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 [LYSIS PROTOCOL].

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 [DESPHOPHORYLATION PROTOCOL].

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 [PCR PROTOCOL].
>Left digesting with E,S LIQUID CULTURE.

07/04/12

>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL].
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL].
>Transformed ligation and left overnight plated TRANSFORMATION PROTOCOL.
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
>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
[PCR PROTOCOL]
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”.

Ran gel with:
1 PCR omega P
2 PCR omega I
3 PCR AraC P
4 PCR AraC I
5 ladder 1 Kb
(11.1)