Analysis of the cloning of OmpR and EnvZ

PCR screening

• positive control: S158 (pSB3K3)
• negative control: no template
• screening PCR programm
• elongation time: 1 min 30
• primers: O18 & O19

Reaction mixture (25 µL total volume)

• 12,5 µL Quick load PCR Mix
• 0,5 µL O18
• 0,5 µL O19
• 12 µL water

We wanted to analyse the transformants of the ligations L133 (OmpR in pSB1A2) and L134 (EnvZ in pSB1A2) from the experience of august 9th/11th.
3 clones of L133 and 3 clones of L134 were analysed.
Each clone tested was then cultured in 7,5 mL LB + ampicilline.

Miniprep and stock glycerol of L151

• Protocol stocks

Stock number	Ligation name	Biobricks	Description
S162.1	L151.1		rbs-LasR - Double terminator
S162.2	L151.2
S162.3	L151.3

• Protocol minprep

Miniprep Name	Ligation name	Antibio	Biobricks	Description
MP162.1	L151.1	Kan		rbs-LasR - Double terminator
MP162.2	L151.2
MP162.3	L151.3

Digestion of MP162

Measurement of the concentration of MPs 162

Digestion

Protocol

Ligation

Protocol

Culture of stable strains with biobrocks 2008

Protocol

Biobrick Name	Part	Description
[http://partsregistry.org/Part:BBa_B0032 B0032] in [http://partsregistry.org/Part:BBa_pSB1A2 pSB1A2]	rbs (efficiency=0.3)
[http://partsregistry.org/Part:BBa_E0422 E0422]	ECFP (LVA+)
[http://partsregistry.org/Part:BBa_E0420 E0420]	ECFP (LVA-)
[http://partsregistry.org/Part:BBa_E0430 E0430]	EYFP (LVA-)
[http://partsregistry.org/Part:BBa_E0432 E0432]	EYFP (LVA+)
[http://partsregistry.org/Part:BBa_I732078 I732078]	mRFP1 (LVA+)
[http://partsregistry.org/Part:BBa_I13507 I13507]	mRFP1 (LVA-)