From 2008.igem.org

(Difference between revisions)

Revision as of 16:38, 21 August 2008

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Strain	Resistance	Ligation	DNA cloned	vector	expected size of the fragment amplified by VF & VR	mesured size
S159.1	kanamycine	L139.1	E0240 (GFP tripart)	pSB3K3	1192 bp	1,5 kb 1,1 kb 0,6 kb
S161.1	ampicilline	L142.7	FlhDC+promotor	pSB1A2	1403 bp	1,4 0,4 kb 0,3 kb

The plates obtained from the speading of yesterday can't be used because there are not single colonies.
We have to try again, but with a stronger dilution of the bacteria or with a smaller volume of spreading.

Resuspension of some bacteria from the glycerol stock into 1 mL of LB+antibiotic
Dilution 10 and dilution 100
Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
Overnight incubation (37°C)

Miniprep and stock glycerol

Stock Glycerol of New Biobrick

Protocol stocks

Stock number	Biobricks	Description
S163.1	B0032	RBS
S163.2
S164.1	E0422	RBS+ ECFP+ LVA+ term
S164.2
S165.1	E0430	RBS+ YFP+ LVA- term
S165.2
S166.1	E0432	RBS+ YFP LVA+ term
S166.2
S167.1	E0420	RBS+ ECFP LVA- term
S167.2
S168.1	I732078	RBS+ mRFP LVA+ term
S168.2

Miniprep of New Biobricks

Protocol Miniprep

Miniprep	Biobricks	Description
MP163.1	B0032	RBS
MP163.2
MP164.1	E0422	RBS+ ECFP+ LVA+ term
MP164.2
MP165.1	E0430	RBS+ YFP+ LVA- term
MP165.2
MP166.1	E0432	RBS+ YFP LVA+ term
MP166.2
MP167.1	E0420	RBS+ ECFP LVA- term
MP167.2
MP168.1	I732078	RBS+ mRFP LVA+ term
MP168.2

Construction of pFlgA - YFP tripart (+/- LVA)

Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)

Digestion

Measurement of concentration of minipreps

standard protocol

Miniprep	Biobrick	C° (µg/mL)	ratio 260/280
MP164.1	E0422	95	1.69
MP164.2	E422	90	1.76
MP165.1	E0430	131	1.74
MP165.2	E0430	nd	nd
MP166.1	E0432	112	1.65
MP166.2	E0432	79	1.6
MP167.1	E0420	199	1.72
MP167.2	E0420	194	1.73
MP168.1	I732078	111	1.65
MP168.2	I732078	104	1.67
MP122.1	E0840	56	1.58
MP122.2	E0840	98	1.63

Digestion

Protocol Digestion

Name	Template DNA	Description	Vol MP (µl)	Vol H2O (µl)	Enzymes
D166	MP165.1	RBS+ YFP LVA- term - FV	7.63	17	EcoRI and XbaI
D167	MP166.1	RBS+ YFP LVA+ term - FV	8.9	15.8	EcoRI and XbaI
D131	MP122.2	GFP tripart - I	10.2	14.5	XbaI and PstI

Gel Extraction

Protocol

Gel Extraction of D166-D167-D131

Well	1	2	3	4	5	6	7	8	9	10	11	12
Sample	1kb ladder	MP165.1	MP166.1	MP122.2	no sample	D166	no sample	D167	no sample	D131	no sample	100pb ladder
Expected size (pb)		2 957	2 996	2 957		2942		914		900
Measured size (pb)		3 000	3 000	3 000		2500		2500		950

Results of the transformation we did yesterday

Number of colonies

Ligation name	Description	Antibio	Number Colonies observed	Fluorescence	Comments
Ligations
L153	D123(BV) - D165(BI) J23100 - rbs-LasR-Double terminator	Amp	768	No	OK
L154	D103(BV) - D165(FV) J23101 - rbs-LasR - Double terminator	Amp	236	No	OK
Controls
C1	D123(BV)	Amp	23	No	OK
C2	D103(FV)	Amp	144	No	OK
Positive Control	pUC19	Amp	2264 (efficiency 4,5.10^8)	No	OK

PCR Screening

==> Protocol

Ligations results J23100+D165 and J23101+D165

Well	Sample	Expected size	Measured size
1	153.1	1194	1200
2	153.2
3	153.3
4	L153.4
5	L153.5
6	L153.6
7	L153.7
8	L153.8
9	1kb ladder
10	L154.1	1194	1200
11	L154.2
12	L154.3
13	L154.4
14	L154.5
15	L154.6
16	L154.7
17	L154.8

Minipreps

Miniprep Name	Ligation name	Antibio	Biobricks	Description
MP169.1	L153.1	Amp		J23100 - rbs-LasR-Double terminator
MP169.2	L153.2			J23100 - rbs-LasR-Double terminator
MP170.1	L154.1			J23101 - rbs-LasR - Double terminator
MP170.2	L154.2			J23101 - rbs-LasR - Double terminator

Promoter characterization plasmids

Ligation

Our ligations from yesterday didn't work. The positive control for transformation worked.

Digestion

We had a problem with a gel extraction so we have to make again the digestions from yesterday

Other digestions made:

Digestion

Protocol

D176 : pFlgB digested with ApoI
D177 : pFlgB digested with NruI
D178 : pFlhB digested with BstAPI

Digestion

to be modified

Protocol Digestion

Plasmid	Description	Miniprep used	Enzymes
MP3.3 ?	B0015 (double terminator B0010-B0012) - FV	4	EcoRI and XbaI
MP119	promoter J23101 - BV	1	SpeI and PstI
MP104	PTet (Tet promoter) - BV	1	SpeI and PstI
MP114	TetR - FI	1	EcoRI and SpeI
MP143	gfp generator - BI	2	SpeI and PstI

We had a problem with a gel and we lost these digestions.

Ligation

Protocol

Ligation name	Vector digestion	Vector description	Vector volume	Insert digestion	Insert description	Insert volume	Product description	Antibiotic
L155	D164	J23101 promoter	10	D163	gfp generator	2	J23101 promoter-gfp generator	Amp
L156	D161	pTet promoter	1	D163	gfp generator	4	pTet promoter-gfp generator	Kana
	D161	1					Vector autoligation control	Kana
L157	D125.2	B0015	3	D162	4	tetR	tetR-B0015	Amp
	D125.2	3					Vector autoligation control	Amp

Sequencing

We received results of our sequencing from COCHIN
We succeed for FlgA promoter
FlgB and flhB don't match with our expected sequences. We decided to cut our Miniprep product with other enzymes to check our sequence.

Screening

Gel 1‎

Gel 1

Well	1	2	3	4	5	6	7	8
Sample	1kb ladder	D176.1	D176.2	D176.3	D176.4	D177.1	D177.2	100pb ladder
Expected size		2970 194 48				3212
Measured size		2900 200 /				Plamsid not digested

Gel 2

Well	1	2	3	4	5	6	7	8
Sample	nothing	D177.3	D177.4	D178.1	D178.2	D178.3	D178.4	1kb ladder
Expected size		3212		3213
Measured size		Plamsid not digested

Conclusion => the sequence of pFlgB and pFlhB are not good we will tried to isolate pFlhB again.

@@ Line 472: / Line 472: @@
 ==Digestion==
-===Measurement of concentration of minipreps===
-to be modified
-[[Team:Paris/Notebook/Protocols#Concentration of the Miniprep|standard protocol]]
-{| border="1" style="text-align: center"
-|'''Plasmid'''
-|'''Miniprep'''
-|'''Concentration (µg/mL)'''
-|'''ratio 260/280'''
-|-
-|MP3
-|3
-|38
-|1.72
-|-
-|MP3
-|4
-|30
-|1.70
-|-
-|MP101
-|1
-|333
-|1.68
-|-
-|MP101
-|2
-|416
-|1.66
-|-
-|MP101
-|4
-|200
-|1.74
-|-
-|MP104
-|1
-|145
-|1.63
-|-
-|MP104
-|3
-|147
-|1.29
-|-
-|MP104
-|4
-|51
-|1.66
-|-
-|MP114
-|1
-|173
-|1.75
-|-
-|MP114
-|2
-|263
-|1.43
-|-
-|MP119
-|1
-|42
-|1.62
-|-
-|MP119
-|2
-|26
-|1.56
-|-
-|MP119
-|3
-|39
-|1.64
-|-
-|MP143
-|1
-|138
-|1.69
-|-
-|MP143
-|2
-|150
-|1.61
-|-
-|MP163
-|1
-|80
-|1.61
-|-
-|MP163
-|2
-|79
-|1.69
-|}
 ===Digestion===

Team:Paris/August 20

From 2008.igem.org

Revision as of 16:38, 21 August 2008

Contents

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Miniprep and stock glycerol

Stock Glycerol of New Biobrick

Miniprep of New Biobricks

Construction of pFlgA - YFP tripart (+/- LVA)

Digestion

Measurement of concentration of minipreps

Digestion

Gel Extraction

Results of the transformation we did yesterday

Number of colonies

PCR Screening

Minipreps

Promoter characterization plasmids

Ligation

Digestion

Digestion

Digestion

Digestion

Ligation

Sequencing

Screening