Team:Paris/August 13

Sequencing Minipreps we did yesterday

• O18 has been diluted to have a concentration of 5µM
• In each tube : 1µL of O18 diluted
• Pure water qsp 15µL

Minipreps: Plasmid extraction

• Protocol (see # 3) Experiments done by QIAcube

• List of Minipreps

Name	Ligation	Biobricks	Description
MP147.1	L100.1	rbs TetR - ECFP D110 (BV) - D130 (BI)
MP147.2	L100.2
MP147.3	L100.3
MP148.1	L101.1	rbs TetR - GFP tripart D110 (BV) - D131 (BI)
MP148.2	L101.2
MP148.3	L101.3
MP149.1	L114.1	AracpBAD - gfp tripart D126 (BV) - D131 (BI)
MP149.2	L114.2
MP149.3	L114.3
MP150.1	L120.1	tetR repressible promoter - ECFP D106 (BV) - D130 (BI)
MP150.2	L120.2
MP150.3	L120.3
MP151.1	L122.1	RBS-lasI - ECFP D107 (BV) - D130 (BI)
MP152.1	L123.1	RBS lasI - gfp tripart D107 (BV) - D131 (BI)
MP151.2	L123.2
MP152.3	L123.3
MP153.1	L126.1	Strongest RBS (1)- LasR activator (+LVA) D102 (BV) - D114 (BI)
MP153.2	L126.2
MP153.3	L126.3
MP154.1	L132.1	pSB1A2 - flhDC D142(FV) - D139(FI)
MP154.2	L132.2
MP154.3	L132.3
MP155.1	L133.1	pSB1A2 - OmpR* D142(FV) - D140(FI)
MP156.1	L134.1	pSB1A2 - EnvZ* D142(FV) - D141(FI)
MP156.2	L134.2
MP156.3	L134.3
MP157.1	L138.1	pSB3K3 - gfp generator (E0240) D137(FV) - D138(FI)

Glycerol Stocks

• Protocol (see #2)

• List of Stocks

Strain	Ligation	Biobricks	Description
S146.1	L100.1	rbs TetR - ECFP D110 (BV) - D130 (BI)
S146.2	L100.2
S146.3	L100.3
S147.1	L101.1	rbs TetR - GFP tripart D110 (BV) - D131 (BI)
S147.2	L101.2
S147.3	L101.3
S148.1	L114.1	AracpBAD - gfp tripart D126 (BV) - D131 (BI)
S148.2	L114.2
S148.3	L114.3
S149.1	L120.1	tetR repressible promoter - ECFP D106 (BV) - D130 (BI)
S149.2	L120.2
S149.3	L120.3
S150.1	L122.1	RBS-lasI - ECFP D107 (BV) - D130 (BI)
S151.1	L123.1	RBS lasI - ECFP D107 (BV) - D131 (BI)
S151.2	L123.2
S151.3	L123.3
S152.1	L126.1	Strongest RBS (1)- LasR activator (+LVA) D102 (BV) - D114 (BI)
S152.2	L126.2
S152.3	L126.3

PCR screening

4 more transformants of L130 (pFlhB into J61002) are screened by PCR.

• PCR screening programm
• elogation time : 1 min 30
• template: colonies from the transformation plate
• positive control: S142 (J61002)
• negative control: no template
• primers: O18 and O19

Electrophoresis

• 1% agarose gel
• 10 µL loaded

Gel 1: PCR screening (2-7) & PCR product purification using the Qiaquick Gel Extraction kit

Promoter Characterization: work plan