Team:BGU ISRAEL/Notebook

PlastiCure

Notebook



October
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30 31
September
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11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30
August
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28 29 30 31
July
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10 11 12 13 14 15 16
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31
June
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
May
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
April
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
March
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20 21 22 23 24 25 26
27 28 29 30 31

When - 18/10/16

Who's In the lab today: Eyal and Dor

Colony PCR for the dialysis bag experiment.
We used 2 genes to identify if e.coli and putida can grow together, 

aldA (1440bp) gene for e.coli and pcaD (792bp) gene for putida.

 

aldA gene of E. coli, plates 3,7,10,1,6 were positive

pcaD gene of P. putida, plates 8,2,3,5,1 were positive

When - 17/10/16

Who's In the lab today: Eyal, Dor, Liran and Tomer

Ligation of 7K transporter inserts and pSEVA 434 vector

 

50ng vectur cut with EcorI and PstI

142ng insert cut with EcorI and PstI

2ul T4 DNA ligase Buffer X10

1ul T4 DNA ligase

add UPW for total volum of 20ul

 

16C over night

 

Transformation of 7K trensporter + pSEVA 434 Ligation mix

Tubes of compatent BL21

1ul from each plasmid for each tubes and one tube without plasmid.

Mix with tip.

Incubation in ice for 20 min.

Heat shock 30 sec 42C

Incubation in ice for 3 min

800ul SOC without antibiotic

1h in 37C

-centrifugation 5000 rpm 2min

-take off 500ul from the liquid.

-mix the residue with the rest liquid.

-sowing in LB+antibiotic

Incubation 37C O/N

When - 15/10/16

Who's In the lab today: Eyal and Dor

TPA detection assay

Took 2 sample of 200uL one inside the bag, and one outside the bag into 96 wellplate

 

TPA detection for kinetics measurements:

We transformed WT and variants LC-Cutinase to E. coli BL21 and we raised each one of them in M9+PET medium+CMP+IPTG.

We took samples of 200uL daily from each Erlenmeyer into 96 wellplate anti UV.

We took the plate to a UV table for 1 hour, and insert it into a plate reader for checking the absorption according to the time and date samples, and the change in TPA concentration in time according to our TPA detection protocol. The results show significant raising in the absorption but we didn’t succeed to detect the TPA.

The reasons why we didn’t succeed to detect TPA can be contamination of the medium with another carbon source or maybe because we didn’t filter the samples so there are some materials that can cause quenching and disturb the fluorescence.

 

When - 14/10/16

Who's In the lab today: Eyal and Dor

TPA detection assay

Took 2 sample of 200uL one inside the bag, and one outside the bag into 96 wellplate.

When - 13/10/16

Who's In the lab today: Tomer

Restriction reaction for pSEVA 434

The total reaction mix volum were 50ul.

every reaction contain:

1000ng DNA

1ul of every enzyme.

5ul buffer

 

 

Plasmid

E1

E2

Buffer

1

434

AvrII

-

3.1

2

434

EcorI

-

3.1

3

434

PstI

-

3.1

4

434

EcorI

PstI

3.1

5

434

-

-

3.1

When - 11/10/16

Who's In the lab today: Liran and Tomer

Produced pSEVA plasmids from starters using mini prep kit.

 

 

Concentration ng/ul

224

100

434

118

414

126

 

Restriction reaction for pSEVA 242, pSEVA 414 and pSEVA 434:

The total reaction mix volum were 50ul.

every reaction contain:

1000ng DNA

1ul of every enzyme.

5ul buffer

 

 

Plasmid

E1

E2

Buffer

1

434

AvrII

-

cutsmart

2

434

EcorI

-

cutsmart

3

434

PstI

-

cutsmart

4

434

EcorI

PstI

cutsmart

5

434

SpeI

SacII

cutsmart

6

434

-

-

cutsmart

9

414

SpeI

-

cutsmart

10

414

SacII

-

cutsmart

11

414

SacII

SpeI

cutsmart

12

414

-

-

cutsmart

13

224

AvrII

-

cutsmart

14

224

EcorI

-

cutsmart

15

224

PstI

-

cutsmart

16

224

EcorI

PstI

cutsmart

17

224

-

-

cutsmart

 

1h 37C

20min 65C

 

When - 10/10/16

Who's In the lab today: Liran and Inbar B

Colony-PCR for DH10B competent cells after transformation with ligation mix of 7K trensporter + pSEVA 434

 

 

Templat

pr Rev

pr Fw

taq readymix

UPW

colony

colony

1ul

1ul

10ul

7ul

7K insert posetiv control

5ul 7K insert

1ul

1ul

10ul

3ul

no templat

-

1ul

1ul

10ul

8ul

 

Gibson assembly of EG insert and pSEVA434 vector with Gibson assembly kit

Total volum 5ul

 

EG insert after DpnI

pSEVA434

posetive control mix

Gibson assembly mix

UPW

EG + 434

45ng

27ng

-

2.5ul

-

434 no insert- negativ control

-

27ng

-

2.5ul

1ul

Posetive control from the kit

-

-

2.5ul

2.5ul

-

 

20min 50C

 

Transformation of GA reaction mix:

Tubes of compatent DH10B

1ul from each plasmid for each tubes.

Mix with tip.

Incubation in ice for 20 min.

Heat shock 30 sec 42C

Incubation in ice for 3 min

800ul SOC without antibiotic

1h in 37C

-centrifugation 5000 rpm 2min

-take off 500ul from the liquid.

-mix the residue with the rest liquid.

-sowing in LB+antibiotic

Incubation 37C O/N

 

5 colonys in posetive control, no colonys on the other plates.

When - 09/10/16

Who's In the lab today: Liran, Eyal and Dor

We sent 4 samples contain cutinase WT, R7, F4 and CO for mass spec.

Transformation of 7K trensporter + pSEVA 434 Ligation mix

Tubes of compatent DH10B

1ul from each plasmid for each tubes and one tube without plasmid.

Mix with tip.

Incubation in ice for 20 min.

Heat shock 30 sec 42C

Incubation in ice for 3 min

800ul SOC without antibiotic

1h in 37C

-centrifugation 5000 rpm 2min

-take off 500ul from the liquid.

-mix the residue with the rest liquid.

-sowing in LB+antibiotic

Incubation 37C O/N

 

Produced pSEVA plasmids from starters using mini prep kit.

 

 

Concentration ng/ul

414 stab 2

69

414 col2

72

414 col2

61

414 stab 2

130

434 stab

136

224 gly

195

 

Restriction reaction for pSEVA 242, pSEVA 414 and pSEVA 434:

The total reaction mix volum were 50ul.

every reaction contain:

1000ng DNA

1ul of every enzyme.

5ul buffer

 

 

Plasmid

E1

E2

Buffer

1

434 stab

AvrII

-

cutsmart

2

434 stab

EcorI

-

2.1

3

434 stab

PstI

-

3.1

4

434 stab

EcorI

PstI

2.1

5

434 stab

SpeI

-

cutsmart

6

434 stab

SacII

-

cutsmart

7

434 stab

SacII

SpeI

cutsmart

8

434 stab

-

-

cutsmart

9

414 stab 2

SpeI

-

cutsmart

10

414 stab 2

SacII

-

cutsmart

11

414 stab 2

SacII

SpeI

cutsmart

12

414 stab 2

-

-

cutsmart

13

224 gly

AvrII

-

cutsmart

14

224 gly

EcorI

-

2.1

15

224 gly

PstI

-

3.1

16

224 gly

EcorI

PstI

2.1

17

224 gly

-

-

cutsmart

 

1h 37C

20min 65C

 

When - 08/10/16

Who's In the lab today: Eyal, Dor, Liran and Efrat

We prepared the dialysis bag experiment one again according to our protocol for kinetics measurements using our TPA detection assay- we will take sample from inside and outside the bag twice a day for 1 week and then we will expose it to UV light according to the TPA detection protocol to measure the kinetics index of cutinase CO on PET.

We used dialysis bag to check if e coli can live with P. putida- e coli inside the dialysis bag and putida outside of the dialysis bag in LB medium.- we will take samples inside the dialysis bag and outside the dialysis bag and sow it on  cam, kan, amp plate to see if they are still alive together (putida has a endogenic resistant to cam and amp, and we insert pACYC to e coli for cam resistant).

Restriction reaction for pSEVA 242, pSEVA 414 and pSEVA 434:

The total reaction mix volum were 50ul.

every reaction contain:

1000ng DNA

1ul of every enzyme.

5ul buffer

 

 

Plasmid

E1

E2

Buffer

1

434 stab 2

AvrII

-

cutsmart

2

434 stab 2

EcorI

-

2.1

3

434 stab 2

PstI

-

3.1

4

434 stab 2

EcorI

PstI

2.1

5

434 stab 2

-

-

cutsmart

6

434 stab 2

SpeI

-

cutsmart

7

434 stab 2

SacII

-

cutsmart

8

434 stab 2

SacII

SpeI

cutsmart

9

414 stab 2

SpeI

-

cutsmart

10

414 stab 2

SacII

-

cutsmart

11

414 stab 2

SacII

SpeI

cutsmart

12

414 stab 2

-

-

cutsmart

13

224 gly1

PstI

-

cutsmart

14

224 gly2

EcorI

-

2.1

 

1h 37C

20min 65C

 

Ligation of 7K transporter inserts and pSEVA 434 vector

 

50ng vectur cut with EcorI and PstI

142ng insert cut with EcorI and PstI

2ul T4 DNA ligase Buffer X10

1ul T4 DNA ligase

add UPW for total volum of 20ul

 

16C over nighet.

 

BioBricks: produced plasmids from starters using mini prep kit.

 

plasmid

concentration(ng/ul)

260\230

260\280

F4 col1

301

2.06

1.85

F4 col3

283

2.04

1.87

F7 col3

327

2.23

1.87

F7 col4

218

1.80

1.87

R4 col2

291

2.10

1.90

R4 col4

314

2.10

1.87

R7 col5

313

2.07

1.86

R7 col6

313

2.07

1.86

PcaC col1

267

1.92

1.88

When - 07/10/16

Who's In the lab today: Tomer, Efrat, Liran and Inbar B

Examine PET degradation bay LCC CO:

3 PET bids were incubate with 0.5L LB contaning cmp and BL21 with LCC CO on pACYC plasmid.

When OD reach 0.5-0.7 add IPTG.

For control, repeat with pACYC plasmid without insert, and without bacteria.

 

C.O

pAYAC

 

Testing BL21 that contains LCC variants groth on PET plates:

PET plates:

For 1L:

200ml M9 salts

2ml 1M mgSO4

20ml glucose 20%

100ul 1M CaCl2

(5ml soft agar for each plate)

The 19 plates:

 

IPTG &PET

IPTG no PET

PET no IPTG

Glucose

F4

V

V

-

-

F7

V

V

-

-

R4

V

V

-

-

R7

V

V

V

-

CO

V

V

V

-

pACYC

V

V

V

-

WT

V

V

V

V

 

Produced pSEVA plasmids from starters using mini prep kit.

 

Concentration ng/ul

 

47

434 stab 1

85

434 stab 2

36

414 stab 1

159

414 stab 2

38

414 col1

121

414 col2

62

414 col3

370

414 col4

32

224 gly1

24

224 gly2

 

transformation of pSEVA 224 from gly1, pSEVA 414 from col2, pSEVA 414 from stab 2 and pSEVA 434 from stab 2:

Tubes of compatent DH10B

1ul from each plasmid for each tubes and one tube without plasmid.

Mix with tip.

Incubation in ice for 20 min.

Heat shock 30 sec 42C

Incubation in ice for 3 min

800ul SOC without antibiotic

1h in 37C

-centrifugation 5000 rpm 2min

-take off 500ul from the liquid.

-mix the residue with the rest liquid.

-sowing in LB+antibiotic

Incubation 37C O/N

 

PCR for amplification EG insert for Gibson with KAPA HIFI Hot Start, after previous reaction ended with Unspecific product

Template –EG 1ul to 49 ul UPW (EG first concentration- 572 ng\ul)

Primers- 10uM

Annealing temperature 70C, 1ul DMSO.

 

 

Upw (ul)

Pr fwd (ul)

Pr rev

template

KAPA Hiti hot start (ul)

EG

20

1.5

1.5

1

25

 

PCR to amplify TphA1, TphA2, TphA3 and TphB for Gibson assembly

Primers- 10uM

Annealing temperature 68C  

 

Upw (ul)

Pr fwd (ul)

Pr rev

template

KAPA Hiti hot start (ul)

TphA1

21

1.5

1.5

1

25

TphA2

21

1.5

1.5

1

25

TphA3

21

1.5

1.5

1

25

TphB

21

1.5

1.5

1

25

 

Using DpnI on EG, TphA1, TphA2, TphA3 and TphB to eliminate closed plasmid from the PCR product. pSB1C3 as control

 

44ul of PCR reaction mix

5ul x10 CutSmart buffer

1ul DpnI

37C for 1h

80C for 20min

 

TphA1- Unspecific product TphA2 -no product

When - 06/10/16

Who's In the lab today: Inbar B and Liran

Testing bacteriom growth on antibiotics stocks:

 

antibiotics

KT2440 from Glycerol stock

KT2440 from petri dish

Cmp

V

V

Cmp+Amp

V

V

Cmp+Strep

-

V

Spect

-

V

Cmp+Strep+Amp

-

-

Km

-

-

Strep X2

-

-

Spect X2

-

V

Control-no antibiotic

V

V

Amp

V

V

 

antibiotics

W.T

Control – no antibiotic

V

Cmp+Amp

V

Amp

V

Strep

-

 

 

antibiotics

E.Coli 224

Control – no antibiotic

V

Km 25 ug\ul

V

Km 50 ug\ul

V

Strep

-

Spect

-

 

Restriction reaction for pAC75 in order to get EG insert for Gibson after PCR product was Unspecific 

 

 

reaction 1

reaction 2

reaction 3

reaction 4

reaction 5

pAC75

3.5ul

3.5ul

3.5ul

3.5ul

5ul

cutsmart x10

10ul

10ul

10ul

10ul

-

UPW

83.5ul

85.5ul

85.5ul

85.5ul

-

KpnI

1ul

1ul

-

-

-

HindIII

1ul

-

1ul

-

-

AatII

1ul

-

-

1ul

-

 

1h 37C

20min 65C

 

When - 05/10/16

Who's In the lab today: Efrat and Liran

Preparing antibiotics stocks:

Spectinomycin 100mg\ml

cmp 34mg\ml

amp 100mg\ml

Kn 25mg\ml

 

Colony-PCR for DH10B competent cells after transformation with ligation mix of pSB1C3 and the inserts F4, F7, R4, R7 and CO:

 

template- toch colony, then reaction

 

Annealing temperature

 (C°)

Upw (ul)

Pr fwd (ul)

Pr rev

KAPA Hiti hot start (ul)

C.O

58.4

22

1.5

1.5

25

F4

58.4

22

1.5

1.5

25

F7

58.4

22

1.5

1.5

25

R4

58.4

22

1.5

1.5

25

R7

58.4

22

1.5

1.5

25

When - 04/10/16

Who's In the lab today: Inbar B, Tomer and Liran

PCR for amplification EG insert for Gibson with KAPA HIFI Hot Start, after previous reaction ended with Unspecific product

Template –EG 1ul to 49 ul UPW (EG first concentration- 572 ng\ul)

Primers- 10uM

Annealing temperature 68C

Using DpnI on EG insert to eliminate closed plasmid from the PCR product

 

44ul of EG PCR reaction mix

5ul x10 CutSmart buffer

1ul DpnI

37C for 1h

80C for 20min

 

Unspecific product

 

Restriction reaction for pSEVA 242, pSEVA 414, pSB1C3 and pUC19 as control:

The total reaction mix volum were 50ul.

every reaction contain:

1000ng DNA

1ul of every enzyme.

5ul buffer

Duplicates for every reaction.

 

 

Plasmid

E1

E2

Buffer

1

224

AvrII

-

cutsmart

2

224

EcorI

-

2.1

3

224

PstI

-

3.1

4

224

EcorI

PstI

2.1

5

224

DpnI

-

cutsmart

C1

224

-

-

cutsmart

6

414

SpeI

-

cutsmart

7

414

SacII

-

cutsmart

8

414

SpeI

SacII

cutsmart

9

414

DpnI

-

cutsmart

C2

414

-

-

cutsmart

10

pSB1C3

PstI

-

3.1

11

pSB1C3

EcorI

-

2.1

12

pSB1C3

EcorI

PstI

2.1

C3

pSB1C3

-

-

cutsmart

13

pUC19

PstI

-

3.1

14

pUC19

DpnI

-

cutsmart

C4

pUC19

-

-

cutsmart

 

1h 37C

20min 65C

 

 

transformation of LCC varients:

Tubes of compatent BL21

1ul from each plasmid for each tubes and one tube without plasmid.

Mix with tip.

Incubation in ice for 20 min.

Heat shock 30 sec 42C

Incubation in ice for 3 min

800ul SOC without antibiotic

1h in 37C

100ul from each tube to 5ml LB+5ul cmp.

Incubation 37C O/N

When - 03/10/16

Who's In the lab today: Efrat

BioBricks after PCR cleanup:

 

Concentration ng/ul

260/230

260/280

F7

313

1.96

1.88

R4

374

1.92

1.88

R7

294

2.00

1.9

F4

232

2.01

1.88

 

Restriction reaction for BioBriks inserts

 

250ng DNA (insert or PSB1C3 vector)

3ul NEB Buffer II

0.5ul PstI

0.5ul EcorI

add UPW for total volum of 20ul

 

1h 37C

20min 80C

 

BioBricks restriction product after PCR cleanup:

 

Concentration ng/ul

260/230

260/280

F7

92

1.49

1.88

R4

70

1.06

1.89

R7

91

2.01

1.88

F4

74

1.53

1.85

 

ligation of BioBriks F7, R4, R7 and F4 inserts and pSB1C3 vector

 

50ng vectur cut with EcorI and PstI

150ng insert cut with EcorI and PstI

0.5ul T4 DNA ligase Buffer

1ul T4 DNA ligase

add UPW for total volum of 50ul

 

16C over nighet.

 

Colony-PCR for DH10B competent cells after transformation with ligation mix of pSB1C3 and the inserts F4, F7, R4, R7 and CO:

 

template- toch colony, then reaction

 

Annealing temperature

 (C°)

Upw (ul)

Pr fwd (ul)

Pr rev

KAPA Hiti hot start (ul)

C.O

58.4

22

1.5

1.5

25

F4

58.4

22

1.5

1.5

25

F7

58.4

22

1.5

1.5

25

R4

58.4

22

1.5

1.5

25

R7

58.4

22

1.5

1.5

25

pACYC as negative control

58.4

22

1.5

1.5

25

 

When - 01/10/16

Who's In the lab today: Inbar B Eyal and Dor

Cutinase kinetics measurement:

We were trying to get the kinetics index of our cutinase- WT. F4, CO using the first velocity way (calculating the shape of the graph in the linear measurement) and exposing the V by that way.

After that, using the linear burk plot to reveal the kinetics index.

We tried to do this tests for more than 10 days, but we didn’t succeed to get a stable indexes. We tried to change the enzyme concertation to see measure the process more accurately, but still, we didn’t succeed to get stable indexes.

Using DpnI on EG insert to eliminate closed plasmid from the PCR product

 

44ul of EG PCR reaction mix

5ul x10 CutSmart buffer

1ul DpnI

37C for 1h

80C for 20min

 

When - 30/09/16

Who's In the lab today: Inbar B Liran and Tomer

PCR for amplification EG insert for doing Gibson reaction with KAPA HIFI Hot Start

Template –EG 1ul to 49 ul UPW (EG first concentration- 572 ng\ul)

Primers- 10uM

 

Restriction for pSEVA 224 and pSEVA414:

 

Plasmids sizes:

pSEVA 224 5250bp

pSEVA 414 3479bp

Transporter 4368bp

pUC19 2568bp

pSB1C3 2070 bp

 

For Gibson reactions:

Enzyme

Plasmid

Concentration

AvrII

pSEVA 224

112ng/ul

SpeI+SacII

pSEVA 414

173ng/ul

 

For restriction and ligation:

Enzyme

Plasmid

Concentration

EcoRI +PstI

pSEVA 224

112ng/ul

 

Enzyme

Buffer

EcoRI

3.1-100%

2.1-100% + star activity

PstI

3.1-100%

2.1-75%

AvrII

Cut smart-100%

1.1-100%

SacII

Cut smart-100%

2.1-100%

SpeI

Cut smart-100%

2.1-100%

 

Reactions:      

DNA

pSEVA224 10ul

pSEVA414 6ul

Buffer 3.1

5ul

Enzyme

1ul

DDW

pSEVA224 34ul

pSEVA414 38ul

Total

50ul

When - 27/09/16

Who's In the lab today: Eyal, Dor and Inbar B

Activity test for cutinase:

We tasted cleaned cutinase F4, CO and WT, and uncleaned cutinse F4, CO and WT in this test.

We used 96DW and a plate reader.

We added 50ul pNPB without cutinase, Uncleaned cutinase WT with pNPB (50ul and 50ul), Uncleaned cutinase R7 with pNPB (50ul and 50ul), Uncleaned cutinase F4 with pNPB (50ul and 50ul), pNPB and cleaned cutinase WT(50ul an 50ul), pNPB and cleaned cutinase

 

Restriction for pSEVA

 

Concentration ug/ul

260/230

260/280

pSEVA 224 EcoRI

50

3.14

1.72

pSEVA 224 EcoRI and PstI

67

0.82

1.70

pSEVA 414 EcoRI and PstI

90

1.99

1.86

PcaG EcoRI and PstI

63

0.97

1.74

pSEVA 514 EcoRI and PstI

19

0.30

1.41

EG

9

0.17

1.62

When - 26/09/16

Who's In the lab today: Liran and Tomer

Preparing Strep stock for P.putida

 

Stock concentration 100mg/ml

 

2g powder

20 ml UPW

 

Preparing TET stock for P.putida:

 

Stock concentration 10mg\ml

0.8g powder

30ml UPW

 

pSEVA plasmids concentration after restriction and cleanup:

 

pSEVA224 8.68 ng\ul

pSEVA224 after mini prep 84.48 ng\ul

pSEVA414 6.31 ng\ul

pSEVA414 after mini prep 3.01 ng\ul

pSEVA514 after mini prep 63.13 ng\ul

When - 25/09/16

Who's In the lab today: Efrat Liran and Tomer

BioBricks after PCR cleanup:

 

Concentration ug/ul

260/230

260/280

C.O

11.2

0.73

1.92

F4

235.05

1

1.98

F7

371.92

1.63

1.93

R4

57.18

0.38

2.18

R7

146.84

0.81

1.93

pCaH

106.59

0.86

1.98

pCaC

61.25

0.42

1.88

pCaD

27.55

0.12

2.32

pCaG

-

-

-

Transporter

254.54

1.55

1.93

Pseva 514

8.98

0.06

8.91

 

Ligation for pcaD\pcaH\pacC

 

PcaC

PcaD

PcaH

Control

Vector

2.8ul

28ul

28ul

28ul

Insert

1.4ul

2ul

3.1ul

-

Buffer

2ul

2ul

2ul

2ul

DDW

12.8ul

12.2ul

11.1ul

14.2ul

Ligase

1ul

1ul

1ul

1ul

 

Overnight 10C

Restriction- Transporter 7k (TPA)

DNA

10ul

Buffer 3.1

10ul

EcoRI

1ul

PstI

1ul

DDW

78ul

Total

100ul

 

Restriction time- 60 min in 37 °C

Inactivation- 20 min in 65°C

When - 24/09/16

Who's In the lab today: Inbar B Liran and Tomer

Produced the pSBIC3 plasmid from the starter and cleaned it using mini prep kit in 6 eppendorf:

 

Concentration ug/ul

260/230

260/280

1

215

2.04

1.85

2

401

2.09

1.88

3

272

1.88

1.84

4

346

2.14

1.87

5

265

2.15

1.89

6

263

2.12

1.87

 

PCR-Transporter for TPA (location 7K from iGEM plates)

 

Transporter

Control

Template

0.5ul

-

P.RW

1.5ul

0.75ul

P.FOW

1.5ul

0.75ul

Hot start

25ul

12.5ul

DDW

21.5ul

11ul

Total

50ul

25ul

 

PCR Program:

 

Temp

Time

Cycle

1.

95°C

3 min

1

2.

98°C

20 sec

25

3.

65°C

15 sec

25

4.

72°C

180 sec

25

5.

72°C

4 min

1

When - 22/09/16

Who's In the lab today: Efrat

Restriction:

Plasmid: pSB1C3

 

Plasmids

Buffer

EcoRI

PstI

SacI

DDW

Total

7.06ul

10ul

1ul

1ul

1ul

79.94ul

100ul

7.06ul

10ul

1ul

1ul

-

80.94ul

100ul

7.06ul

10ul

-

1ul

-

81.94ul

100ul

7.06ul

10ul

1ul

-

-

81.94ul

100ul

7.06ul

10ul

1ul

-

1ul

80.94ul

100ul

Gene

Buffer

EcoRI

PstI

SacI

DDW

Total

10ul

10ul

1ul

1ul

-

78ul

100ul

 

Concentration after clean up:

Gene

Concentration ng/ul

pcaH

5.34

pcaG

8

pcaD

-5.34

pcaC

19.99

C.O

-5.91

F4

9.59

F7

18.55

R4

12.10

R7

44.28

When - 21/09/16

Who's In the lab today: Ben and Inbar B

Runing pSEVA vectors on gel in order to confirm thire exsistens in old sempels

 

PCR clean up

 

gene

concentration(ng/ul)

260\230

pSB1C3 cut EcorI PstI

7

0.02

When - 20/09/16

Who's In the lab today: Inbar B, Ben, Liran Eyal and Dor

Prepering XL1 heat shock competent cells, after the previous XL1 competent cells fail at the test kit.

Repeat PCR to amplify TphA2 for Gibson assembly

template- old PCR product

 

Annealing temperature

(C°)

Upw (ul)

Pr fwd (ul)

Pr rev

template

KAPA Hiti hot start (ul)

TphA2

68

21

1.5

1.5

1

25

TphA2X

68

5.5

0.38

0.38

-

6.25

 

 

Runing pSEVA vectors on gel in order to confirm thire exsistens in old sempels

 

Repeat PCR to amplify LCC variants for BioBriks because efret used all the sempels

Primers 10 uM.

 

 

Annealing temperature

 (C°)

Upw (ul)

Pr fwd (ul)

Pr rev

template

KAPA Hiti hot start (ul)

C.O

58.4

21

1.5

1.5

1

25

F4

58.4

21

1.5

1.5

1

25

F7

58.4

21

1.5

1.5

1

25

R4

58.4

21

1.5

1.5

1

25

R7

58.4

21

1.5

1.5

1

25

 

Activity test for cutinase:

We tasted cleaned cutinase F4, R7 and WT, and uncleaned cutinse F4, R7 and WT in this test.

We used 96DW and a plate reader.

We added 100ul pNPB without cutinase, Uncleaned cutinase WT with pNPB (100ul and 100ul), Uncleaned cutinase R7 with pNPB (100ul and 100ul), Uncleaned cutinase F4 with pNPB (100ul and 100ul), pNPB and cleaned cutinase WT(100ul an 100ul), pNPB and cleaned cutinase R7(1 00ul an 100ul) and pNPB and cleaned cutinase F4(100ul an 100ul). ). [We duplicate every sample]

25/9

  • We prepared a stock of TPA 50 ml 20mM according to our protocol (TPA detection protocol)/ after that, we diluted the stock to get concentration of 15mM, 10mM, 5mM, 1mM, 0.5mM, 0.1mM, 0.05mM, 0.01mM, 0.005mM, 0.001mM.

We added 200uL from each concentration to a different wall in 96W anti-UV plate.

We exposed the plate to UV according to the protocol and search for fluorescence emission we saw in the literature.

After few days of testing this method we wrote the TPA detecting assay.

When - 19/09/16

Who's In the lab today: Inbar B, Ben and Efret

Produced plasmids from the starters using mini prep kit.

 

plasmid

concentration(ng/ul)

260\230

260\280

pSEVA 514

151

2.00

2.05

pSB1C col1

272

2.15

1.93

pSB1C col2

282

2.19

1.91

 

PCR to amplify TphA1, TphA2, TphA3 and TphB for Gibson assembly

 

iGEM competent cell test kit

XL1

BL21

 

Ligation of BioBriks inserts and pSB1C3 vector

 

50ng vectur cut with EcorI and PstI

150ng insert cut with EcorI and PstI

0.5ul T4 DNA ligase Buffer

1ul T4 DNA ligase

add UPW for total volum of 50ul

 

16C over nighet.

When - 18/09/16

Who's In the lab today: Tomer and Liran

PCR to amplify 7K transporter from plasmid

 

 

Annealing temperature

 (C°)

Upw (ul)

Pr fwd (ul)

Pr rev

template

KAPA Hiti hot start (ul)

7K transporter

68

21

1.5

1.5

1

25

RFP as posetiv control with pSV1C as template

68

21

1.5

1.5

1

25

When - 17/09/16

Who's In the lab today: Eyal and Dor

Dialysis experiment:

We prepared the dialysis experiment according to the “Dialysis bag experiment” using the starters we raised two day ago. We tested OD every day.

 

Date

time

Outside the dialysis bag

Inside the dialysis bag

17/9

13:00

0.09

0.108

18/9

13:30

0.1

0.31

19/9

12:15

0.1

0.4

20/9

14:00

0.107

0.42

21/9

15:00

0.1

0.24( the bag was fallen)

22/9

11:00

0.2

0.4

23/9

14:00

0.2

0.6

24/9

13:30

0.5

0.64

When - 15/09/16

Who's In the lab today: Idit, Eyal and Dor

the dialysis bag content was loded onto a cation seperation colon

 

We prepared starters with e-coli and pACYC.

 After one week we saw that the M9 outside of the dialysis bag was contaminated with e coli.

 

Transformation of pSEVA plasmids to P.putida KT2440 competent cells and groing cells on diffrent antibiotics in order to check the competent cells

  • Compatent P.putida KT2440 from -80C to ice for 10min.
  • 1 ul from each plasmid to each tube and a few tubes without plasmid(negative control on diffrent antibiotics)
  • mix with tip
  • incubate in ice for 20 min
  • heat shock 42C 3min.
  • incubate in ice for 3min
  • SOC 900ul without antibiotic
  • 37C for 2h
  • centrifugate 500rpm 2min
  • carrying of 900ul liquid from each tube.
  • mix the residue with the liquid
  • sowin on LB plates with relavent antibiotic.

 

number of times that bacterias grows on difrent antibiotics, after trensformesuon of diffrent plasmids.

When - 14/09/16

Who's In the lab today: Efrat

protein seperation

 

the solution was centefuges in 16000g  for an hour

the sediment was re-suspendent in TAE buffer pH 7.

the solution was inserted into a dialysis bag over night.

When - 13/09/16

Who's In the lab today: Efrat

Restriction reaction for BioBriks inserts

 

250ng DNA (insert or PSB1C3 vector)

3ul NEB Buffer II

0.5ul PstI

0.5ul EcorI

add UPW for total volum of 20ul

 

1h 37C

20min 80C

 

protein seperation

 

500 ml of each varient was centrifuged in 8000g for 10 minutes.

amonium solfate was added to the supernatant and incubated over night on a magnetic plate with a stirerer.

When - 12/09/16

Who's In the lab today: Efrat

Repeat PCR to amplify PcaH, PcaG ,PcaD, PcaC from P.putida KT2440

 

PCR clean up

 

gene

concentration(ng/ul)

260\230

PcaH

86

1.03

PcaG

96

0.98

PcaD

25

1.32

PcaC

81

1.39

protein seperation

 

the starters were diluted in 500 ml LB

After achieving 0.9-1 OD values IPTG was added and the incubated overnight in 37 degrees Celsius and agitation of 300 rpm

When - 11/09/16

Who's In the lab today: Inbar B and Efrat

Prepering P.putida KT2440 competent cells:

protein seperation

 

Three variants were chosen CO, F4, R7 for separation.

First a transformation into BL21 competents was performed

1 µl of plasmid was added into 50 µl of competent batch and incubated on ice for 30 min.

Heat shock for 30 sec in 42 degrees Celsius

Incubation for 3 min.

950 µl of SOC medium was add and placed into incubation for an hour in 37 degrees Celsius.

After incubation the bacteria were precipitated and them re-suspended in 200 µl in LB

Starters of 5 ml were made from the transformation to use in the following day

 

PCR clean up

 

gene

concentration(ng/ul)

260\230

PcaH

1

0.01

PcaG

28

0.6

PcaD

33

0.27

PcaC

30

0.2

CO

133

0.99

F4

123

0.93

F7

152

0.63

R4

140

0.38

R7

157

0.47

When - 10/09/16

Who's In the lab today: Eyal and Dor

Activity test for cutinase:

We tasted cleaned cutinase WT and uncleaned cutinase WT in this test.

We used 96DW and a plate reader.

In one wall we added 100ul pNPB without cutinase. In the second wall we added uncleaned cutinase WT with pNPB (100ul and 100ul), and in the third wall we added pNPB and cleaned cutinase WT (100ul an 100ul). [We duplicate every sample]

 

Dialysis experiment:

Transformation was done to insert pACYC with CMP resistant and cutinase WT gene into BL21 e-coli.

We prepared starters with this e-coli.

We prepared M9+PET (we crashed the PET with coffee grinder).

When - 06/09/16

Who's In the lab today: Inbar B and Efrat

PCR to amplify PcaH, PcaG ,PcaD, PcaC from P.putida KT2440 (05.09.16): for BioBriks

Primers 10 uM template- toch colony, then reaction.

KAPA Hiti hot start (ul)

Pr rev

Pr fwd (ul)

Upw (ul)

Annealing temperature

(C°)

    

25

1.5 PcaH

1.5 PcaH

22

61.6

PcaH

1

25

1.5

1.5

22

58.4

PcaG

2

25

1.5

1.5

22

62.3

PcaD

3

25

1.5

1.5

22

58.4

PcaC

4

6.25

0.38

0.38

5.5

61.6

HX

5

6.25

0.38

0.38

5.5

58.4

GX

6

6.25

0.38

0.38

5.5

62.3

DX

7

6.25

0.38

0.38

5.5

58.4

CX

8

KAPA Hiti hot start (ul)

template

Pr rev

Pr fwd (ul)

Upw (ul)

Annealing temperature

(C°)

  

25

1

1.5

1.5

21

58.4

C.O

25

1

1.5

1.5

21

58.4

F4

25

1

1.5

1.5

21

58.4

F7

25

1

1.5

1.5

21

58.4

R4

25

1

1.5

1.5

21

58.4

R7

6.25

-

0.38

0.38

5.5

58.4

C.OX

6.25

-

0.38

0.38

5.5

58.4

F4X

6.25

-

0.38

0.38

5.5

58.4

F7X

6.25

-

0.38

0.38

5.5

58.4

R4X

6.25

-

0.38

0.38

5.5

58.4

R7X

When - 04/09/16

Who's In the lab today: Everybody!

Successfully ligated:

 

Sample

Ligated to pSB1C3

pcaC

 

pcaD

Yes (Sample 2)

pcaG

 

pcaH

 

CO

 

R4

 

R7

Yes (Sample 6)

F4

 

F7

 

When - 29/08/16

Who's In the lab today: Everybody!

we made PET plates: For 1L:
  1. 200ml M9 salts
  2. 2ml 1M mgSO4
  3. 20ml glucose 20%
  4. 100ul 1M CaCl2
  5. (5ml soft agar for each plate)
the plate that we made:
50 100 250 500 no PET Glucose

When - 17/08/16

Who's In the lab today: Everybody!

We concentrated the cutinase with centricons.

When - 11/08/16

Who's In the lab today: Everybody!

  • we made M9 plates without carbon source.
  • we made a new stock for chloramphenicol.
  • we sow from the rhodococus experiment on natrient broth plates.
gel weight:
pcaC pcaD pcaG pcaH CO R4 R7 F4 F7
0.2816 0.2801 0.1882 0.2811 0.3890 0.2891 0.2882 0.3887 0.2855

Nano drop sup: (calibration by pACYC)
Cutinase variantsProtein concentration
WT 0.83
CO 0.43
R4 -0.21
F4 -0.37
R7 -0.09
F7 -0.95

protein concentration after centrecon:
Cutinase variantsProtein concentration
WT 4.46
R4 2.41
F4 2.23
R7 2.54
F7 2.41
pACYC 3.9

When - 10/08/16

Who's In the lab today: Everybody!

BL21 growth and induction for expression LCC for activity test:
Cutinase variants14:3715:15
pACYC 0.215 0.458
WT 0.267 0.497
CO 0.236 0.440
R4 0.221 0.413
F4 0.236 0.446
R7 0.195 0.376
F7 0.208 0.401
15:35-5ul IPTG 1M for each 5ml preparation of IPTG:
  • 0.1gr +420ul DDW
  • fiber with 0.22um filter.

When - 09/08/16

Who's In the lab today: Everybody

Cutinase experiment:
nano-drop protein measurement (calibration by pACYC)
Cutinase variantsProtein concentration
pACYC 0
WT 0.22
CO 2.16
R4 -1.68
F4 -0.37
R7 0.25
F7 0.4
F4 0.14
BL21 0.76

When - 070816

Who's In the lab today: Everybody!

Competent cells
  1. 5ml LB were taken from erlenmeyer 500ml for calibration the spectophotometer before O.D test.
  2. dilution 5ml BL21 culture starters to 500 ml LB.
  3. when the O.D reach to 0.4-0.6 we mix and save in ice for 10min
  4. we transfer the LB to surbal tubes.
  5. centrifuge 10 min, 6000 rpm, 4C
  6. we removed the sup and gentle fluidization in 25ml solution A for 125ml culture.
  7. 30min incubation in ice.
  8. centrifuge 5min, 5000, 4C
  9. gentle fluidization all the plet in 10ml solution B.
  10. we divided the solution to ependorf, 50 ul for each ependorf and throw it quickly to liquid nitrogen.
transformation for each LCC variants and control for activity test:
  1. we took 8 tubes of competent BL21.
  2. 1ul from each plasmid for each tubes and one tube without plasmid.
  3. mix with tip.
  4. incubation in ice for 20 min.
  5. heat shock 30 sec 42C.
  6. incubation in ice for 3 min.
  7. 800ul SOC without antibiotic.
  8. 1h in 37C.
  9. 100ul from each tube to 5ml LB+5ul cmp. for negative control-plate without antibiotic.
  10. incubation 37C O/N.

When - 01/08/16

Who's In the lab today: Everybody!

the starters diluted to a new starter. O.D tests before the induction:
Cutinase variantsO.D 14:00
pACYC 0.286
WT 0.240
CO 0.239
R4 0.244
F4 0.250
R7 0.252
F7 0.225
F4 0.14
BL21 0.438

when we checked the O.D at 15:30 all the samples O.D where more than 1.0. we assumed that something is wrong with this experiment, maybe the O.D check in 14:00

When - 31/07/16

Who's In the lab today: Ben

we made starters for LCC activity tests. we are doing the same test like we have done before.

When - 27/07/16

Who's In the lab today: Ben

protocol for comasi x10:

  1. 30 gr Tris base.
  2. 144 gr glycine
  3. 10 gr SDS

we dissolve those component in 800ml.
after we dissolove all the component we complete the solution to 1000 ml.

Experiment order:

  1. we need to grow up 5ml varients, W.T, and only plasmid(according to the protocol)
  2. after 1 day we disolve and collect the sup and transfer it to centricon.
  3. we concentrated till 0.5ml(x10)
  4. we swiched the buffer with another Tris
  5. we centrifuge every 5 min for keep the materials in the solution

When - 26/07/16

Who's In the lab today: Tomer

pNPB testing according to the protcol

WT 0.489
CO 0.54
F7 0.5
R7 0.52

O.D testing for LCC varients

WT 0.514
F7 0.525
BL21 0.67

biobrick primers arrived:
for each primer we added DDW as writen in the form to get the concentration of 100mM.
we diluted each primer to a new eppendorf to get a concentration of 10mM by mix 5ul primer+45ul DDW.

When - 18/07/16

Who's In the lab today: Tomer

activity test like we had done before.

When - 17/07/16

Who's In the lab today: Tomer

transformation for all the varients +BL21 without control.
we added 16 starters ( 2 for each plasmid+2 control)- 8 pNPB tubes, 8 PET tubes.

When - 11/07/16

Who's In the lab today: Eyal

Turbidity check and induction:
7 ml LB+ 7ul CP+ 70ul from each starter. 200 rpm, 37C.
dilution of x2→ 1 ml culture+ 1 ml LB

 11:4012:40
LCC WT 0.117x2 0.371x2
pACYC 0.116x2 0.360x2
BL21 0.153x2 0.450x2

at 12:40→ we added 1M IPTG for each tube.

Activity test:

  1. centrifuge each culture with 4C 30min 7100g. we transfer thw sup for new tubes.
  2. A. we had made stock of pNPB 100 mM→ 17.5 ul pNPB+ 982.5 ul analytical ethanol
    B. we had made Tris pH=8 0.25mM→ dilution Tris 1M- 1ml to 39ml UPW
  3. we added 100 ul of the sup near the plate reader with multichannel
  4. we prepared pNBP:
    for concentration A- 50 ul from the stock to 9.95ml Tris
    for concentration B-100 ul from the stock to 9.9ml Tris
  5. we added pNPB for each tube. 100 ul for each tube
  6. checking absorption in 405nm per min for 50 min

When - 10/07/16

Who's In the lab today: Eyal

transformation for activity test LCC, plasmid pACYC without insert and pACYC LCC WT C6:
The aim of this test- transformation for compatent bacteria BL21 and activity test will check how much pNPB we need to use in further experiment.

  • 3 tubes, compatent BL21 for -80C to ice for 10min.
  • 1 ul from each plasmid to each tube and 1 tube without plasmid(negative control)
  • mix with tip
  • incubate in ice for 20 min
  • heat shock 42C 30sec
  • incubate in ice for 3min
  • SOC 900ul without antibiotic
  • 37C for 1h
  • 100ul from each tubes to LB 5ml+cmp 5 ul
  • centrifugate 500rpm 2min
  • carrying of 800ul liquid from each tube
  • mix the residue with the liquid
  • sowing LB+cmp
  • 10 min absorption RT
  • reverse the plates and incubate in 37C

When - 23/06/16

Who's In the lab today: Ben and Dar

glycerol stock protocol:

  • preparing glycerol:
    • add 10 ml LB to 10 ml glycerol
  • preparing the stock:
    1. take 500 ul of starter (with required plasmid transfected)
    2. add 500 ul of the glycerol (from stck)
    3. freeze in -80 C

When - 15/06/16

Who's In the lab today: Ben and Dar

We isolated the plasmids from each cell culture using Geneaid
Again we incubated them with XhoI and XbaI (same protocol as on 14/06)
We ran the samples on a 1% agarose gel with EtBr but using a new loading buffer.

In lanes 6,7,8 we can see good results.
Lane 6 shows the plasmid circular and uncut.
Lane 7 shows the plasmid cut in one location using XbaI.
Lane 8 shows the plasmid circular and uncut (as it does not have a XhoI restriction site).

starters growth for activity testing of LCC:
LB 50 ml+ CP 50 ul.
each tube will get 5 ml.
In addition, LB 5ml without antibiotic for BL21 growth without insertion.
For each tube. we added colony with another varient+2 control tube.
the colony that we are testing:
pACYC LCC WT C6
pACYC LCC C.O C4
pACYC LCC F4 C2
pACYC LCC F7 C3
pACYC LCC R4 C7
pACYC LCC R7 C2
pACYC no insert
BL21 (without antibiotic)

Turbidity check and induction:
For LB 7 ml + CP 7ul we added 70 ul for each starter. ( 37C 200 rpm)

 

PET degradation test:
we cheked if there is any change in PET wight after one day:

 

When - 14/06/16

Who's In the lab today: Ben and Dar

We isolated the plasmids from each cell culture using Geneaid Presto mini plasmid kit.
We Incubated each of the plasmids with XhoI and XbaI restriction enzymes (2 hours incubation in 37 degrees and 20 minutes in 65 degrees).
We ran the results in a 1% agarose gel with EtBr. Both plasmids show on the gel uncut (lanes 2-3). We have no ability to determine their size (as they are circular).
We put new starters for another preparation of plasmid DNA.

When - 13/06/16

Who's In the lab today: Ben and Dar

We incubated starters for the pSEVA224 and pSEVA414 in 30 degrees.

When - 11/06/16

Who's In the lab today: Eyal and inbar b

compentent cells prepretion

 

When - 10/06/16

Who's In the lab today: Eyal

preparation of TRIS-HCL:

  1. for prvpB degredation---> C1V1=C2V2→ 1M*V1= 25mM*9.9ml→ V1=247.5 ul, DDW=9.6525ml
  2. for PET degredation---> C1V1=C2V2→ 1M*V1= 500mM*2ml→ V1=1ml, DDW=1ml

preparation of pNP-B:

  • pNPB - 35ul -----------> 1 ml of pNPB ---------> 100 mM into
  • acetonitrite - 965 ul --------> in 200 mM conc. ----->9.9 TRIS 25 nM

we left 8 tubes with LCC variants for incubation at 10:30 AM for the weekend (50 C)

When - 08/06/16

Who's In the lab today: Eyal

 
 

When - 06/06/16

Who's In the lab today: Eyal

we designed an expriment to test differnet carbon surce from our metabolic pathway for putida.
We used 96DW and we orgenized it as seen in the picture:

 
 

this experiment will last for one week we will check the change in the turbidity with plate-reader.

When - 31/05/16

Who's In the lab today: Eyal

we cut De-lorenzo plasmids (414+224), we run thus plasmids on agar gel for analazing if thus plasmids were cut.

When - 28/05/16

Who's In the lab today: Eyal

starters for Putida KT2440.
5 ml LB+5 ul amp, 100ng/ul, 30C

transformetion for pACYC plasmid without insertion(negative control for coplement cells) and with insertion LCC WT C6, LCC CO C4, LCC F4C2, LCCF7C3, LCC R4C7, LCC R7C2 to BL21 bacteria for protein exprection.

  • 8 tubes with compatent BL21 bacteria, 80C,10 min.
  • 1 ul from each plasmid for each tube.
  • mixed with tip.
  • keep in ice for 20 min.
  • keep in ice for 3 min more.
  • 500 ul SOC without antbiotic.
  • 27C 1h
  • centrifugation 5000 rpm 2min
  • take off 500ul from the liquid.
  • mix the residue with the rest liquid.
  • sowing in LB+CMP

When - 24/05/16

Who's In the lab today: Eyal

seq of LCC variants 3 ul primer in each reaction 300 ng DNA.
(each with Rw\Fw primer).

When - 19/05/16

Who's In the lab today: Roee and Neomi

We made plates with : LB+TET, LB+Strep. we took the starters with De-Lorenzo bacteria to swo on thus plates.

After all the starters and glycerol stocks were found contaminated a new plate of rhodococcus ruber was recieved from alex sivans lab. a new SM medium was made and we have set the tools inorder to grow new starters on sunday may the 22nd.

 
 

When - 13/05/16

Who's In the lab today: Eyal

Constructing a growth-curve for the Putida on Cathecol. The experiment was contaminated. we will have to repete it.

 

 

When - 11/05/16

Who's In the lab today: Eyal

pSEVA starters from 10.5, and sowing on LB+KM 2 ml starters 300 rpm, 2 min, RT, using sup and sowing the output.

When - 03/05/16

Who's In the lab today: Eyal, Roee and Dor

one colony from each plate was sent fot seq(because of the bw concentration, 15 ul from each stock was sent)

2 starters of rhodococcus ruber were made from two different glycerol stocks to check for contamination. each sample was plated on a nutrient agar plate that was divided in to two parts one for each glycerol stock. the starters were put in ramons lab in the incubator shaker 37 degrees and the plate was put in our incubator at 37 degrees as well.

we prepered glycerol stocks of BL21.

When - 30/04/16

Who's In the lab today: Eyal

 

When - 29/04/16

Who's In the lab today: Ben

 

When - 27/04/16

Who's In the lab today: Ben

 

When - 25/04/16

Who's In the lab today: Eyal

 reaction 1reaction 2reaction 3reaction 4reaction 5reaction 6reaction 7
 LCC W.TLCC C.OLCC F4LCC R4LCC F7LCC R7control
pACYC 1.8 1.8 1.8 1.8 1.8 1.8 1.8
Insert 0.9 1.3 0.6 0.8 0.9 0.8 -
Buffer 2 2 2 2 2 2 1.4
DDW 14.3 13.9 14.6 14.4 14.3 14.4 15.2
Ligase 1 1 1 1 1 1 1

incubeted at 16C

M9 salts add to 800 ml H2O:

  • 31.64 gr Na2HPO4*2H20
  • 15 gr Kh2PO4
  • 2.5 gr NaCl
  • 5.0 gr NH4Cl

stirr until dissolved
adjust to 100 ml with distilled H2O

make M9:

  • 700 ml DDW
  • 200ml M9 salts
  • 2 ml 1M MgSO4
  • 100ul 1M CaCl2
  • adjust to 980 ml with DDW

When - 19/04/16

Who's In the lab today: Efrat

Cyclic Voltametry was preformed in oreder to mesure Cathecol’s redox porential conditions:

  • 2% wt Catechol solution in M9. Potential range:
  • 0-0.5 V.E step: 0.005 V
  • scan rate: 0.01, 0.005, 0.001 V. Room temperature, pH 7
 0.005 V0.01 V0.001 V
E for I max 0.324797 0.334791 0.41574
E for I min 0.119925 0.099937 0.131918
Delta E 0.204872 0.234854 0.283822
E 0 0.222361 0.217364 0.273829

When - 18/04/16

Who's In the lab today: Ben

I loaded the PCR products from yesterday onto an agarose gel (1.5%).

The results:

  • Lane 1: 1kb plus ladder
  • Lane 2: pcaG (~660bp)
  • Lane 3: pcaH (~760bp)
  • Lane 4: pcaC (~450bp)
  • Lane 5: pcaD (~840bp)

When - 17/04/16

Who's In the lab today: Ben

I ran a colony-PCR on P. putida with primers for the 4 protocatechuate degredation genes (pcaG/H, pcaC/D)

The reaction mix was:

  • 25μL of ReadyMix Taq PCR reaction mix
  • 22μL of UPW
  • 1.5μL of each primer
  • A swab from a P. putida colony.

(total reaction volume 50μL)

Cycle parameters were according to Sigma-Aldrich protocol.

ligation of LCC variants into pACYC vector.
Using a ligation calculator in 3:1 ration: we will use about 36 ng of insert for each ligation reaction:

 reaction 1reaction 2reaction 3reaction 4reaction 5reaction 6
 LCC W,TLCC C.OLCC F4LCC R4LCC F7LCC R7
pACYC 1.4 1.4 1.4 1.4 1.4 1.4
Insert 0.9 1.3 0.6 0.8 0.9 0.8
Buffer 2 2 2 2 2 2
DDW 14.7 14.3 15 14.8 14.7 14.8
Ligase 1 1 1 1 1 1

+control: pACYC 1.4, Buffer 2, DDW 1, Ligase 1
-Incubated overnight at 16C

prepered LB agar plates whith CAM resistance.

When - 16/04/16

Who's In the lab today: Eyal

 

When - 13/04/16

Who's In the lab today: Inbar B, Inbar S and Eyal

we took Rodococus rubber from glycerol stock and plated on te LB plate at the biological hood.

When - 12/04/16

Who's In the lab today: Eyal, Inbar B and Inbar S

we have made 2 plates containing only LB.
we have made 8 plates containing LB+CRB.

colonies were present.
two- 5 ml starters from CAM plate (pACYC) for midiprep and miniprep.

starter for midiprep 200 ml
midiprep.

When - 11/04/16

Who's In the lab today: Inbar B, Inbar S and Eyal

we took new pACYC from eyal arbely lab, we transformed E. coli it to mg1655 and seed it on CAM plate.

When - 08/04/16

Who's In the lab today: Tomer

we have made 3 medium with glucose, PET, PolyEthylene and palced them in 37C shaker.

When - 06/04/16

Who's In the lab today: Tomer and Ben

restriction of pACYC and E1B GFP

    • E1B GFp 1 ul
    • 3.1 buffer 5 ul
    • XhoI 1ul
    • DDW 43 ul

    • pACYC 5 ul
    • 3.1 5 ul
    • XhaI 1 ul
    • DDW 39 u
  2. Added 1ul BglII incubeted at 37 C
  3. Glycerol stock made for putida:
    • 500 ul from starters
    • 500 ul glycerol 30 % +LB

making a startersfor Rodococus rubber:
We took 50 ml of SM+Glucose and added to it some of the glycerol stock.

When - 05/04/16

Who's In the lab today: Dar, Tomer and Ben

we prepared new LB medium (1L), gathered and prepared glassware for autoclave.
we set up startes for the Putida - for growth experiments and for preparing new glycerol stocks.

We prepared a 20% (w) Catechol solution using 10gr of Catechol (Pyrocatechol from Sigma-Aldrich) and 50ml of DDW.

We prepared M9 Catechol medium using:

  • 10ml M9 Salts
  • 5μL CaCl2 (1M)
  • 100μL MgSO4
  • 39ml DDW
  • 165μL Catechol 20%

When - 31/03/16

Who's In the lab today: Dar

checking whether the Putida from the experiment last week (Growth curves for Putida and E.Coli on EG) are still alive (and that the OD we’ve measured isn’t the result of dead bacteria).
at both times samples were taken from the liquid solution in the tub and put on plates (LB+amp). at both times growth on the plates was observed - the bacteria were still alive.

When - 28/03/16

Who's In the lab today: Inbar S and Eyal

Try #3 of the pACYC restriction
This time - 1 hour with each enzyme and run on agarose gel 2ul after each cut.

  • pACYC 2000ng 30ul(66ng/ul)
  • DDW 58ul
  • 3.1 Buffer 10ul
  • XhoI 1ul
  1. 1 hour 37 deg
  2. 20 min 65 deg
  3. 1 ul BalII
  4. 1hour -> run on agarose gel 2ul.

When - 28/03/16

Who's In the lab today: Inbar S and Eyal

Another restriction reaction for the pACYC plasmid.
We used the pACYC2 product from 25/03(68ng/ul) - Restriction requires 2000ng of plasmid DNA so we used a volume 29.4ul.

Reaction protocol:

  • DNA 29.4ul
  • 3.1 Buffer (NEB) 10ul
  • BalII 1ul
  • XhoI 1ul
  • DDW 58.8ul

No Bands again.

When - 27/03/16

Who's In the lab today: Dar

Setting up the growth-curve experiment for both the Putida and E.Coli (MG1655) on Ethylene Glycol.
This time the experiment will take a week - 2-3 measurements per day.

When - 25/03/16

Who's In the lab today: Inbar

Mini-prep for pACYC.

 Concentration (ng/uL)260/280nm
pACYC1 66 1.81
pACYC2 68 1.78

When - 24/03/16

Who's In the lab today: Tomer and Inbar Bariah

Removing the putida starter from the incubator to the fridge.

DNA extraction from reaction mix- LC-cutinase variants.
total volume 30ul

LC-cutinase variantconcentration(ng/ul)260\280
lc-cutinase - WT 43 1.63
lc-cutinase - WT CO 28 1.52
FreeRun4 61 1.71
FreeRun7 47 1.66
ResRun4 41 1.69
ResRun7 46 1.64
 

WT, WTCO, F4, F7, R4, R7, 1KbPlus led

 

In addition, starters for pACYC were prepared:
10ml LB+10uL CAM for 2 starters.

When - 23/03/16

Who's In the lab today: Inbar Segal, Dar, Ben, Inbar B, Roee, Dor, Tomer and Eyal

Produced the pACYC plasmid from the starter and cleaned it using mini prep kit.
Concentration: 52ng/ul 260\280: 1.83

another try at constructing a growth-curve for the Putida and E.coli on Ethylene glycol and Glucose.

 

Setting up new starters for the Putida and E.coli (MG1655)

When - 22/03/16

Who's In the lab today: Eyal and Inbar Segal

Cutting LC-cutinase and pACYC vector with restriction enzymes, no bands were visible on electrophoresis gel.

 

When - 18/03/16

PCR extraction of LC-cutinase variants
total volume 30ul

LC-cutinase variantconcentration(ng/ul)260\280
lc-cutinase - WT CO 134 1.81
FreeRun4 143 1.79
FreeRun7 133 1.80
ResRun4 110 1.78
ResRun7 138 1.81

When - 17/03/16

Who's In the lab today: Inbar Bariah

PCR to amplify LC-cutinase variants: WT CO, FreeRun4, FreeRun7, ResRun4, ResRun7.
WTCO, WTCOX,F4,F4X,F7,F7X,R4,R4X,R7,R7X,1KbPlus led

 

When - 15/03/16

Who's In the lab today: Ben, Dar, Roee and Tomer

We tried constructing a growth curve for E. coli and P. putida on Glucose and Ethylene Glycol.

The Experiment failed (the O.D was declining instead of increasing), probably because the starters we used were kept in the refrigerator for more than 4 days (the bacteria were probably dead).

When - 08/03/16

Who's In the lab today: Eyal and Inbar Segal

we did a transformation to PACYC, using 50microliter competent bacteria.

100 microliters were used for plating.

an activity test was made of DpnI - we did a transformation in order to check that the plasmid indeed dissolved (nothing is supposed to grow).

concentrations of the genes we received from Weizmann institute:

  • LC-cutinase - WT - 26.55 ng/ul
  • LC-cutinase - FreeRun4 - 19.15 ng/ul
  • LC-cutinase - FreeRun7 - 15.575 ng/ul
  • LC-cutinase - ResRun4 - 20.175 ng/ul
  • LC-cutinase - ResRun7 - 25.575 ng/ul

When - 07/03/16

Who's In the lab today: Ben, Dar, Eyal and Inbar Segal

Starters did not grow.

12 Petri Dishes of LB+CAM were prepared.

When - 06/03/16

Who's In the lab today: Ben, Tomer and Dar

Preparation of starters for growth curve experiment. (P. putida and E. coli) Glucose and Ethylene Glycol.

When - 25/02/16

Who's In the lab today: Ben

went to the supply room to bring in supplies:
  1. one measuring cup 2L made of glass, and one 1L measuring cup made of plastic.
  2. 20ml syringes and 10ml syringes - 10 from each volume. and a few more 2.5 ml because they didn't have 5ml syringes.
  3. around 30 minisart filters.
  4. latex gloves Medium size x5.
  5. A bundle of glass pipettes 10ml (they didn't have 20ml ones) we need to order especially.
  6. big tank for water (needs a wash and then we can use it for ddw or upw).

When - 24/02/16

Who's In the lab today: Neomi, Tomer and Dor

a glycerol stock of rhodoccocus ruber was prepared from the starter made yesterday. I didn't know if the glycerol in our fridge was autoclaved so I borrowed from Ramon's lab, the stock is at (-80) degrees Celsius at Ramon's shelf.

Removed the putida from the incubator after growing on ethylene glycol and glucose and checked the OD (600nm)
Results:

TubeOD (600nm)
Glucose 20% 0.686
Glucose 15% + Ethylene Glycol 5% 0.420
Glucose 10% + EG 10% 0.859
Glucose 5% + EG 15% 0.112
Ethylene Glycol 20% 0.465

because the measurements were inconsistent I made several measurements. especially tubes 1 and 5:

TubeOD (600nm)
Glucose 20% first re-measurement 0.459 second re-measurement 0.611
Glucose 15% + Ethylene Glycol 5% first re-measurement 0.019 second re-measurement 0.032

When - 23/02/16

Who's In the lab today: Ben and Roee

  • a starter of Rhodococcus ruber was made. it included 50ml SM(medium) + glucose + microelements. the work was done in a biological hood afterwards it was placed in a shaker at 37 degrees Celsius overnight.
  • taking the putida out of the shaker after growing it on glucose and ethylene glycol and checking OD (600nm).
    Results:
TubeOD (600nm)
Glucose 20% 0.560
Glucose 15% + Ethylene Glycol 5% 0.671
Glucose 10% + EG 10% 0.465
Glucose 5% + EG 15% 0
Ethylene Glycol 20% 0.09

When - 21/02/16

Who's In the lab today: Roee, Dar, Dor and Neomi

how to make competent bacteria?
  • all the process need to occur on ice, in a centrifuge set to 4degree Celsius, and the glycerol need to be cold.
  • first of all, we grow the bacteria over night with antibiotics -> in 50 ml falcon tube.
  1. centrifuge - maximum speed for 15 min and remove the supernatant.
  2. we filled the falcon tube again with glycerol 10%. centrifuge once again for 15 min at max speed and again remove the supernatant.
  3. repeat step 2.
  4. we combine both tubes (if we have more than one, if not we wash it again with glycerol as we did in 2+3) after we fluidize the bacteria in glycerol we complete the volume with 10% glycerol, we centrifuge and remove the supernatant.
  5. fluidize the sediment with 2 ml glycerol and divide into tubes 50 microliter in each one and freeze with liquid nitrogen.

a medium for growing Rhodococcus Ruber was made in the solution are missing microelements that we are supposed to receive from Valentina and there is no carbon source. the solution is in the fridge door with a sticker “rhodococus ruber solution”.

 
there was no CaCl2 2H2O si we used 680microliter of CaCl2 1M from the fridge.

When - 22/02/16

Who's In the lab today: Roee, Dar, Dor, Tomer, Inbar Segal and Eyal

competent bacteria were made from the putida KT2440 strain. the bacteria were divided into 25 Eppendorf's with 50microliter each (the volume needed for each transformation) the Eppendorf's are in the freezer (-80 degrees) of Ramons lab in a red box the with the inscription “Ashalim”.

starting the experiment of growing the Pseudomonas putida on ethylene glycol:
  1. we made the minimal medium where each one has different rates of carbon sources.
  2. we inserted to each medium one colony of Pseudomonas Putida.
  3. we made a blank of 1ml to measure OD.
  4. we put the bacteria in a Shaker set to 37 degrees Celsius in Naama's lab.

We did a PCR reaction for the LC-Cutinase + leader Sequence Gene. in order to take it from the iGEM plasmid and insert it into a PACYC vector. we ran the PCR products on an agarose gel using electrophoresis and took a picture. we got one big band in the size we wanted 1KBP~ and a second band, weaker one in the size of the entire plasmid.

When - 15/02/16

Who's In the lab today: Inbar Bariah

main action - growing two strains of Pseudomonas putida (EM383, KT2440) on SOC :

Inserting a Q-tip that touched the glycerol stock of the relevant culture into 3ml of SOC with ampicillin (near fire) and grow overnight. -results- the bacteria grew and the medium became very murky. the bacteria were afterwards grown on petri dishes with LB + amp from every strain we inserted 20 microliters and we also made plates where we isolated via striking.

colonies from every strain (KT2440, EM383) were moved into a liquid LB medium for incubation overnight in 37 degrees Celsius.

When - 14/02/16

Who's In the lab today: Inbar Bariah, Neomi and Tomer

main action - making M9 medium for growing Pseudomonas Putida

Instead of glucose as a carbon source we planned an experiment where the ratios between glucose and ethylene glycol vary in order to check if the bacteria can use ethylene glycol as a carbon source. if so, we won't need to insert the two plasmids we took from E. coli.

planning the experiment - we divided the medium into 5 different test tubes where the carbon source will be different in each one:
  1. 100% glucose.
  2. 75% glucose, 25% ethylene glycol.
  3. 50% glucose, 50% ethylene glycol.
  4. 75% ethylene glycol, 25% glucose.
  5. 100% ethylene glycol.
making the medium:
  1. with the assistance of Idit we made MgSo4 and a Salt sterile solutions using autoclave.
  2. we used Sterile CaCl that already gone through autoclave and also DDW that we already had in the lab.
  3. we made 20% glucose and filtered it.
  4. we filtered ethylene glycol and made a 20% ethylene glycol solution.
concentrations:
main action - making ampicillin:
  1. Weighing 1g ampicillin.
  2. Adding 10ml DDW.
  3. Filtering with syringe 0.24 wide.
  4. Dividing it into 20 Eppendorf's (500microliter each).
A protocol for making petri dishes with LB medium:
 

When - 02/02/16

Who's In the lab today: Roee, Inbar Bariah and Tomer

main action - re-sending the two plasmids that got low concentrations to sequencing again.

After checking once again the sequences in blast of the two plasmids that got low percentage and after consulting with Dr.Birnbaum and Naama we understood that the way we looked at the percentage wasn't correct and that the sequencing was actually correct and we had the sequence we wanted on the plasmid. (before we knew about the sequencing being correct we made Chloramphenicol in working dosage in order to grow the bacteria that carried the plasmid on the medium we wanted).

instructions for sending DNA to sequencing:
  1. Plasmid Dosage need to be between 75-100 ng/ul.
  2. volume needed: ten microliters per plasmid.
  3. Primers - 2 microliters per sequenced plasmid (minimum volume 5 ul).

When - 18/01/16

Who's In the lab today: Inbar Segal

Preparation of competent bacteria: E. coli MG1655
  1. Add all of the starter to 500 ml of LB
  2. Use 1 ml of LB as blank, measure OD at time zero at wavelength of 600 nm. (time zero was 0.064 nm)
  3. Measure OD every hour until the bacteria gets to the logarithmic phase (about 3 hours).
  4. The Incubater, Shaker, Spectofotometer were used in Ofer yifrach lab (3 floor).
  5. At 11:20 OD was 0.54 nm, we took it out of the shaker and put it in ice.
  6. We checked if the bacteria can grow on agar with kan and amp - we did not see any colony.

When - 17/01/17

Who's In the lab today: Dor, Inbar Barih and Neomi

How to make a solution for competent bacteria:
 

When - 16/01/16

Who's In the lab today: Dor, Roee and Tomer

Checking the results of the sequencing:
 
 

When - 14/01/16

Who's In the lab today: Dor, Roee, Tomer and Naama

How to check the results of sequencing:
  1. Open the file with the graphs displaying the output.
  2. Go to edit->copy seq->FASTA format.
  3. Go to Blast and paste what we copied in the first window, if we copied first the sequence of the foreword primer after we will do for the reverse primer and vice-versa.
  4. Open the file with the graphs displaying the output, but this time for the reverse primer if we opened its first for the foreword and vice-versa.
  5. Go to edit->copy seq->FASTA format.
  6. Go to Blast press enter, and paste this part in the same window one row below.
  7. Take the reference and put in a different box/one row below.
  8. Press Blast
Pay attention:
  1. In the top of the page in Blast you can check if R or F fits the reference.
  2. It's better to check if R and F match (we expect it to be similar not the same).
  3. Notice the values of query-how similar are the samples, ident-percentage of identical bases.
  4. Look where most of the different bases are located (hopefully in the end or the beginning).
  5. Small letters instead of big ones (atg and not ATG) marks that this sequence repeats itself.
  6. If you to write comments, write according to the locations of the reference.
Planning a Vector:
  1. We looked at the map of the vector that was sent (for the Putida) and tried to see which restriction enzymes are relevant.
  2. we looked at the nebcutter website to check for each of the fragments which restriction enzymes are relevant (each fragment separately, according to the reference).
  3. press custom digest for the list of enzymes, check that this enzyme don't cut inside of the fragments.
  4. choose restriction enzymes.
  5. Plan primers with tails that will match the enzymes.
  6. pcr and ligation.
  7. transformation.

When - 04/01/16

Who's In the lab today: Dor and Roee

We prepared working stock primers to 10μM, and sent 11 plasmids to sequencing:
  • LLK - lc cutinase + led seq + his tag
  • LL - lc cutinase + led seq

When - 31/12/15

Who's In the lab today: Liran and Tomer

Measuring DNA concentration of the plasmids (the numbers match the ones from above):
Plasmid 1 2 3 4 5 6 7 8 9 10 11
concentration (ng/ul) 222.96 188.87 159.67 125.30 98.86 293.56 153.54 189.91 155.54 157.45 166.29

When - 30/12/15

Who's In the lab today: Ben, Liran, Dar, Tomer and Dor

PCR for two genes (aldA, flco) of the E.coli strain MG1655:
  1. Prepration of a DNA gel
Well 1 3 4 6 7
Sample ladder flco flco control (no template) aldA control (no template) aldA
Extraction of plasmids (using the yellow/orange kit):

NumberPlasmid
1 tph A1
2 tctA A1
3 tph A3
4 LC cutinase
5 LC cut + lead seq + his tag
6 tph A2
NumberPlasmid
7 tphB
8 tctA A2
9 rctB B1
10 tph C
11 LC cut + lead seq

When - 29/12/15

Who's In the lab today: Inbar Barih, Inbar Segal, Eyal, Liran and Ben

Making starters for bacteria after Transformation:
  1. Add 5 ml of LB to a 50ml falcon.
  2. Add 5 ul of the matching antibiotic (the ratio is 1:1000).
  3. With a toothpick stab one colony spread it on another Agar plate (in case the starter wont work we will be able to use that colony again) and then throw it inside of the 50 ml flacon.

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