Team:NCTU Formosa/notes

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Notes

The experimental log that records down the purpose, method, result and date of each experiment that we have conducted.

About the notes

Our notes are the abbreviations of the experimental process, including titles, genes we transformed, plasmids we used, and resistances on the plasmids. We categorized these notes by date. Moreover, team members followed the iGEM protocol in each experimental step in order to get better experimental efficiency and the results we had expected.

March 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

24

25

26

27

28

29

Transformation of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) and cultivation on three LB plates.
transformation of PompC and cultivation on LB-A plate

30

Single colony isolation from three plates and cultivation them in liquid LB
Single colony isolation from PompC LB-C plate and cultivation in liquid LB-A
mini-prep of cultivated PompC E.coli

31

Transformation of RBS B0030+PSB1A3&B0034+PSB1A3 and cultivation on LB-A plate.
Mini-prep for cultivated ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) PCR of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar).
Electrophoresis of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) ──NOT OK.
PCR of insert fragment [PompC+psB1A2]

April 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

2

Mini-prep of cultivated B0030&B0034 E.coli and then digestion: [B0030]XP&[B0034]XP.

3

4

5

6

Electrophoresis of [B0030]XP&[B0034]XP Not OK.
PCR of ho1 to check ho1 and transform to test the resistance.

7

Electrophoresis of [B0030]mini&dig XP and [B0034]mini&dig XP OK

Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

Cultivation of ho1 in liquid LB-K and LB-K plate

Cultivation of Cph8+RBS in liquid LB-C.

8

Mini-prep of cultivated B0030&B0034 Ar E.coli. Mini-prep of ho1 & Cph8+RBS.

9

Electrophoresis of mini ho1&Cph8+RBS.

10

11

PCR of mini ho1&Cph8+RBS
Electrophoresis of ho1&Cph8+RBS(After PCR)
Digestion: [ho1]EP&[Cph8+RBS]EP.

12

Transformation of Plac&Ptet& pcyA but there is no colony appearing in pcyA plate.
Electrophoresis of digested ho1&Cph8+RBS (NOT OK).

13

14

Cultivation of Ptet&Plac E.coli in LB tubes.
Transformation: LuxR,B0015,J61048 and cultivation on LB-A plate

15

Mini-prep of cultivated Ptet&Plac E.coli
Digestion:[Ptet]ES&[Plac]ES PCR of insert fragment pcyA
Electrophoresis of [Ptet]dig ES&[pcyA]PCR&[Plac]dig ES.
Single colony isolation from J61048,B0015,LuxR plate and cultivation in liquid LB-A mini-prep of cultivated LuxR,J61048,B0015

16

PCR of mini Cph8+RBS
Electrophoresis of Cph8+RBS(After PCR)──NOT OK.
digestion: [J61048]EP,[B0015]EP,[LuxR]EP
Electrophoresis of ter. mini [J61048] dig E/EP and t er. mini [B0015] dig E/EP and luxR mini dig E/EP

17

PCR of mini Cph8+RBS Electrophoresis of Cph8+RBS(After PCR)──NOT OK.
Electrophoresis of the products of digestion of ter. [J61048], ter. [B0015] , luxR
transformation of tetR plasmid and cultivation on LB –A plate 3. Digestion: ter. [J61048], ter. [B0015] dig EP

18

19

Cultivation of pcyA Kr E.coli on LB-K plates
mini-prep of cultivated tetR E.coli
digestion: tetR+pSB1A2 dig EP
electrophoresis of tetR dig EP,ter.[J61048 ] dig EP and ter.[B0015] dig EP

20

Cultivation of pcyA Kr E.coli in liquid LB-K tubes.
PCR of tetR,ter.[J61048] and ter.[B0015 ]mini
electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini

21

Single colony isolation of pcyA Kr E.coli
Mini-prep of cultivated pcyA Kr E.coli
Digestion:[pcyA]ES
Electrophoresis of [pcyA]mini&dig ES OK
PCR of tetR,ter.[J61048] and ter.[B0015 ]mini
electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini
electrophoresis of the digestion products of tetR dig EP and B0015 dig EP
aliquot every 20ul of PompC,J61048 and LuxR

22

23

24

25

26

27

28

Transformation of PompC mini and cultivation on LB-A plate

29

transformation of PompC , Ar mini and cultivation on LB-A plate
single colony isolation from PompC , Ar

30

Transformation of 37ﾟC RBS and ho1.
Mini-prep of cultivated PompC , Ar E.coli

May 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

digestion : pLac [EP] & pSB1K3 [EP]
ligation :insert pLac [EP]/Vector pSB1K3 [EP]
transformation of pLac+pSB1K3 and cultivation on LB-K plate

2

PCR of insert fragment [pLac+pSB1K3] OK
DNA Sequencing OK
transformation of B0034_new and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

3

mini-prep of cultivated B0034 E. coli
digestion : B0034 [SP]
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS
cultivation on LB-A plate

4

PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation
transformation of 37℃ RBS and cultivation on LB-C plate

5

6

7

8

transformation of 37℃ RBS and cultivation on LB-Kplate

9

Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K

10

digestion : B0034 [ES] & pSB1C3 [EP]
mini-prep of cultivated 37℃ RBS E. coli

11

ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]
transformation of B0034+Zif268+AlsS and cultivation on LB-C plate

12

digestion : pLac [ES] & pLac [SP]
digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]
ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]
ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]

13

14

15

PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing OK
transformation of B0034+ HivC and cultivation on LB-A & LB-K plate

16

17

18

Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A
Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C
Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K

19

mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS
digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP]
ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]
ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]
transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate
mini-prep of cultivated B0034+HivC (Ar & Kr)

20

PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK
DNA Sequencing 3 parts OK

21

digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]

22

23

digestion : HivC+ilvD-3,18 [EP]
ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]

24

Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A

25

mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli
transformation of ilvD+37℃ RBS and cultivation on LB-C plate

26

PCR of insert fragment [ilvD+37℃]-----OK
DNA Sequencing OK

27

28

29

30

Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K
ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]

31

mini-prep of cultivated PBSII+ilvC E. coli
digestion : pSB1C3 [EP] & PBSII+ilvC [XP]
ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]
transformation of B0034+PBSII+ilvC and cultivation on LB-C plate
transformation of B0034+HivC and cultivation on LB-A plate

June 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

PCR of insert fragment [B0034+PBSII+ilvC] OK!
DNA Sequencing OK!

2

3

mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli

mini-prep of cultivated HivC+ilvD-12,20 E. coli

4

digestion : HivC+ilvD-12,20 [XP]

5

Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)

6

digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]
ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]
ligation : insert HivC [EP]/vector pSB1C3 [EP]
transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate

7

PCR of insert fragment [B0034+ HivC] & [HivC] OK

8

Design the primer for B0034+PBSII+ilvC point mutation (pm)
Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C

9

mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]

10

transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed

11

DNA Sequencing B0034+HivC & HivC OK
PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK

12

Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A
Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A

13

mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better

14

PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK

15

DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK

16

17

18

19

PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK
digestion: pLac+B0034+Zif268+AlsS [DPn1]

20

transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate

21

Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A

22

mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli
digestion : pLac+B0034+Zif268+AlsS (pm) [EP]

23

DNA sequencing OK

24

25

PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK
digestion : B0034+ PBSII+ilvC [DPn1]

26

transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail

27

PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

28

digestion : B0034+ PBSII+ilvC [DPn1]

29

transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate

30

PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK
Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C

July 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli
digestion : B0034+ PBSII+ilvC (pm) [EP]

2

DNA sequencing OK

3

4

5

6

7

digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]

8

9

Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
digestion : pSB1K3 [EP]
ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]
transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate

10

Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR
Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]
PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK
SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K

11

mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli
DNA sequencing OK
culture condition test: activation DH5αovernight

12

transfer to new medium(1/100), OD0.2 start counting culture time
transfer to 30゜C and 27゜C

13

transfer to 30゜C and 27゜C

14

15

transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate

16

PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK

17

Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]
Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]

18

19

transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)

20

PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK
Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A

21

mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli
Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]
ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]

22

transformation of G1+G2,and cultivation on LB- C plate failed

23

ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]

24

transformation of G1+G2,and cultivation on LB- C plate
Digestion: G2’(B0034+HIVC+ilvD) [XP]
ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

25

PCR of insert fragment [kivD+B0015]
transformation of G1+G2’, and cultivation on LB- C plate failed

26

Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C
sample and do GC

27

mini-prep of cultivated kivD+B0015 E. coli

28

29

Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]
Digestion: G2’ [DPn1]

30

transformation of G2’, and cultivation on LB- A plate

31

Digestion: kivD+B0015 [EP] (checking bp----failed)
Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A

August 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

mini-prep of cultivated G2’ E. coli

2

3

4

5

6

7

8

9

Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]
ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

10

strain test: activation of different strains overnight

11

transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C

12

Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C
transfer to 27゜C

13

mini-prep of cultivated G1+G2’ E. coli

14

15

Digestion: Ptet+B0032[ES] & GliI+KivD[XP]
sample and do GC

16

ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]
Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C

17

transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate
mini-prep of cultivated G1+G2 E. coli

18

PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK
DNA sequencing------NOT OK

19

20

21

22

23

24

carbon source test: activation DH5α overnight

25

transfer to new medium(1/100), OD0.2 start counting culture time

26

culturing for 72hours, inject feeding solution per 24hours

27

culturing for 72hours, inject feeding solution per 24hours

28

transformation of DNA program, and cultivation on LB- A plate
culturing for 72hours, inject feeding solution per 24hours

29

ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]
Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C
sample & do GC

30

transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate
mini-prep of cultivated DNA program E. coli
Do GC

31

PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK
DNA sequencing NOT OK

September 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]

2

transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate

3

4

digestion : [pSB1K3] EP
digestion : [pSB1K3] EP

5

6

7

PCR of insert fragment [Zif268+AlsS] OK
DNA Sequencing OK
ligation : point mutation HivC
TA cloning : point mutation HivC

8

9

Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K

10

mini-prep of cultivated Zif268+ AlsS E. coli
transformation of point mutation HivC and cultivation on LB-A plate
transformation of B0034 and cultivation on LB-A plate

11

12

Single colony isolation from HivC LB-A plate, and in liquid LB-A
Single colony isolation from B0034 LB-A plate, and in liquid LB-A

13

mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli

14

digestion : Zif268+AlsS [XP]

15

16

17

Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A

18

mini-prep of cultivated ilvD E. coli
digestion : ilvD [EP] & pSB1K3 [EP]
transformation of 37℃ RBS and cultivation on LB-C plate

19

Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

20

digestion : B0034 [SP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]
mini-prep of cultivated Hivc E. coli

21

transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
transformation of HivC and cultivation on LB-A plate

22

PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing NOT OK
digestion : B0034 [SP] & Zif268+AlsS [XP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A
SCI from ilvD LB-K plate, and cultivation in liquid LB-K

23

PCR of insert fragment [B0034+Zif268+AlsS] NOT OK
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
mini-prep of cultivated B0034 and ilvD E. coli
Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

24

mini-prep of cultivated HivC E. coli
digestion : B0034 [SP] & ilvD [XP]

25

digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]
ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]
ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]

26

transformation of HivC+ilvD and cultivation on LB-A & LB-C plate

27

PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

28

transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate
PCR of insert fragment [HivC+ilvD] OK
DNA sequencing NOT OK

29

Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C

30

mini-prep of cultivated & pSB1C3 E. coli