Notebook

「Notebook」

Sensing 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 & Si-tag→OprF-3×LBT X(or Si-tag)-pET28a

7.22

PCR

Ziyang Xiao & Zichun Weng

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

7.27

PCR

Efan Wang & Ziyang Xiao

Name	Total
3*LBT5_1	50μl
3*LBT5_2	50μl
3*LBT6_1	50μl
3*LBT6_2	50μl
3*LBT7_1	50μl
3*LBT7_2	50μl
3*LBT8_1	50μl
3*LBT8_2	50μl
Sitag 1	50μl
Sitag2	50μl

7.27

Restriction Enzyme Digestion

Ziyang Xiao & Ting Peng

Name	Enzyme
OprF-pET28a	HindIII and BamHI
3*LBT2	HindIII and BamHI
3*LBT3	HindIII and BamHI
3*LBT4	HindIII and BamHI
3*LBT8	HindIII and BamHI
Sitag	HindIII and BamHI

7.27

Restriction Enzyme Digestion

Ziyang Xiao & Ting Peng

Name	Enzyme
OprF-pET28a	HindIII and BamHI
3*LBT2	HindIII and BamHI
3*LBT3	HindIII and BamHI
3*LBT4	HindIII and BamHI
3*LBT8	HindIII and BamHI
Sitag	HindIII and BamHI

8.4

Restriction Enzyme Digestion

Xiao Deng

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

7.28

DNA ligation & transformation

Xiao Deng & Ting Peng

Name	Backbone	Stain	Medium
3*LBT2	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT3	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT4	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*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*LBT1	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT5	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT6	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT7	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT9	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT10	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*LBT11	OprF-pET28a(BamHI and HindIII cut)	Top10	LB(kan added)
3*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

Recycling 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-LBT X-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

8.10

Bypass PCR

Yating Hu

Name	Total
pmrA-pmrB-Fe&LBT 2	50 μl
pmrA-pmrB-Fe&LBT 3	50 μl
pmrA-pmrB-Fe&LBT 4	50 μl
pmrA-pmrB-Fe&LBT 5	50 μl
pmrA-pmrB-Fe&LBT 6	50 μl
pmrA-pmrB-Fe&LBT 7	50 μl
pmrA-pmrB-Fe&LBT 8	50 μl
pmrA-pmrB-Fe&LBT 9	50 μl
pmrA-pmrB-Fe&LBT 10	50 μl
pmrA-pmrB-Fe&LBT 12	50 μl

9.2

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

8.20

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-LBT 2
2	pmrA-pmrB-LBT 3
3	pmrA-pmrB-LBT 4
4	pmrA-pmrB-LBT 5
5	pmrA-pmrB-LBT 6
6	pmrA-pmrB-LBT 7
7	pmrA-pmrB-LBT 8
8	pmrA-pmrB-LBT 9
9	pmrA-pmrB-LBT 10
10	pmrA-pmrB-LBT 12

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

Bypass 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

Standardization Part

pmrA-pmrB-LBT X-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pBAD30

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

Bypass 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	Pst I and Spe I
OprF-3 X LBT 4	Pst I and Spe I
OprF-3 X LBT 10	Pst I and Spe I
OprF-3 X LBT 11	Pst I and Spe I
pSB1C3	Pst I and Spe I

9.20

Restriction Enzyme Digestion

Huiping Shi

Name	Enzyme
OprF-3 X LBT 3	Pst I and Spe I
OprF-3 X LBT 6	Pst I and Spe I
OprF-3 X LBT 7	Pst I and Spe I
OprF-3 X LBT 8	Pst I and Spe I
OprF-3 X LBT 11	Pst I and Spe I
OprF-3 X LBT 12	Pst I and Spe I

9.27

DNA ligation & transformation

Yubei Sun & Huiping Shi

Name	Backbone	Strain	Medium
OprF-3 X LBT 1	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 3	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 4	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 8	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-Sitag	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)

9.27

DNA ligation & transformation

Ziyang Xiao

Name	Backbone	Strain	Medium
OprF-3 X LBT 6	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 7	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 10	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 11	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
OprF-3 X LBT 12	pSB1C3(Pst I and Spe 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

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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

pmrA-pmrB-LBT X-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pBAD30

10.2

PCR

Ziyang Xiao

Name	Total
pmrC-GFP	30 μl

9.5

Restriction Enzyme Digestion

Yubei Sun

Name	Enzyme
pmrC-GFP	Pst I and XbaI

9.27

DNA ligation & transformation

Ziyang Xiao

Name	Backbone	Strain	Medium
pmrC-GFP	pSB1C3(Pst I and Spe I 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 Spe I cut)	DH5a	LB(chl added)
pmrA-pmrB-LBT 9-pmrC-GFP	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)
pmrA-pmrB-LBT 11-pmrC-GFP	pSB1C3(Pst I and Spe I cut)	DH5a	LB(chl added)

10.6

Colony PCR

Ziyang Xiao

Number	Name
1	pmrA-pmrB-LBT 5-pmrC-GFP
2	pmrA-pmrB-LBT 9-pmrC-GFP
3	pmrA-pmrB-LBT 11-pmrC-GFP

Team:HUST-China/Notebook

「Notebook」

Sensing part

OprF→OprF-pET28a

3×LBT1-12 & Si-tag→OprF-3×LBT X(or Si-tag)-pET28a

Recycling part

pmrC-GFP→pmrC-GFP-pBAD30

pmrA-pmrB-Fe→pmrA-pmrB-Fe-pBAD30

pmrA-pmrB-Fe-pBAD30→pmrA-pmrB-LBT X-pBAD30

pmrA-pmrB-LBT X-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pBAD30

Standardization Part

pmrA-pmrB-LBT X-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pBAD30

pmrA-pmrB-LBT X-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pBAD30

pmrA-pmrB-LBT X-pmrC-GFP-pBAD30→pmrA-pmrB-LBT X-pmrC-GFP-pSB1C3