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Arabinose/Xylose AND Gate Notebook

2 JUNE 2.1 06/07/12 2.2 06/12/12 2.3 06/13/12 2.4 06/14/12 2.5 06/15/12 2.6 06/18/12 2.7 06/19/12 2.8 06/22/12 2.9 06/25/12 2.10 06/26/12 2.11 06/27/12 2.12 06/29/12	3 JULY 3.1 07/03/12 3.2 07/04/12 3.3 07/06/12 3.4 07/07/12 3.5 07/08/12 3.6 07/09/12 3.7 07/10/12 3.8 07/11/12 3.9 07/12/12 3.10 07/13/12 3.11 07/14/12 3.12 07/16/12 3.13 07/17/12 3.14 07/18/12 3.15 07/19/12 3.16 07/20/12 3.17 07/23/12 3.18 07/24/12 3.19 07/25/12 3.20 07/26/12 3.21 07/27/12 3.22 07/30/12 3.23 07/31/12	4 AUGUST 4.1 08/01/12 4.2 08/02/12 4.3 08/03/12 4.4 08/06/12 4.5 08/08/12 4.6 08/09/12 4.7 08/10/12 4.8 08/13/12 4.9 08/16/12 4.10 08/20/12 4.11 08/21/12 4.12 08/22/12 4.13 08/23/12 4.14 08/24/12 4.15 08/25/12 4.16 08/28/12 4.17 08/29/12 4.18 08/30/12 4.19 08/31/12
5 SEPTEMBER 5.1 09/01/12 5.2 09/03/12 5.3 09/04/12 5.4 09/05/12 5.5 09/06/12 5.6 09/14/12 5.7 09/16/12 5.8 09/17/12 5.9 09/18/12 5.10 09/19/12 5.11 09/19/12 5.12 09/20/12 5.13 09/21/12 5.14 09/22/12 5.15 09/23/12 5.16 09/24/12 5.17 09/26/12 5.18 09/27/12	OCTOBER 5.19 10/09/12 5.20 10/10/12 5.21 10/11/12 5.22 10/12/12 5.23 10/13/12	On hover the images to see descriptions

JUNE

06/07/12

PY BROTH 10g salt/L
PSB2K3 kanamicin
We transformed RFP E1010.
plate 1 18F
plate 2 17E
Stock 50 mg/mL
E.coli 1/1000
TRANSFORMATION PROTOCOL.
We made liquid cultures with colonies LIQUID CULTURE.
We extracted plasmids PLASMID EXTRACTION PROTOCOL.
We ran a gel to check the extraction GEL ELECTROPHORESIS PROTOCOL.

06/12/12

We made glycerols of the bacteria
GLYCEROL PROTOCOL .
We transformed plasmid pHp45 TRANSFORMATION PROTOCOL.

06/13/12

We ran gel and extracted from gel LIQUID CULTURE.
We made liquid cultures LIQUID CULTURE and lysed.

06/14/12

We extracted from gel LIQUID CULTURE.
We made glycerols of pHp45 Ω GLYCEROL PROTOCOL .
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450 PSB4A5 Am (ampicillin) 1I BBa_J04450 AraC BBa_C0080
2012 14L plate 1 pSB2K3 Km+
2011 14L plate 1 pSB2K3 Km+
2012 14L plate 1 pSB2K3 Km+
TRANSFORMATION PROTOCOL.

06/15/12

> Digested pHp45 Ω with E/P LIQUID CULTURE.
> We extracted plasmid with kit.
> Ran a gel GEL ELECTROPHORESIS PROTOCOL . (5)
>Extracted from gel LIQUID CULTURE.

ARAC
>From the overnight plate we prepared liquid culture LIQUID CULTURE.
>We left them incubating overnight.
>We left a plate (LB Km DH5α C0080 and a control).

06/18/12

>Plasmid extraction AraC with kit.
>C0080-psB2K3 915 bp
>Streaked 4 LB Km 30 plates DH5α psB2K3.
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red.
>Ran gel with Spr/Strr (eppendorf -20ºC) GEL ELECTROPHORESIS PROTOCOL.
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04.
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 GLYCEROL PROTOCOL.
>Digested C0080 X,S.
>Digested B0014 E,P LIQUID CULTURE.

06/19/12

>Digested B0014 with E, P and with E,X. LIQUID CULTURE.
>Ran gels with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL .
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation.
C0080 is in the first lane.
>Extracted from gel C0080 X,S LIQUID CULTURE.
>Made glycerols from 4 plates LB Km DH5α pSB2K3 GLYCEROL PROTOCOL .
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS].
AmyE 5’ 18K plate 3 2010, 2011, 2012
AmyE 3’ 18M plate 3 2010, 2011, 2012
>Digested B0014 with E,X and E,P again LIQUID CULTURE.

06/22/12

>Ran gels with digestions B0014 with E,X and E,P GEL ELECTROPHORESIS PROTOCOL.

>Dephosphated B0014 E,X and B0014 E,P.

06/25/12

>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS].

06/26/12

>Ran gel with psB2K3 and psB4A5 GEL ELECTROPHORESIS PROTOCOL .

06/27/12

>Transformed with plasmid B0079 1576 bp psB1A2 12A TRANSFORMATION PROTOCOL.
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011
AmyE 5’ grew 2 colonies.

06/29/12

>We did a DH5α K143001 Km30 Amp 100 glycerol GLYCEROL PROTOCOL 07.
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control
These were both plated in 2 plates each.
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made.
>Liquid cultures LIQUID CULTURE.

2 tubes DH5α K143001 Km30 Amp 100
2 tubes DH5α K143002 Km30 Amp 100
2 tubes DH5α B0079 Amp 100
1 tube LB Km 30 Amp 100 control
1 tube LB Amp 100 control
>From the 6 tubes we extracted plasmid from kit.

JULY

07/02/12
>Digestions LIQUID CULTURE.
B0079 digestion with S,P
K143001 with S,P
K143002 with S,P
>PCR’s
•AraC
•Cassete ΩSpr/Strr
PCR PROTOCOL

07/03/12

>After Cassete ΩSpr/Strr PCR we ran a gel GEL ELECTROPHORESIS PROTOCOL . (8)
>Ran gel with digestions from yesterday. (9)
>Did band extractions LIQUID CULTURE.
>Stored at -20ºC.
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix.
>Left digesting with E,S LIQUID CULTURE.

07/04/12

>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1).
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S).
>Transformed ligation and left overnight plated TRANSFORMATION PROTOCOL.
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs.
>Digested with PstI LIQUID CULTURE.

07/06/12

4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown.
Ran a gel with yesterday’s digestions to chek if they were done properly (10)
GEL ELECTROPHORESIS PROTOCOL .
Ran another gel with the rest of the samples.
Extracted GusA fragment
LIQUID CULTURE.
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated LIGATION PROTOCOL.
Repeated ΩSpr/Strr PCR.

07/07/12

Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 .

07/08/12

Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002.

07/09/12

From yesterday’s transformations only one colony grew.
From the previous transformation only 2 colonies grew.
These 3 were streaked in 3 plates:
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km 30 Sp 100 control.
Did liquid cultures in 3 tubes:
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km30 Sp 100 control.
LIQUID CULTURE.

Ran a gel with: (11)
GusA P
PBBR1 GusA
Ω E PCR P
Ω PCR P
Ω PCR I
Ω E
Ω PCR I
GEL ELECTROPHORESIS PROTOCOL
Did GusA primers dissolution for PCR.
GusA PCR
Gel Extraction by kit of lanes 3 and 5 LIQUID CULTURE.

PCR omega P, PCR omega I, PCR AraC P, PCR AraC I
Add 10 μl of each primer (LW and UP).
Add 3 μl of plasmid (P).
Add 30.4 μl buffer.
Add 5 μl Mg.
Add 8 μl DNTp’s.
Add 42.6μl H2O miliQ.
Add 1 μl RTTG polymerase.
Centrifuge (spin) 8 secs.
Add vegetable oil till the eppendorf is full.
Place eppendorf 1 mL in thermocycler.
Run PCR with program “BERNA”.

07/10/12

•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times) DIGESTIONS. (11.2)

07/11/12

•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X LIQUID CULTURE.
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation LIGATION PROTOCOL.

07/12/12

•Ran a gel with yesterday’s digestions: (12)

GEL ELECTROPHORESIS PROTOCOL .

•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes TRANSFORMATION PROTOCOL.

•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid.
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control GLYCEROL PROTOCOL .
•Ligated GusA PCR with B0014 E,X desphophorylated LIGATION PROTOCOL.

•Digested Pfrc54 (A3) with S,P LIQUID CULTURE.

•Desphophorylated Ω+AmyE 3’ E,X.

07/13/12

•Ran gel with pfrc54 S,P GEL ELECTROPHORESIS PROTOCOL . (13)
•Transformation of GusA PCR + B0014 ligation TRANSFORMATION PROTOCOL.
•Transformed with GFP E0040 psBIA2.
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night TRANSFORMATION PROTOCOL.
•B. Subtitils competent cells.

07/14/12

•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes:
•Extracted pellets
•Extracted plasmids.
•Ran gel GEL ELECTROPHORESIS PROTOCOL .

•From transformed DH5α km 30:
GusA+B0014 DH5α Km50 24 pellets
E0040 DH5α LB Amp100
From these two we:
•Did liquid cultures LIQUID CULTURE.
•Extracted plasmid.
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid LIQUID CULTURE.
•Ran gel with this transformation GEL ELECTROPHORESIS PROTOCOL .

07/16/12

•Digested E,P AraC+ Ω+AmyE 3’
•Ω+AmyE 3’ E,P LIQUID CULTURE.

07/17/12

•Ran gel with yesterday’s digestions LIQUID CULTURE. (14)
•The gel we ran didn’t work, probably because the agarose was not prepared correctly.
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S LIQUID CULTURE.

07/18/12

•From yesterday’s digestions we ran a gel GEL ELECTROPHORESIS PROTOCOL . (15)

•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 LIQUID CULTURE.

•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ LIGATION PROTOCOL.

•We digested 1 + Ω+AmyE 3’ E,P LIQUID CULTURE.

07/19/12

•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes LIQUID CULTURE.

•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E TRANSFORMATION PROTOCOL.

•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21.

•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P LIQUID CULTURE.

07/20/12

•Ran gel with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL . (16)
•Transformed GusA+B0014 in two tubes TRANSFORMATION PROTOCOL.
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 LIQUID CULTURE.

07/23/12

•From 24 GusA+B0014 tubes (-) we didn’t do anything.
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid.
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P LIQUID CULTURE.

07/24/12

•Digested B0014 E with X B0014 X with E
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.

07/25/12

•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.
•Joined B0014 E with X B0014 X with E digestions.
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid LIQUID CULTURE. Transformed TRANSFORMATION PROTOCOL.
•From LasR DH5α make liquid cultures and plate LIQUID CULTURE.
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P LIQUID CULTURE.
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated LIGATION PROTOCOL.

07/26/12

•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria LIQUID CULTURE.

•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE.

•Due to problems with the way we did the transformations of ligations we repeated them:
GusAPCR X,P+ B0079 S,P dephosphorylated

GusAPCR X,P+ B0079 S,P dephosphorylated (-)

GusAPCR X,P+ A3 S,P dephosphorylated

GusAPCR X,P+ A3 S,P dephosphorylated (-)
TRANSFORMATION PROTOCOL.

•Did the following digestions LIQUID CULTURE.
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4
•K143002 X,P
•AraC+ Ω S,P
•C0179 X,S

07/27/12

Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P LIGATION PROTOCOL.

07/30/12

•Transformed with ligations:
•AraC+ Ω dephosphprylated +K143002 X,P
•GusAPCR X,P+ A3 S,P dephosphorylated
•GusAPCR X,P+ B0079 S,P dephosphorylated
•Transformed the following sythesis:
•91996 Pveg 140 bp
•91997 ArsR-CzrA_promoter 1 194 bp
•91998 ArsR-CzrA_promoter 2 221 bp
•91999 ArsR-CzrA_promoter 3 213 bp
•92000 pBad-pXyl 387 bp
•92001 XylR 1117pb
•92002 CI_pro_(NAND_INHIBITOR) 774
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked. TRANSFORMATION PROTOCOL.

•Make liquid cultures of the following transformations for tomorrow LIQUID CULTURE:
•AraC+ Ω+K143002
•GusA+A3
•GusA+B0079
•Synthesis

07/31/12

Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab.

AUGUST

08/01/12

Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P LIQUID CULTURE.

08/02/12

•Extracted plasmids from liquid cultures:
AraC+ Ω+K143002
•GusA+A3
•GusA+BBR1

PCR GusA, GusA I, PCR GusA P, PCR GusA I

•Add 10 μl of each primer (LW and UP).
•Add 3 μl of plasmid (P).
•Add 30.4 μl buffer.
•Add 5 μl Mg.
•Add 8 μl DNTp’s.
•Add 42.6μl H2O miliQ.
•Add 1 μl RTTG polymerase.
•Centrifuge (spin) 8 secs.
•Add mineral oil till the eppendorf is full.
•Place eppendorf 1 mL in thermocycler.
•Run PCR with program “BERNA”.

08/03/12

• Ran a gel with: (18)

GEL ELECTROPHORESIS PROTOCOL .
•Ran another gel to extract with:
1.GusA PCR 1
2.GusA PCR 2
3.00
4.01
GEL ELECTROPHORESIS PROTOCOL .
•Did the following digestions LIQUID CULTURE:

• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P

•Extracted GusAPCR X,P digestion LIQUID CULTURE.

•Left the following ligation: AraC+ Ω+K143002 LIGATION PROTOCOL.

08/06/12

•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) LIQUID CULTURE.
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated LIGATION PROTOCOL.
•Ran a Gel with: (19)

08/08/12

BBa_B0040 6I psB1A2 Amp+ plate 1
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P LIQUID CULTURE. (19.1)

08/09/12

•From yesterday’s PCR’s :
• E0040 plasmid 1
E0040 plasmid 2
E0040 digested 1
E0040 digested 2
We purified with PCR kit

•Ran a gel GEL ELECTROPHORESIS PROTOCOL .
•Made 14 PCR’s from AraC+ Ω+AmyE 3’.
•From B0079+GusA ligation and B0040 transformation: •Grew colonies.
•Made liquid cultures LIQUID CULTURE.
•Streaked these in a new plate.
•Diluted plasmid E0080 2 1/50.

08/10/12

•Yesterday’s gel did not come out as expected so we repeated the PCR.

08/13/12

•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) GEL ELECTROPHORESIS PROTOCOL : (20)

•Extracted pasmids form liquid cultures: B0049, B0079+GusA.

•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω

• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P LIGATION PROTOCOL.

• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.

• We ran a gel to extract.

08/16/12

•Ran a gel with yesterday’s digestions:

•Digested:
ArSR-CzrA 97 with S,P
ArSR-CzrA 98 with S,P
ArSR-CzrA 99 with S,P
E0040 PCR with E,S
Digestion Protocol.

08/20/12

•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P LIGATION PROTOCOL.

08/21/12

•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated.
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR.
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC LIQUID CULTURE.

08/22/12

•We did 2 PCR’s for A3.

•Digested K143001+PBad, pXyl with E,S
LIQUID CULTURE. (22.1)

GEL ELECTROPHORESIS PROTOCOL .
•Did band extraction of:
1. K143001+PBad, pXyl E,S 10
2.K143001+PBad, pXyl E,S 11
3.K143001+PBad, pXyl E,S 12

LIQUID CULTURE.
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ LIQUID CULTURE
GLYCEROL PROTOCOL .

•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ GLYCEROL PROTOCOL .

•Transformed ligations:
B0079+GusA Amp+
Pveg+XylR Chloramphenicol+ TRANSFORMATION PROTOCOL..

08/23/12

•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this.
•Ran gel with GEL ELECTROPHORESIS PROTOCOL : (23.01)
Digestion Protocol.

08/24/12

From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation.
Repeated ligation B0079+GusA.

Ligated E0040 PCR E,S+B0014 E,X dephospohylated LIGATION PROTOCOL.

Made 2 new plates from Pveg+XylR ligation and liquid cultures LIQUID CULTURE.

08/25/12

Extracted pellet from 16 tubes of pVeg+XylR that were left overnight.

Ligated B0079+GusA and B0040+B0014 LIGATION PROTOCOL.

08/28/12

Made liquid cultures from transformations tht were left overnight -R0079+GusA
-R0079+GusA (-)
-E0040+B0014
-E0040+B0014 (-)
LIQUID CULTURE.

08/29/12

Digested Pveg+XylR 2,3,4 with E,S.

Digested Ω+AmyE 3’ with S,P DIGESTION.

Ran a PCR with A3 PCR (2 .6 ml tubes).

Ran a gel with
GEL ELECTROPHORESIS PROTOCOL : (23.01)

08/30/12

•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (24)

•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S LIQUID CULTURE.

08/31/12

•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (25)

•Dephosphorylated:
Ω+AmyE 3’ S,P
Ω+AmyE 3’ II S,P
6 AraC+ Ω+AmyE 3’ E,X
8 AraC+ Ω+AmyE 3’ E,X
11 AraC+ Ω+AmyE 3’ E,X
12 AraC+ Ω+AmyE 3’ E,X

•Ran a gel with A3 PCR GEL ELECTROPHORESIS PROTOCOL : (26)

•It was the second time we didn’t obtain a band from A3’s PCR.

•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification.

•Ran a gel woth E0040+B0014 X,P 4 to extract
Gen extraction protocol.

•Did an A3 PCR.

SEPTEMBER

09/01/12

•Ran a gel with GEL ELECTROPHORESIS PROTOCOL : (28)

•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P LIGATION PROTOCOL.

09/03/12

•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 LIQUID CULTURE.

09/04/12

•Ran gel with digestions:
B0079+GusA E,S 3,4,5
PVeg+XylR E,S 1,5,7,10,13,16
GEL ELECTROPHORESIS PROTOCOL .

•Purified A3 PCR 1,2.

•Ran a gel of A3 PCR
GEL ELECTROPHORESIS PROTOCOL and digested with X,P LIQUID CULTURE.

•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.

•Plated colonies that grew in a new plate.

•Made liquid cultures of these
LIQUID CULTURE.

•Repeated the ones that did not grow.

•Digested E0040+B0014 4 with X,P LIQUID CULTURE.

•Ran a gel GEL ELECTROPHORESIS PROTOCOL .

•Extracted from gel LIQUID CULTURE.
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+

09/05/12

•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel LIQUID CULTURE.

•Extracted plasmid from 2 tubes of E0040+B0014.

•Ran a gel with yesterday’s digestions:
1.E0040+B0014 X,P
2.E0040+B0014 X,P
3.PVeg+XylR E
4.B0079+GusA S
5.1 kb plus ladder

•Extracted band from 1. and 2. LIQUID CULTURE.

•Purified A3 PCR from 2 0.6ml tubes 1 and 2.

•1 colony grew from ligation pVeg+E0040+B0014.

•We streaked this in another plate and did liquid cultures
LIQUID CULTURE.

•To do:
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P.
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31)

09/06/12

•A3 PCR and gel A3 PCR X,P 1,2.

09/14/12

•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X.
•Ligate:
PVeg S,P dephosphorylated+XylR X,P.
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P.
pSB13C3 E,P dephosphorylated + A3 PCR E,P.
pSB13C3 E,P dephosphorylated + GusA PCR E,P.
pSB13C3 E,P dephosphorylated + XylR E,P.
pSB13C3 E,P dephosphorylated + pVeg E,P.
pSB13C3 E,P dephosphorylated + pBad pXyl E,P.
pSB13C3 E,P dephosphorylated + Ω PCR E,P.

09/16/12

• Colonies in these plates grew:
- R0079+E0040+B0014
- pBad/pXyl
- XylR
- pVeg
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony.
- pVeg+XylR 1,2
- pBad/pXyl+pSB1C3 1,2
- XylR+pSB1C3 1,2
- pVeg+pSB1C3 1,2
- A3+pSB1C3 1,2
- GusA+pSB1C3 1,2
- omegacassette+pSB1C3 1,2

• Ran gel from the plasmid extractions that we did yesterday:
- A3+E0040+B0014 1-16
- pVeg+E0040+B0014 1-16, 18
- omega cassette+AmyE 3’ 11,13,18,22
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated.
• Digested omega cassette with E,P.
• Digested pSB1C3 with E,P.
• Made liquid cultures from AmyE 5’ and AmyE 3’.
• Did plasmid extraction from pSB1C3 colonies 3 and 6.

09/17/12

• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S.
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S.
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X.
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated.
• Digested AmyE 3’ with E,X. Then dephosphorylated.

09/18/12

• Ran gel with:
1. A3 2 E,S
2. A3 3 E,S
3. A3 4 E,S
4. A3 5 E,S
5. A3 6 E,S
6. A3 11 E,S
7. A3 16 E,S
8. E0040+B0014 colony 1 E,X
9. AmyE 5’ E,P
10. AmyE 3’ E,X
11. Omega cassette E,S
12. pSB1C3 E,P
13. ladder
Bottom:
1. pVeg 1 E,S
2. pVeg 2 E,S
3. pVeg 3 E,S
4. pVeg 4 E,S
5. pVeg 5 E,S
6. pVeg 9 E,S
7. pVeg 10 E,S
8. pVeg S,P dephosphorylated
9. pVeg 2 S,P
10. pBad/pXyl S,P
11. XylR X,P
12. R0079+GFP+TT E,S
13. R0079 S,P
14. Ladder
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P.

09/19/12

• Did PCR from A3 and a 1/50 dilution.
• Ran gel with:
1. pVeg 3 E,P.
2. pVeg 9 E,P.
3. pVeg 10 E,P.
4. pBAd/pXyl 3 E,P.
5. pBAd/pXyl 5 E,P.
6. pBAd/pXyl 1 E,P.
7. A3 3 E,P.
8. A3 4 E,P.
9. XylR 1 E,P.
10. XylR 3 E,P.
11. XylR 8 E,P.
12. pSB1C3 6 E,P.
13. pSB1C3 6 E,P.
14. AmyE 5’ E,S.
15. AmyE 5’ E,S.
16. A3 1/50 PCR.
17. A3 PCR.
18. Ladder
• We did transformations:
- 2 of R0079+GusA
- R0079+GusA control
- 2 of A3+pSB1AK3
- A3+pSB1AK3 control
- 2 of omega cassette+pSB1C3
- 2 of GusA+pSB1C3
• Digested:
- pVeg+pSB1C3
- pBad/pXyl+pSB1C3
- XylR+pSB1C3
- A3+pSB1C3
With E,P.
• Ran gel with these digestions.

09/19/12

• Made liquids cultures from:
- Glicerol of AmyE 5’
- Glicerol of AmyE 3’
- pBad/pXyl+pSB1C3 colony 5
• Made liquid cultures from yesterday transformations:
- R0079+GusA 2 and control
- A3+pSB1AK3 2 and control
- Omega cassette+pSB1C3 2
- GusA+pSB1C3 2
• We transformed yesterday ligations:
- pBad/pXyl+pSB1C3
- XylR+pSB1C3
- pVeg+pSB1C3
- A3+pSB1C3
• Purified A3 PCR with kit, and then, digested with E,P and E,S.
• Ran gel

09/20/12

• Made liquid cultures from transformations:
- Omega cassette+pSB1C3 colony 7
- 2 of GusA+pSB1C3 colony 32
- R0079+GusA colony 12
• Digested A3 1/50 PCR with E,P and E,S.
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014.
• Ligated:
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P
- pVeg S,P dephosphorylated + E0040+B0014 X,P
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated.

09/21/12

• Ran gel with:
1. R0079+GFP+TT 1
2. R0079+GFP+TT
3. pBad/pXyl+pSB1C3 1
4. pBad/pXyl+pSB1C3
5. omega cassette+psB1C3 1
6. omega cassette+psB1C3
7. pSB1C3 E,P 1
8. pSB1C3 E,P
9. pSB1C3 E
10. pSB1C3 P
11. A3 1/50 PCR E,P

09/22/12

• Ran gel with lysis extractions:
1. GusA+pSB1C3 1
2. GusA+pSB1C3 2
3. GusA+pSB1C3 3
4. GusA+pSB1C3 4
5. GusA+pSB1C3 5
6. GusA+pSB1C3 6
7. GusA+pSB1C3 7
8. GusA+pSB1C3 8
9. GusA+pSB1C3 9
10. GusA+pSB1C3 10
11. GusA+pSB1C3 11
12. GusA+pSB1C3 12
13. pSB1C3
14. A3+E0040+B0014 1
15. A3+E0040+B0014 2
16. A3+E0040+B0014 3
17. A3+E0040+B0014 4
18. A3+E0040+B0014 5
19. A3+E0040+B0014 6
20. A3+E0040+B0014 7
21. A3+E0040+B0014 8
22. A3+E0040+B0014 9
23. pVeg+E0040+B0014 3
24. pVeg+E0040+B0014 4
25. pVeg+E0040+B0014 5
26. pVeg+E0040+B0014 6
27. pVeg+E0040+B0014 7
28. pVeg+E0040+B0014 8
29. pVeg+E0040+B0014 9
30. pVeg
31. pBad/pXyl+E0040+B0014 1
32. pBad/pXyl+E0040+B0014 2
33. pBad/pXyl+E0040+B0014 3
34. pBad/pXyl+E0040+B0014 4
35. pBad/pXyl+E0040+B0014 5
36. pBad/pXyl+E0040+B0014 6
37. pBad/pXyl+E0040+B0014 7
38. pBad/pXyl+E0040+B0014 8
39. pBad/pXyl+E0040+B0014 9
40. pBad/pXyl+E0040+B0014 10
41. pBad/pXyl
42. pVeg+XylR 1
43. pVeg+XylR 2
44. pVeg+XylR 3
45. pVeg+XylR 4
46. pVeg+XylR 5
47. pVeg+XylR 6
48. pVeg+XylR 7
49. pVeg+XylR 8
50. omega cassette

• Ran gel with yesterday digestions:
1. R0079+GFP+TT E,P
2. R0079+GFP+TT E,P
3. Omega cassette+pSB1C3 E,P
4. Omega cassette+pSB1C3 E,P
5. Ladder
6. pBad/pXyl+pSB1C3
7. pBad/pXyl+pSB1C3
8. pSB1C3 6 E,P.
9. A3 1/50 PCR E,S
10. pBbak C011
11. pBbak C012
12. ladder

• Did pellet from R0079+GusA.
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7.
• Digested them with E,P.
• Digested:
- GusA+pSB1C3 1,3,4,10 and 11
- A3+E0040+B0014 2,4,7 and 9
- pVeg+E0040+B0014 4,5 and 6
- pBad/pXyl+E0040+B0014 3,7 and 10
- pVeg+XylR 2,4 and 5
with E,P.
• Made liquid cultures from pVeg+pSB1C3
• Ligated:
- XylR E,P + pSB1C3 E,P dephosphorylated.
- pVeg E,P + pSB1C3 E,P dephosphorylated.
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated.
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P.
- pVeg S,P dephosphorylated + E0040+B0014 X,P.
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated.
- pVeg S,P dephosphorylated + XylR X,P.

09/23/12

• Ran gel with yesterday digestions:
1. Omega cassette+pSB1C3 1 E,P
2. Omega cassette+pSB1C3 4 E,P
3. Omega cassette+pSB1C3 6 E,P
4. Omega cassette+pSB1C3 7 E,P
5. GusA+pSB1C3 1 E,P
6. GusA+pSB1C3 3 E,P
7. GusA+pSB1C3 4 E,P
8. GusA+pSB1C3 10 E,P
9. GusA+pSB1C3 11 E,P
10. pSB1C3 E,P dephosphorylated
11. pVeg+XylR 2 E,P
12. pVeg+XylR 4 E,P
13. pVeg+XylR 5 E,P
14. pVeg S,P dephosphorylated
15. pVeg+GFP+TT 4 E,P.
16. pVeg+GFP+TT 5 E,P.
17. pVeg+GFP+TT 6 E,P.
18. ladder
Bottom
1. pBad/pXyl+GFP+TT 3 E,P
2. pBad/pXyl+GFP+TT 7 E,P
3. pBad/pXyl+GFP+TT 10 E,P
4. pBad/pXyl S,P dephosphorylated
5. A3+GFP+TT 2 E,P
6. A3+GFP+TT 4 E,P
7. A3+GFP+TT 7 E,P
8. A3+GFP+TT 9 E,P
9. Ladder

09/24/12

• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7.
• Made 1/50 dilutions from these PCR products.
• Ran gel with:
1. Omega cassette+pSB1C3 1/50 PCR 1
2. Omega cassette+pSB1C3 PCR 1
3. Omega cassette+pSB1C3 1/50 PCR 4
4. Omega cassette+pSB1C3 PCR 4
5. Omega cassette+pSB1C3 1/50 PCR 6
6. Omega cassette+pSB1C3 PCR 6
7. Omega cassette+pSB1C3 1/50 PCR 7
8. Omega cassette+pSB1C3 PCR 7
9. GusA+pSB1C3 1 P
10. GusA+pSB1C3 3 P
11. GusA+pSB1C3 4 P
12. GusA+pSB1C3 10 P
13. GusA+pSB1C3 11 P
14. pVeg+pSB1C3 6 E,P
15. pVeg+pSB1C3 5 E,P
16. pVeg+pSB1C3 4 E,P
17. pVeg+pSB1C3 3 E,P
18. pVeg+pSB1C3 2 E,P
19. pVeg+pSB1C3 1 E,P
20. ladder

• From yesterday transformations, these didn’t grow:
- pBad/pXyl+E0040+B0014 1 and 2.
- A3+E0040+B0014.
• Made liquid cultures from transformations that did grow:
- pVeg+E0040+B0014.
- pVeg+XylR.
- A3+pSB1C3
- pVeg+pSB1C3
- XylR+pSB1C3

09/26/12

• Did plasmid extractions form liquids cultures:
- 8 of pVeg+pSB1C3
- 8 of A3
- 8 of XylR
• Digested them with E,P.
• Ran gel with:
1. Ladder
2. XylR+pSB1C3 1 E,P
3. XylR+pSB1C3 2 E,P
4. XylR+pSB1C3 3 E,P
5. pVeg+pSB1C3 1 E,P
6. pVeg+pSB1C3 3 E,P
7. pVeg+pSB1C3 4 E,P
8. pVeg+pSB1C3 5 E,P
9. pVeg+pSB1C3 6 E,P
10. pVeg+pSB1C3 8 E,P
11. pSB1C3 6 E,P dephosphorylated
12. A3+pSB1C3 1 E,P
13. A3+pSB1C3 3 E,P
14. A3+pSB1C3 4 E,P
15. A3+pSB1C3 5 E,P
16. A3+pSB1C3 6 E,P
17. A3+pSB1C3 8 E,P
18. Ladder
19. P4+E0040+B0014 1 E,X
20. P4+E0040+B0014 7 E,X

09/27/12

• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours.
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P.

10/09/12

• Ligated:
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated.
- pVeg S,P dephosphorylated + XylR X,P.
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S.

10/10/12

• Ligated:
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated.
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P.
- pVeg S,P dephosphorylated + E0040+B0014 X,P.
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated.
• Digested R0079+E0040+B0014 with E,S.
• Digested AmyE 3’ with E,X. Then dephosphorylated.
• Digested pVeg and R0079, A3 with X,P.
• Digested pBad/pSB1C3 with S,P.

10/11/12

• Did plasmid extraction from liquid cultures:
- 2 of AmyE 5’
- 2 of AmyE 3’
- 2 of E0040+B0014
• Ran gel with:
1. A3 1/50 PCR X,P
2. A3 1/50 PCR X,P
3. A3+pSB1C3 6 X,P
4. A3+pSB1C3 6 X,P
5. A3+pSB1C3 8 X,P
6. A3+pSB1C3 8 X,P
7. pVeg X,P
8. pVeg 96 X,P
9. pVeg 96 X,P
10. pVeg+pSB1C3 1 X,P
11. pVeg+pSB1C3 1 X,P
12. pVeg+pSB1C3 3 X,P
13. pVeg+pSB1C3 3 X,P
14. R0079 X,P
15. Ladder

10/12/12

• Ligated: - AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated.
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated.
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated.
- pVeg S,P dephosphorylated + XylR X,P.
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated.

10/12/12

• Digested AmyE 5’ with S,P
• Dephosphorylated it.
• Ligated:
- AmyE 5’ S,P dephosphorylated with
- R0079 X,P
- pVeg 96 X,P
- A3 1/50 PCR X,P
- A3+pSB1C3 6 X,P
- R0079+E0040+B0014
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P.
• Ran gel with:
1. A3+pSB1C3 1
2. A3+pSB1C3 2
3. A3+pSB1C3 3
4. A3+pSB1C3 4
5. A3+pSB1C3 5
6. A3+pSB1C3 6
7. A3+pSB1C3 7
8. A3+pSB1C3 8
9. A3+pSB1C3 9
10. A3+pSB1C3 10
11. A3+pSB1C3 11
12. A3+pSB1C3 6 control
13. AmyE 5’+P4+CI+E1010+B0014 1
14. AmyE 5’+P4+CI+E1010+B0014 2
15. AmyE 5’+P4+CI+E1010+B0014 3
16. AmyE 5’+P4+CI+E1010+B0014 4
17. AmyE 5’+P4+CI+E1010+B0014 5
18. AmyE 5’+P4+CI+E1010+B0014 6
19. AmyE 5’+P4+CI+E1010+B0014 7
20. P4+CI+E1010+B0014 1 control

10/09/12

• Ligate:
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated.
• Ligate:
pVeg S,P dephosphorylated + XylR X,P.
• Ligate:
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated

10/13/12

• Digested AmyE 5’ with S,P. Then dephosphorylated.
• Ligate:
AmyE 5’ S,P dephosphorylated +
- R0079 X,P
- pVeg 96 X,P
- A3 PCR 1/50 X,P
- A3+pSB1C3 colony 6 X,P
- R0079+E0040+B0014 E,X
• Ligate:
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P

• Ran a gel with:
1. A3+pSB1C3 1
2. A3+pSB1C3 2
3. A3+pSB1C3 3
4. A3+pSB1C3 4
5. A3+pSB1C3 5
6. A3+pSB1C3 6
7. A3+pSB1C3 7
8. A3+pSB1C3 8
9. A3+pSB1C3 9
10. A3+pSB1C3 10
11. A3+pSB1C3 11
12. A3+pSB1C3 6 control
13. AmyE 5’+P4+CI+E1010+B0014 8
14. AmyE 5’+P4+CI+E1010+B0014 9
15. AmyE 5’+P4+CI+E1010+B0014 10
16. P4+CI+E1010+B0014 1 control
17. pVeg+XylR 1
18. pVeg+XylR 2
19. pVeg+XylR 3
20. pVeg+XylR 4
21. pVeg+XylR 5
22. pVeg+XylR 6
23. pVeg+XylR 7
24. pVeg+XylR 8
25. pVeg+XylR 10
26. pVeg+XylR 11
27. pVeg+XylR 12
28. pVeg 96 control

• Ran gel with:
1. A3 1/50 PCR X,P
2. A3+pSB1C3 6 X,P
3. pVeg 96 X,P
4. pVeg+pSB1C3 1 X,P
5. pVeg+pSB1C3 3 X,P
6. R0079 X,P
7. pBad/pXyl+pSB1C3 S,P dephosphorylated
8. ladder
9. Digested:
- A3+pSB1C3 colonies 3,4,5 and 10 with E,P.
- AmyE 5’+pBad/pXyl+P4+CI+E1010*B0014 colonies 1,2,7 and 8 with E,P.
- pVeg+XylR 1,4,5 and 12 with E,P.
• Ran gel with:
1. GFP+TT 1 X,P
2. GFP+TT 1 X,P
3. GFP+TT 1 E,S
4. GFP+TT 1 E,S
5. GFP+TT 2 X,P
6. GFP+TT 2 X,P
7. GFP+TT 2 E,S
8. GFP+TT 2 E,S
9. Ladder
10. A3 1/50 PCR X,P
11. A3+pSB1C3 6 X,P
12. pVeg 96 X,P
13. pVeg+pSB1C3 1 X,P
14. R0079 X,P
15. AmyE 5’ S,P