Team:Paris/August 11

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Transformation

Protocol for Top 10 cells : (see # 13)

Digestion

PCR

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR amplification

Protocol

List of Oligos :

Number	Name	Sequence	Length	Comments
O110	FlhDC-Uri-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG	58	Don't amplify the both OmpR binding site
O111	FlhDC-Total-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA	58	Amplify the both OmpR binding site
O113	FlhDC(nu)-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG	54	Don't amplify the natural rbs of FlhD (only promoter)
O124	Oligo-pfliL-Forward-TRO	TCGAATTCGCGGCCGCTTCTAGAGCAAGGGCGTGTAACAGGCAAC	45
O125	Oligo-pfliL-Reverse-TRO	TCCTGCAGCGGCCGCTACTAGTAGTCATGTGTTGCGGTCTTCCTGTG	47
O130	Gene-FlhC-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAGTGAAAAAAGCATTGTTCAGG	53
O131	Gene-FlhC-R-TRO	GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAAACAGCCTGTACTCTCTGTTCATCC	60
O132	Gene-FlhD-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCATACCTCCGAGTTGCTGAAAC	53	Don't amplify the natural rbs of FlhD
O133	Gene-FlhD-R-TRO	GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGGCCCTTTTCTTGCGCAGCGCTTCT	60
O140	PlasTest_GFP_Rv	CCCTGCAGTTAATTAATATAAACGCAGAAAGGCCAC	36	Amplify E0240 with a PacI and a PstI restriction site at the end
O141	PTst_GFPrbs+_F	GCCTGCAGTCACACAGGAAAGTACTAGATGC	31	Amplify E0240 with the promoter and a PstI restriction site at the beginning

Preparation of the templates :
Resuspension of 1 colony in 100µl of water.

Preparation of PCR mix :

For each sample,

1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

Name	genes	Oligo	templates
PCR_130	E0240 RBS+	O141_O140	MP143
PCR_130'	-	O141_O140	Water
PCR_131	flhD RBS-	O132_O133	MG1655
PCR_131'	-	O132_O133	Water
PCR_132	flhC RBS-	O130_O131	MG1655
PCR_132'	-	O130_O131	Water
PCR_133	flhDC + prom	O110_O131	MG1655
PCR_133'	-	O110_O131	Water
PCR_134	flhDC + prom	O111_O131	MG1655
PCR_134'	-	O111_O131	Water
PCR_135	pfliL	O124_O125	MG1655
PCR_135'	-	O124_O125	Water
PCR_136	pflhDC	O110_O113	MG1655
PCR_136'	-	O110_O113	Water
PCR_137	pflhDC	O111_O113	MG1655
PCR_137'	-	O111_O113	Water

Program PCR_Screening : Annealing 30" at 60°C - Time élongation 1'30" at 72°C - Number cycle : 30

PCR verification/Analysis

Analysis of PCR product (Gel 1)

Analysis of PCR product (Gel 2)

After the PCR :

2*3µl have been analysed by electrophoresis
the other 44µl of PCR products have been purified by the Promega kit.

Electrophoresis

ladder : 10µl ladder 1 kb
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 1% agarose gel

Results :

Name	Promotor	Gel	Band	Expected size	Measured size
PCR_130	E0240	Gel 1	2	876 bp	900 bp
PCR_130'	Negative Control	Gel 1	3	0 bp	0 bp
PCR_131	flhD RBS-	Gel 1	4	351 bp	350 bp
PCR_131'	Negative Control	Gel 1	5	0 bp	0 bp
PCR_132	flhC RBS-	Gel 1	6	579 bp	600 bp
PCR_132'	Negative Control	Gel 1	7	0 bp	0 bp
PCR_133	flhDC + prom	Gel 2	2	1165 bp	1300 bp
PCR_133'	Negative Control	Gel 2	3	0 bp	0 bp
PCR_134	flhDC + prom	Gel 2	4	1311 bp	2100 pb
PCR_134'	Negative Control	Gel 2	5	0 bp	0 pb

==> Conclusion : We observed the size expected for the PCR products, but not for pflhDC (PCR_138), is right. We hypothesis for PCR_138 that the size is longer that expected due to the aspecific fixation of Oligo O111 (upper to the real site).

Electrophoresis

Analysis of PCR product (Gel 3)

ladder : 10µl ladder 100 bp
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 2% agarose gel

Results :

Name	Promotor	Gel	Band	Expected size	Measured size
PCR_140	pfliL	Gel 3	2	124 bp	0 bp
PCR_141	Negative Control	Gel 3	3	0 bp	0 bp
PCR_142	pflhDC	Gel 3	4	223	0 bp
PCR_143	Negative Control	Gel 3	5	0 bp	0 bp
PCR_144	pflhDC	Gel 3	6	369	0 bp
PCR_145	Negative Control	Gel 3	7	0 bp	0 bp

==> Conclusion : We need to do again the experience

Culture of ligation transformants

4 clones of each transformation were cultured in 7,5 mL LB + ampicilline. The colonies picked up were the rest of those already picked up from the transformation plate (for the PCR screening of August 8).
37°C overnight

Ligation

L128

L129

L130

Name

pFlgA

pFlgB

pFlhB

Clone N°

1

2

3

4

1

2

6

7

1

2

7

8

Red fluorescence

yes

no

yes

no

yes

no

@@ Line 230: / Line 230: @@
 |style="background: #cbff7B"|<center> 900 bp </center>
 |- style="text-align: center;"
-|PCR_131
+|PCR_130'
 |Negative Control
 |Gel 1
@@ Line 237: / Line 237: @@
 |style="background: #cbff7B"|<center> 0 bp </center>
 |- style="text-align: center;"
-|PCR_132
+|PCR_131
 |flhD RBS-
 |Gel 1
@@ Line 244: / Line 244: @@
 |style="background: #cbff7B"|<center> 350 bp </center>
 |- style="text-align: center;"
-|PCR_133
+|PCR_131'
 |Negative Control
 |Gel 1
@@ Line 251: / Line 251: @@
 |style="background: #cbff7B"|<center> 0 bp </center>
 |- style="text-align: center;"
-|PCR_134
+|PCR_132
 |flhC RBS-
 |Gel 1
@@ Line 258: / Line 258: @@
 |style="background: #cbff7B"|<center> 600 bp </center>
 |- style="text-align: center;"
-|PCR_135
+|PCR_132'
 |Negative Control
 |Gel 1
@@ Line 265: / Line 265: @@
 |style="background: #cbff7B"|<center> 0 bp </center>
 |- style="text-align: center;"
-|PCR_136
+|PCR_133
 |flhDC + prom
 |Gel 2
@@ Line 272: / Line 272: @@
 |style="background: #cbff7B"|<center> 1300 bp </center>
 |- style="text-align: center;"
-|PCR_137
+|PCR_133'
 |Negative Control
 |Gel 2
@@ Line 279: / Line 279: @@
 |style="background: #cbff7B"|<center> 0 bp </center>
 |- style="text-align: center;"
-|PCR_138
+|PCR_134
 |flhDC + prom
 |Gel 2
@@ Line 286: / Line 286: @@
 |style="background: #ff6d73"|<center> 2100 pb </center>
 |- style="text-align: center;"
-|PCR_139
+|PCR_134'
 |Negative Control
 |Gel 2