Team:NTHU Taiwan/Notebook

MENU ?

Lab Notebook

July

7/1

Culture burkholderia acidpuldis

7/4

Try different culture medium

7/8

Successfully culture burkholderia acidpuldis

7/11

PCR for burkholderia acidpuldis (get fac-dex DNA sequence)

Primer: FDPXXBAF

Reverse primer: FDEXPSTR

98^oC/98^oC/57^oC/72^oC/72^oC/12^oC

30 sec/30 sec/30 sec/3.5 min/3.5 min/unlimited

For 30 cycles

-Order facdex DNA sequence from IDT

7/12

PCR clean up

Run gel => PCR result fail

Too many unexpected band

7/13

Learn how to ligation and transform

Try to fix PCR protocol

Add positive and negative control group

1. Only medium (negative control)

2. ddH2O (negative control)

3. another bacteria (positive control)

4. do PCR without taq polymerase (negative control)

PCR set

98^oC/98^oC/57^oC/72^oC/72^oC/12^oC

30 sec/30 sec/1 min/1.5 min/1.5 min/unlimited

For 30 cycles

PCR result success => this protocol can work

7/15

PCR for burkholderia acidpuldis to get fac-dex DNA sequence

7/16

Gradient PCR for burkholderia acidpuldis to get fac-dex DNA sequence

7/17

Try to fix PCR protocol

7/18

Give up PCR and wait for DNA sequence form IDT

Bacterial Culture (B.acidipaludis)

7/20

Measuring growth curve(B.acidipaludis, E. coli)

Bacterial Culture (B.acidipaludis)

7/21

Bacterial Culture(E. coli in LB)

Bacterial Culture (E. coli in LB+DMSO)

7/22

Bacterial Culture (E. coli in LB)

Bacterial Culture (E. coli in LB+DMSO)

7/26

Bacterial Culture (B.acidipaludis)

Bacterial Culture (E. coli in MeOH/PFOA)

Bacterial Culture (E. coli in MeOH)

7/27

Bacterial Culture (E. coli in LB)

7/30

Bacterial Culture (B.acidipaludis)

August

8/1

Transformation (DH5α)

8/2~8/3

Pick up colon and plasmid miniprep:

[BBa_E0040]

[BBa_J04500]

[pSB1C3]

[BBa_k592016]

[BBa_J23119]

Cell test

Making Gel

Bacterial Culture (B.acidipaludis)

Making Gel

Measuring OD(Bacteial culture at 7/21, 7/22, 7.26, 7/27)

Pick up colon

8/4

Change the experiment kit and try again

[BBa_J23119]

[BBa_J04500]

[pSB1C3]

[BBa_k592016]

[BBa_E0040]

Miniprep

8/5

Digestion:

[pSB1C3] cleaved by EcoR1 and Pst1

[pSB1C3] cleaved by Xba1 and Pst1

[Facdex-1] cleaved by Xba1 and Pst1I

[BBa_J04500] cleaved by EcoR1 and Spe1

[Facdex-3] cleaved by Xba1 and Pst1

[BBa_B0012] cleaved by Xba1 and Pst1

[BBa_E0040] cleaved by EcoR1 and Spe1

[J23119] cleaved by EcoR1 and Spe1

[BBa_k592016] cleaved by Xba1 and Pst1

Bacterial Culture (B.acidipaludis)

8/6

Ligation:

[pSB1C3], [BBa_J04500], [Facdex-1]

[pSB1C3], [BBa_J04500], [Facdex-3]

[pSB1C3], [BBa_E0040], [BBa_B0012]

[pSB1C3], [BBa_J23119], [BBa_k592016]

Transformation:

[pSB1C3], [BBa_J04500], [Facdex-1]

[pSB1C3], [BBa_J04500], [Facdex-3]

[pSB1C3], [BBa_E0040], [BBa_B0012]

[pSB1C3], [BBa_J23119], [BBa_k592016]

8/7

Amplify:

[pSB1C3]

[BBa_J23119]

Pick up colony:

[pSB1C3], [BBa_J04500], [Facdex-1]

T[pSB1C3], [BBa_J04500], [Facdex-3]

[pSB1C3], [BBa_E0040], [BBa_B0012]

[pSB1C3], [BBa_J23119], [BBa_k592016]

8/8

plasmid miniprep:

[pSB1C3], [BBa_J04500], [Facdex-1]: ligation 1-1

[pSB1C3], [BBa_J04500], [Facdex-3] : ligation 1-2

[pSB1C3], [BBa¬_E0040], [BBa_B0012] : ligation 2-1

[pSB1C3], [Facdex-1]: ligation 3-1

[pSB1C3], [Facdex-3]: ligation 3-2

[pSB1C3],ddH₂O: ligation 3-3

[pSB1C3], [BBa_J23119], [BBa_k592016]: : ligation 4-1

Pick up colony:

[pSB1C3], [BBa_J23119]

8/9

plasmid miniprep:

[pSB1C3], [BBa_J23119]

Bacterial Culture (B.acidipaludis)

8/15

Amplify:[BBa¬_K1323009],[BBa_E1010],[BBa_J06504],[BBa_J04450],[BBa_E0040],[BBa_K592016]

8/16

Digestion: [Ligation 1-1] cleaved by EcoR1 and Spe1, [Ligation 2-1] cleaved by Xba1 and Pst1, [pSB1C3] cleaved by EcoR1 and Pst1, [Ligation 4-1] cleaved by EcoR1 and Spe1, [BBa_B0012] cleaved by EcoR1 and Xba1

Pick up colony:[BBa¬_K1323009],[BBa_E1010],[BBa_J06504],[BBa_J04450],[BBa_E0040],[BBa_K592016]

8/17

Ligation:
[Ligation 1-1] and [Ligation 2-1]: kit1
[Ligation 4-1] and [BBa_B0012]: ligation 2

Transformation:
[Ligation 1-1] and [Ligation 2-1]: kit 1
[Ligation 4-1] and [BBa_B0012]: ligation 2
[BBa¬_ E0040]
[BBa_B0012]
[BBa_J04450]

8/18

Pick up colony:
[ligation 1]
[ligation 2]
[BBa_B0012]

8/25

Amplify:
[BBa¬_ E0040]
[BBa_J04450]

8/26

Measuring growth curve and GFP (Elisa)

8/30

Bacterial Culture (B.acidipaludis)

September

9/1

Bacterial Culture (B.acidipaludis)

Transform Fac-dex+GFP plasmid to BL21 and MG1655

9/8

Amplify:

[BBa_K1184000]

[eGFP-EC] in BL21 competent cell

[eGFP-EC] in DH5αcompetent cell

Measuring growth curve(DH5α, BL21, MG1655 in PFOA)

9/9

Pick up colony:

[BBa_K1184000]

[eGFP-EC] in BL21 competent cell

[eGFP-EC] in DH5αcompetent cell

9/10

Digestion:

[Facdex-1] cleaved by Spe1 and Pst1

[J04500] cleaved by Xba1 and Pst1

Transformation:

[Kit1] in BL21 competent cell

[Kit1] in mg1655 competent cell

Electrophoresis

9/11

Ligation:

[Facdex-1] cleaved by Spe1 and Pst1 and [J04500] cleaved by Xba1 and Pst1

Transformation:

[Facdex-1] cleaved by Spe1 and Pst1 and [J04500] cleaved by Xba1 and Pst1

9/12

Pick up colony

[Facdex-1] cleaved by Spe1 and Pst1 and [J04500] cleaved by Xba1 and Pst1

9/13

Plasmid miniprep

[Facdex-1] cleaved by Spe1 and Pst1 and [J04500] cleaved by Xba1 and Pst1

9/21

Transformation:

[Kit1] in mg1655 competent cell for protein expression

9/22

Pick up colony and culture overnight:

[Kit1] in mg1655 competent cell for protein expression

9/23

Transfer bacteria to small tube and culture for three hours

Induce [Kit1] protein expression after adding IPTG

SDS page for [Kit1]

October

10/4

Amplify:

[BBa_J04450]

[BBa¬_J61100]

[BBa¬_I13452]

October

10/5

Pick up colony:

[BBa_J04450]

[BBa¬_J61100]

[BBa¬_I13452]

10/6

Plasmid miniprep:

[BBa_J04450]

[BBa¬_J61100]

[BBa¬_I13452]

10/10

Digestion:

[Facdex-1] cleaved by Xba1 and Pst1

[Facdex-3] cleaved by Xba1 and Pst1

[BBa_J04450] cleaved by EcoR1 and Spe1

10/11

Ligation:

[Facdex-1] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1

[Facdex-3] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1

Transformation:

[Facdex-1] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1

[Facdex-3] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1

10/13

Digestion:

[CrcB-La1] cleaved by Xba1 and Pst1, [CrcB-La2] cleaved by Xba1 and Pst1, [CrcB-Bpe] cleaved by Xba1 and Pst1, [CrcB-flc] cleaved by Xba1 and Pst1, [PSB1A2] cleaved by Xba1 and Pst1, [PSB1A3] cleaved by Xba1 and Pst1, [Fac-dex1] cleaved by Xba1 and Pst1, [Fac-dex3] cleaved by Xba1 and Pst1, [PSB1C3] cleaved by Xba1 and Pst1

Plasmid miniprep:

[Facdex-1] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1, [Facdex-3] cleaved by Xba1 and Pst1 and [BBa_J04450] cleaved by EcoR1 and Spe1

10/14

Ligation:

[CrcB-La1] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-La2] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-Bpe] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[Fac-dex1] and [PSB1A2],[Fac-dex3] and [PSB1A2]

Transformation:

[CrcB-La1] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-La2] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-Bpe] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[Fac-dex1] and [PSB1A2],[Fac-dex3] and [PSB1A2]

10/15

Pick up colony:

[CrcB-La1] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-La2] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[CrcB-Bpe] cleaved by Xba1 and Pst1 and [PSB1A2] cleaved by Xba1 and Pst1,[Fac-dex1] and [PSB1A2],[Fac-dex3] and [PSB1A2]

Ligation:

[PSB1C3] and [pure Fac-dex1], [PSB1C3] and [pure Fac-dex3], [PSB1C3] and [Fac-dex1 kit], [PSB1C3] and [Fac-dex3 kit], [PSB1C3] and [CrcB-La1], [PSB1C3] and [CrcB-La2], [PSB1C3] and [CrcB-Bpe], [PSB1C3] and [CrcB-flc],

10/16

Miniprep

Crcb-la1 in PSB1A2, Crcb-la2 in PSB1A2, Crcb-flc in PSB1A2, Crcb-bpe in PSB1A2, Fac-dex1 in PSB1A2, Fac-dex3 in PSB1A2,

Pick up colon

Fac-dex1 in PSB1C3, Fac-dex3 in PSB1C3, Crcb-la1 in PSB1C3, Crcb-la2 in PSB1C3, Crcb-flc in PSB1C3, Crcb-bpe in PSB1C3, Ligation 2-1 (GFP+ter)

Digest check

Fac-dex1 in PSBA3, Fac-dex3 in PSBA3, Fac-dex in PSBA2, Fac-dex in PSBA2

10/17

Miniprep

Fac-dex1 in PSB1C3, Fac-dex3 in PSB1C3, Crcb-la1 in PSB1C3, Crcb-la2 in PSB1C3, Crcb-flc in PSB1C3, Crcb-bpe in PSB1C3, Ligation 2-1 (GFP+ter)

Fac-dex function tset

Crcb function test

10/18

Miniprep

Fac-dex1+GFP, Fac-dex3+GFP