"

From 2012.igem.org

Revision as of 14:04, 23 July 2012 (view source) Elbrich (Talk | contribs) ← Older edit		Revision as of 09:24, 24 July 2012 (view source) Elbrich (Talk | contribs) Newer edit →
Line 9:		Line 9:
	Possible promoter (inducible promoter)		Possible promoter (inducible promoter)
		+
	1. Strong pBAD promoter (BBa_K206000)		1. Strong pBAD promoter (BBa_K206000)
		+
		+
	2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)		2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)
	Allows for strong promotion that can be:		Allows for strong promotion that can be:
		+
	a) repressed by lacI		a) repressed by lacI
		+
	b) induced by IPTG in E.coli DH5a over a >600 fold range		b) induced by IPTG in E.coli DH5a over a >600 fold range
	Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)		Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)
		+
	actvator: CII or luxI		actvator: CII or luxI
		+
	... and CIII cd for promoter leakiness		... and CIII cd for promoter leakiness
		+
		+
		+	For BBa_R0011 (pL hybrid -> inducible promoter)
		+
		+	Forward primer=prefix-Fprimer
		+	Reverse primer= Rprimer-Pactivator homology (pRE, pluxR)
		+
		+
		+	For P actvator-Actvator-GFP
		+
		+	A= RBS+CII - Fwd primer homolgue to P A (pRE, PluxR), Rev primer homologue to RBS+GFP
		+
		+	= RBS+luxI - For RBS use BBA_537034 (exclude terminator)
		+
		+	B=RBS+GFP - Fwd primer homologue to (A), Rev primer+ suffix
		+
		+
		+	Emeraldo
		+
		+	PCR AlsT (150F, 205F, 300F, 500F -R) and GFP Fwd and Rev using Phusion polymerase. Result=OK! Now we have fragments of 150bp, 250bp, 300bp ,500bp and 850bp.
		+
		+	Nanodrop concentrations (ng/ul):
		+
		+	150_alsT = 31,6
		+
		+	250_alsT = 20,5
		+
		+	300_alsT = 22
		+
		+	500_alsT = 27,6
		+
		+	GFP = 39

---

Revision as of 09:24, 24 July 2012

Tom

Incubating Bacillus in LB medium for overnight growth. Next day: growth curves in SMM medium.

Nisa

Possible promoter (inducible promoter)

1. Strong pBAD promoter (BBa_K206000)

2. LacZ regulated lambda pl hybrid promoter (BBa_R0011)

Allows for strong promotion that can be:

a) repressed by lacI

b) induced by IPTG in E.coli DH5a over a >600 fold range

Pactivator: pRE (BBa_K116603) or pluxR (BBa_R0062)

actvator: CII or luxI

... and CIII cd for promoter leakiness

For BBa_R0011 (pL hybrid -> inducible promoter)

Forward primer=prefix-Fprimer Reverse primer= Rprimer-Pactivator homology (pRE, pluxR)

For P actvator-Actvator-GFP

A= RBS+CII - Fwd primer homolgue to P A (pRE, PluxR), Rev primer homologue to RBS+GFP

= RBS+luxI - For RBS use BBA_537034 (exclude terminator)

B=RBS+GFP - Fwd primer homologue to (A), Rev primer+ suffix

Emeraldo

PCR AlsT (150F, 205F, 300F, 500F -R) and GFP Fwd and Rev using Phusion polymerase. Result=OK! Now we have fragments of 150bp, 250bp, 300bp ,500bp and 850bp.

Nanodrop concentrations (ng/ul):

150_alsT = 31,6

250_alsT = 20,5

300_alsT = 22

500_alsT = 27,6

GFP = 39