Team:Paris/August 1

MiniPreps

Conditions

• Use of Quiagen's protocol on all the clones cultivated on the 31th.
• Preparation of 50µl of minipreps in water.

= List of Minipreps

Name	Description	Biobricks
MP L102		Strong rbs - YFP D129 (BV) - D118 (BI)
MP L103		Strong rbs - GFP D129 (BV) - D122 (BI)
MP L104		Strong rbs - lasR activator D129 (BV) - D114 (BI)
MP L105		Strong promoter - ECFP D123 (BV) - D130 (BI)
MP L106		Strong promoter - gfp Tripart D123 (BV) - D131 (BI)
MP L107		Strongest promoter - ECFP D103 (BV) - D131 (BI)
MP L108		Strong promoter - gfp Tripart D103 (BV) - D130 (BI)
MP L110		Medium promoter - gfp Tripart D124 (BV) - D131 (BI)
MP L111		Weak promoter - ECFP D104 (BV) - D130 (BI)
MP L112		Weak promoter - gfp D104 (BV) - D131 (BI)
MP L115		pLas - ECFP D105 (BV) - D130 (BI)
MP L116		pLas - gfp Tripart D105 (BV) - D131 (BI)
MP L117		yfp - Double Terminator D125 (FV) - D117 (FI)
MP L118		rfp - Double Terminator D125 (FV) - D121 (FI)
MP L119		lasR activator + LVA - Double Terminator D125 (FV) - D113 (FI)
MP L121		Strong promoter - gfp tripart D106 (BV) - D130 (BI)
MP L124		Strongest RBS - rfp D102 (BV) - D122 (BI)
MP L125		Strongest RBS - yfp D102 (BV) - D118 (BI)

Stock Glycerol

Protocol

• 1mL glycerol
• 1mL culture in LB

=== List of Strain stocked

Strain	Ligation	Biobricks	Description
S123	L104	Strong rbs - lasR activator D129 (BV) - D114 (BI)
S124	L105	Strong promoter - ECFP D123 (BV) - D130 (BI)
S125	L106	Strong promoter - gfp Tripart D123 (BV) - D131 (BI)
S126	L107	Strongest promoter - ECFP D103 (BV) - D131 (BI)
S127	L108	Strong promoter - gfp Tripart D103 (BV) - D130 (BI)
S128	L110	Medium promoter - gfp Tripart D124 (BV) - D131 (BI)
S129	L111	Weak promoter - ECFP D104 (BV) - D130 (BI)
S130	L112	Weak promoter - gfp D104 (BV) - D131 (BI)
S131	L115	pLas - ECFP D105 (BV) - D130 (BI)
S132	L116	pLas - gfp Tripart D105 (BV) - D131 (BI)
S133	L117	yfp - Double Terminator D125 (FV) - D117 (FI)
S134	L118	rfp - Double Terminator D125 (FV) - D121 (FI)
S135	L119	lasR activator + LVA - Double Terminator D125 (FV) - D113 (FI)
S136	L121	Strong promoter - gfp tripart D106 (BV) - D130 (BI)
S137	L124	Strongest RBS - rfp D102 (BV) - D122 (BI)
S138	L125	Strongest RBS - yfp D102 (BV) - D118 (BI)

Transformations

Protocol

use of TOP10 Chemically competent cells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspent the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

List of transformation

Name	Biobrick	Description
L100	RBS-TetR-ECFP
L101	RBS-tetR-GFP tripart
L113	pBAD-ECFP
L114	pBAD-GFP
L120	ptet-ECFP
L122	RBS-lasI-ECFP
L123	RBS-lasI-GFP-ter
L126	RBS-lasR-LVA