Team:Paris/July 30

From 2008.igem.org

Results of transformations

Ligation name	Description	Antibio	Nb colonies	Comments
L100	rbs TetR - ECFP D110 (BV) - D130 (BI)	Amp	0	After another night nothing was osberved -> To do again
L101	rbs TetR - GFP tripart D110 (BV) - D131 (BI)	Amp	1	After another night nothing was observed -> To do again
L102	Strong rbs - YFP D129 (BV) - D118 (BI)	Amp	+/- 100	ok
L103	Strong rbs - mRFP D129 (BV) - D122 (BI)	Amp	a lot	ok
L104	Strong rbs - lasR activator D129 (BV) - D114 (BI)	Amp	a lot	ok
L105	Strong promoter - ECFP D123 (BV) - D130 (BI)	Amp	a lot	ok
L106	Strong promoter - gfp Tripart D123 (BV) - D131 (BI)	Amp	a lot	ok
L107	Strongest promoter - ECFP D103 (BV) - D130 (BI)	Amp	a lot	ok
L108 n°2 (the right one)	Strong promoter - gfp Tripart D103 (BV) - D131 (BI)	Amp	+/- 100	ok
L109 n°1	Strong promoter - ecfp D124 (BV) - D130 (BI)	Amp	+/- 30	To do again
L109 n°2	Strong promoter - ecfp D124 (BV) - D130 (BI)	Amp	a lot	To do again
L110	Medium promoter - gfp Tripart D124 (BV) - D131 (BI)	Amp	a lot	ok
L111	Weak promoter - ECFP D104 (BV) - D130 (BI)	Amp	a lot	ok
L112	Weak promoter - gfp tripart D104 (BV) - D131 (BI)	Amp	a lot	ok
L113	AracpBAD - ecfp D126 (BV) - D130 (BI)	Kan	1	After another night nothing was observed -> To do again
L114	AracpBAD - gfp tripart D126 (BV) - D131 (BI)	Kan	2	After another night nothing was observed -> To do again
L115	pLas - ECFP D105 (BV) - D130 (BI)	Amp	a lot	ok
L116	pLas - gfp Tripart D105 (BV) - D131 (BI)	Amp	a lot	ok
L117	yfp - Double Terminator D117 (FI) - D125 (FV)	Amp	+/- 30	ok
L118	rfp - Double Terminator D121 (FI) - D125 (FV)	Amp	+/- 30	ok
L119	lasR activator + LVA - Double Terminator D113 (FI) - D125 (FV)	Amp	+/- 30	ok
L120	tetR repressible promoter - ECFP D106 (BV) - D130 (BI)	Amp	a lot	ok
L121	tetR repressible promoter - gfp tripart D106 (BV) - D131 (BI)	Amp	a lot	ok
L122	RBS-lasI - ecfp D107 (BV) - D130 (BI)	Amp	0	After another night nothing was observed -> To do again
L123	RBS lasI - gfp tripart D107 (BV) - D131 (BI)	Amp	1	After another night nothing was observed -> To do again
L124	Strongest RBS - mRFP D102 (BV) - D122 (BI)	Amp	a lot	ok
L125	Strongest RBS - yfp D102 (BV) - D118 (BI)	Amp	a lot	ok
L126	Strongest RBS (1)- LacR activator (+LVA) D102 (BV) - D114 (BI)	Amp	no dish found!!!	To do again
L127	gfp (1)- Double terminator D119 (FI) - D125 (FV)	Amp	no dish found!!!	To do again
Controls
C1	D110	Amp	0	ok
C2	D129	Amp	5	ok
C3	D123	Amp	+/- 100	ok
C4	D103	Amp	+/- 20	ok
C5	D124	Amp	+/- 100	ok
C6	D104	Amp	+/- 100	ok
C7	D126	Kana	0	ok
C8	D105	Amp	+/- 20	ok
C9	D125	Amp	0	ok
C10	D106	Amp	0	ok
C11	D107	Amp	0	ok
C12	D102	Amp	+/- 10	ok
Positive control	puc19	Amp	155 (transformation efficiency:1.5*10^7/ug)	ok

Analysis of yesterday DNA digestion

The digested DNA of yesterday was analysed one more time by electrophoresis on a 0.8% agarose gel (about 30 minutes at 100 W).

The ladder used was the 1 kb DNA ladder (New England Biolabs).
5 µL of each sample with 1 µL of loading dye were loaded.'

D101	B0034	1	EcoRI	XbaI	FV	2076 bp	2000 bp	6	10
Name	BioBrick	Tube N°	Enz 1	Enz 2	Obs	Exp Size	Mea Size	Conc (ng/µl)	Band
D102	B0034	1	SpeI	PstI	BV	2077 bp	2000 bp	8	11
D105	R0079	1	SpeI	PstI	BV	2222 bp	2000 bp	6	13
D106	R0040	1	SpeI	PstI	BV	2119 bp	2000 bp	6	12
D108	S03154	1	XbaI	PstI	BI	707 bp		0 (failed)	14
D111	S03879	1	XbaI	PstI	BI	725 bp	600 bp	2	15
D115	C0179	2	EcoRI	SpeI	FI	746 bp		0 (failed)	4 & 5
D116	C0179	2	XbaI	PstI	BI	745 bp	700 bp	2	2 & 3
D117	E0030	1	EcoRI	SpeI	FI	746 bp	600 bp	10	16
D128	B0030	2	EcoRI	XbaI	FV	2079 bp	2000 bp	8	6 & 7
D129	B0030	2	SpeI	PstI	BV	2080 bp	2000 bp	8	8 & 9

==> Conclusion :Each of the samples was succesfully digested and purified except for the sample D108. It seems that the QIAprep columms (from the QIAGEN Minipreps kit) can be used instead of the QIAquick columms (for DNA Gel Extraction).

PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR

Protocol of screening PCR

Mix

Name	Vol (µl)	Concentration
Quick Load	25µl	2X
OligoF_VF2 (O18)	1µl	10µM
OligoR_VR (O19)	1µl	10µM
water	23µl

50µl of Mix PCR by tube/clone
one toothpick of each clone's colony by tube
Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

10µl of ladder 1 kb
15µl of screening PCR (gel n°1, 2, 3(9-17), 4, 5, 6, 7, 8, 9, 10, 11)
10µl of screening PCR (gel n°3(1), 13, 14)
migration ~30min at 100W on 0,8% gel

Results

PCR1_’’’L102(1-8)’’’			PCR2_’’’L103(1-8)’’’			PCR3_’’’L104(1-8)’’’			PCR4_’’’L105(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-->9	1003 pb	1000 pb	10-->17	1078 pb	1000pb	2-->9	1239 pb	1200 pb	10-->17
Gel 1 : L102-L103						Gel 2 : L104-L105

PCR5_’’’L106(1-8)’’’			PCR6_’’’L107(1-8)’’’			PCR7_’’’L108.1(1-8)’’’			PCR8_’’’L108.2(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1200 pb	2-->9	1239 pb	1200 pb	10-->17	1200 pb	1200 pb	2-->9	1200 pb	1200 pb	10-->17
Gel 3 : L106-L107						Gel 4 : L108.1-L108.2

PCR9_’’’L110(1-8)’’’			PCR10_’’’L111(1-8)’’’			PCR11_’’’L112(1-8)’’’			PCR12_’’’L115(1-7)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1100 pb	2-->9	1239 pb	1100 pb	10-->17	1200 pb	1000 pb	2-->10	1239 pb	700 pb	11-->16
Gel 5 : L110-L111						Gel 6 : L112-L115

PCR13_’’’L115(8)’’’			PCR14_’’’L116(1-8)’’’			PCR15_’’’L117(1-6)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1239 pb	1200 pb	2	1200 pb	1200 pb	3-->10	1046 pb	1000 pb	11-->16
Gel 7 : L115-L116-L117

PCR16_’’’L117(7-8)’’’			PCR17_’’’L118(1-8)’’’			PCR18_’’’L119(1-5)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1046 pb	1000 pb	2-->3	1004 pb	1100 pb	4-->11	1079 pb	1200 pb	12-->16
Gel 8 : L117-L118-L119

PCR19_’’’L119(6-8)’’’			PCR20_’’’L120(1-8)’’’			PCR21_’’’L121(1-4)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1079 pb	1100 pb	2-->4	1239 pb	1000 pb	5-->12	1200 pb	1200 pb	13-->16
Gel 9 : L119-L120-L121

PCR22_’’’L121(5-8)’’’			PCR23_’’’L122(1-8)’’’			PCR24_’’’L125(1-4)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1000 pb	2-->5	1239 pb	1000 pb	6-->12	1045 pb	1000pb	13-->16
Gel 10 : L121-L122-L125

PCR25_’’’L125(5-8)’’’			PCR26_’’’L109.1(1-7)’’’			PCR27_’’’L109.2(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-->5	1239 pb	1100 pb	2-->9	1239 pb	1100 pb	10-->17
Gel 11 : L125			Gel 14 : L109.1-L109.2

==> Conclusion : with the PCR, we have check that the transformant bacteria contain insert. (obtain amplification at the good size).

But we don't observe results for L102(3), L102(6), L103(4), L106(1), L106(2), L106(4), L111(1)

Migration of an another gel for this sample...

Results:

PCR1_’’’L102(3;6))’’’			PCR2_’’’L103(4)’’’			PCR5_’’’L106(1; 2; 4)’’’			PCR10_’’’L111(1)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-3	1003 pb	-	4	1200 pb	1100 pb	5-6-7	1239 pb	1100 pb	8
Gel 13 : to solve Mistakes

==> Conclusion : We can observe a results for the samples : L102, L103, L106(4) and L111. (but not for L106(1; 2))