From 2008.igem.org

(Difference between revisions)

Latest revision as of 14:03, 6 September 2008

1 Screening of the cloning of E0240 and FlhDC+promotor
- 1.1 Spreading the clones in order to obtain single colonies
2 Miniprep and stock glycerol of stable strains with biobricks 2008
3 Construction for FIFO
- 3.1 Digestion
4 Construction of: promotor-rbs-LasR-dbl ter
5 Promoter characterization plasmids
- 5.1 Ligation
- 5.2 Digestion
6 Sequencing
- 6.1 Digestion
- 6.2 Screening

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 of stable strains with biobricks 2008

Stock number	Miniprep number	Biobricks	Description
S163.1	MP163.1	B0032	RBS
S163.2	MP163.2
S164.1	MP164.1	E0422	RBS+ ECFP+ LVA+ term
S164.2	MP164.2
S165.1	MP165.1	E0430	RBS+ YFP+ LVA- term
S165.2	MP165.2
S166.1	MP166.1	E0432	RBS+ YFP LVA+ term
S166.2	MP166.2
S167.1	MP167.1	E0420	RBS+ ECFP LVA- term
S167.2	MP167.2
S168.1	MP168.1	I732078	RBS+ mRFP LVA+ term
S168.2	MP168.2

Construction for FIFO

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "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

=> Following a mistake, the right E0430(D166) and E0432(D167) digestion, have not been purified. 
So we need to repeat the same digestion experiment tomorrow morning.

Construction of: promotor-rbs-LasR-dbl ter

Results of the transformation we did the day before 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	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17
Sample	L153								1kb ladder	L154
Clone	.1	.2	.3	.4	.5	.6	.7	.8		.1	.2	.3	.4	.5	.6	.7	.8
Expected size	1194									1194
Measured size	1200									1200

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:

Protocol Digestion

Digestion name	Plasmid	Description	Miniprep used	Enzymes	Concentration after gel extraction
D179	MP3.4	B0015 (double terminator B0010-B0012) - BV	4	SpeI and PstI	9
D180	MP101.1	promoter J23101- BV	1	SpeI and PstI	7
D181	MP104.2	PTet (TetR repressible promoter) - FV	1	EcoRI and XbaI	1
D182	MP114.1	TetR - BI	1	XbaI and PstI	10
D183	MP119.3	pBad promoter - BI	1	XbaI and PstI	0
D184	MP143.1	gfp generator - FI	2	EcoRI and SpeI	13
D185	MP163.1	B0032 RBS - BV	2	SpeI and PstI	21

D179 D180 D181 D182 D183 D184 D185

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.

Digestion

Protocol

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

Screening

Gel 1‎ Gel 2

	Gel 1								Gel 2
Well	1	2	3	4	5	6	7	8	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	no sample	D177.3	D177.4	D178.1	D178.2	D178.3	D178.4	1kb ladder
Expected size		2970 194 48				3212			3212		3213
Measured size		2900 200 /				Plamsid not digested			Plamsid not digested

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

@@ Line 492: / Line 492: @@
 ==Screening==
-[[Image:Gel1 FlgB.jpg |thumb |Gel 1‎]]
+Gel 1‎ [[Image:Gel1 FlgB.jpg |150px]] Gel 2[[Image:Gel_2_FlhB.2.jpg‎ |150px]]
-Gel 1
 {|border="1" style="text-align: center"
+|
+|colspan="8"|'''Gel 1'''
+|colspan="9"|'''Gel 2'''
+|-
 |'''Well'''
 |1
@@ Line 504: / Line 509: @@
 |7
 |8
-|-
-|'''Sample'''
-|1kb ladder
-|D176.1
-|D176.2
-|D176.3
-|D176.4
-|D177.1
-|D177.2
-|100pb ladder
-|-
-|'''Expected size'''
-|
-|colspan="4" |2970<br>194<br>48
-|colspan="2" |3212
-|-
-|'''Measured size'''
-|
-|style="background: #cbff7B" colspan="4" |2900<br>200<br>/
-|style="background: #ff6d73" colspan="2" |Plamsid not digested
-|
-|}
-[[Image:Gel_2_FlhB.2.jpg‎ |thumb |Gel 2]]
-Gel 2
-{|border="1" style="text-align: center"
-|'''Well'''
 |1
 |2
@@ Line 541: / Line 519: @@
 |-
 |'''Sample'''
-|nothing
+|1kb<br>ladder
+|D176.1
+|D176.2
+|D176.3
+|D176.4
+|D177.1
+|D177.2
+|100pb<br>ladder
+|no sample
 |D177.3
 |D177.4
@@ Line 548: / Line 534: @@
 |D178.3
 |D178.4
-|1kb ladder
+|1kb<br>ladder
 |-
 |'''Expected size'''
 |
+|colspan="4" |2970<br>194<br>48
 |colspan="2" |3212
-|colspan="4" |3213
+|
+|colspan="2" |3212
+|colspan="5" |3213
 |-
 |'''Measured size'''
 |
-|style="background: #ff6d73" colspan="6" |Plamsid not digested
+|style="background: #cbff7B" colspan="4" |2900<br>200<br>/
+|style="background: #ff6d73" colspan="2" |Plamsid not digested
+|
+|style="background: #ff6d73" colspan="7" |Plamsid not digested
 |
 |}
   Conclusion => the sequence of pFlgB and pFlhB are not good we will tried to isolate pFlhB again.

Team:Paris/August 20

From 2008.igem.org

Latest revision as of 14:03, 6 September 2008

Contents

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Miniprep and stock glycerol of stable strains with biobricks 2008

Construction for FIFO

Digestion

Measurement of concentration of minipreps

Digestion

Gel Extraction

Construction of: promotor-rbs-LasR-dbl ter

Results of the transformation we did the day before yesterday

Number of colonies

PCR Screening

Minipreps

Promoter characterization plasmids

Ligation

Digestion

Sequencing

Digestion

Screening