Team:HUST-China/Notebook

「Notebook」

• Sensing part
• Capture part
• Standardization Part

Sensing part

PmrC-GFP→PmrC-GFP-pBAD30

7.10

PCR

Yiyan Yu

Name	Total
PmrC-GFP	50μl

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

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

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

7.25

PCR

Yiyan Yu

Name	Total
LBT 1 to 12	50 μl each

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

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

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

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)

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

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

oprF→oprF-pET28a

7.2

PCR

Efan Wang

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

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

3×LBT1-12 & Sitag→oprF-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

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

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

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

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

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: