DH5a LB(chl added)

10.13

Splicing by overlap extension step 1

Yiyan Yu

Name Total
PmrA-PmrB(LBT5) 50μl
PmrA-PmrB(LBT9) 50μl
PmrA-PmrB(LBT11) 50μl
PmrC-GFP 50μl

10.14

Splicing by overlap extension step 2

Qingyang Yu

Name Total
PmrA-PmrB(LBT5)-PmrC-GFP 50μl
PmrA-PmrB(LBT9)-PmrC-GFP 50μl
PmrA-PmrB(LBT11)-PmrC-GFP 50μl

10.16

Restriction Enzyme Digestion

Yiyan Yu

Name Enzyme
PmrA-PmrB(LBT 5)-PmrC-GFP EcoR I & Pst I
PmrA-PmrB(LBT 9)-PmrC-GFP EcoR I & Pst I
PmrA-PmrB(LBT 11)-PmrC-GFP EcoR I & Pst I

10.19

Restriction Enzyme Digestion

Yiyan Yu

Name Enzyme
psb1c3-PmrC-GFP EcoR I & Pst I

10.21

DNA ligation & transformation

Ziyang Xiao

Name Backbone Strain Medium
PmrA-PmrB(LBT5)-PmrC-GFP pSB1C3(Pstl I and XbaI cut) DH5a LB(chl added)
PmrA-PmrB(LBT9)-PmrC-GFP pSB1C3(Pstl I and XbaI cut) DH5a LB(chl added)
PmrA-PmrB(LBT11)-PmrC-GFP pSB1C3(Pstl I and XbaI cut) DH5a LB(chl added)

10.22

Colony PCR

Kangyuan Yu

Number Name
4 PmrA-PmrB(LBT5)-PmrC-GFP
1 PmrA-PmrB(LBT9)-PmrC-GFP
2 PmrA-PmrB(LBT9)-PmrC-GFP
6 PmrA-PmrB(LBT9)-PmrC-GFP
2 PmrA-PmrB(LBT11)-PmrC-GFP

Acknowledgments: