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Extraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.
- Carried out 2 times (2 tubes)
| name
| Biobrick
| plasmid
|
| MP142
| -
| pSB3K3
|
| MP143
| E0240
| pSB1A2
|
Amplification of Genes of interest (OmpR, EnvZ, FlhDC)
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
| Number
| Name
| Sequence
| Length
| Comments
|
| O126
| Gene-EnvZ-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAGGCGATTGCGCTTCTCGCCAC
| 53
|
|
| O127
| Gene-EnvZ-R
| GTTTCTTCGAATTCGCGGCCGCTTCTAGTTATTACCCTTCTTTTGTCGTGCCCTGCGCC
| 59
|
|
| O131
| Gene-FlhC-R
| GTTTCTTCGAATTCGCGGCCGCTTCTAGTTATTAAACAGCCTGTACTCTCTGTTCATCC
| 59
|
|
| O132
| Gene-FlhD-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCATACCTCCGAGTTGCTGAAAC
| 53
| Don't amplify the natural rbs of FlhD
|
| O138
| Gene-OmpR-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCAAGAGAACTACAAGATTCTGG
| 53
|
|
| O139
| Gene-OmpR-R
| GTTTCTTCGAATTCGCGGCCGCTTCTAGTTATTATGCTTTAGAGCCGTCCGGTACAAAG
| 59
|
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- Preparation of the templates :
Resuspend of 1 colony in 100µl of water.
For each samples,
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_127
| FlhDC
| O131_O132
| MG1655
|
| PCR_128
| OmpR
| O138_O139
| Strain OmpR*
|
| PCR_129
| EnvZ
| O126_O127
| Strain EnvZ*
|
| PCR_Control -
| -
| O126_O127
| Water
|
- Program PCR_Screening : Annealing 55°C - Time élongation 1'30" - Number cycle : 29
PCR verification/Analyse
After the PCR :
- 3µl have been analysed by electrophoresis
- the other 47µl 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
- Conditions : migration 30min at 100W, on a 1% agarose gel
| Name
| Promotor
| Gel
| Band
| Expected size
| Measured size
|
| PCR_127
| FlhDC gene
|
|
| pb
| pb
|
| PCR_128
| OmpR gene
|
|
| pb
| pb
|
| PCR_129
| EnvZ gene
|
|
| pb
| pb
|
Quantification of the PCR products purified
- Blank : 2µl of buffer EB + 98µl of water
- Samples : 2µl of PCR purified + 98µl of water.
| Name
| Genes
| C° (µg/ml)
| DO 260/280
|
| PCR_127
| FlhDC
| 350
| 1.68
|
| PCR_128
| OmpR
| 150
| 1.97
|
| PCR_129
| EnvZ
| 100
| 1.77
|
| MP 108 cl2 (23 july)
| -vector-
| 150
| 1.84
|
Digestion of PCR products
Protocol :
| Name
| Genes
| Water
| DNA
| Buffer n°2 10X
| BSA 100X
| EcoRI
| PstI
|
| D139
| FlhDC
| 23.7µl
| 1µl
| 3.0µl
| 0.30µl
| 1µl
| 1µl
|
| D140
| OmpR
| 22.7µl
| 2µl
| 3.0µl
| 0.30µl
| 1µl
| 1µl
|
| D141
| EnvZ
| 21.2µl
| 3.5µl
| 3.0µl
| 0.30µl
| 1µl
| 1µl
|
| D142
| -vector-
| 22.7µl
| 2µl
| 3.0µl
| 0.30µl
| 1µl
| 1µl
|
Purification/Analysis
Electrophoresis
- ladder : 10µl ladder 1 kb (for 1%) or ladder 100pb ( for 1.5%)
- samples : 3µl of insert + 2µl of Loading Dye
- Conditions : migration 30min at 100W, on a 1% agarose gel for the vector and 1.5% for the insert.
| Name
| Promotor
| Gel
| Band
| Expected size
| Measured size
|
| D139
| FlhDC gene
|
|
| pb
| pb
|
| D140
| OmpR gene
|
|
| pb
| pb
|
| D141
| EnvZ gene
|
|
| pb
| pb
|
| D142
| -vector-
|
|
| pb
| pb
|
Transformation results
| Name
| Description
| Antibio
| Number of colonies
| Number of red fluorescent colonies
|
| Ligation
|
| L128
| J61002-pFlgA
D136 (FV) - D132 (FI)
| Amp
| ~ 400
| 2
|
| L129
| J61002-pFlgB
D136 (FV) - D133 (FI)
| Amp
| 39
| 5
|
| L130
| J61002-pFlhB
D136 (FV) - D134 (FI)
| Amp
| ~ 1000
| 4 (but 3 are on the edge of the petri dishe)
|
| L131
| J61002-pFlhDC
D136 (FV) - D135 (FI)
| Amp
| 39
| 38
|
| Control
|
| Control 1
| D136
| Amp
| 0
| 0
|
| Positive control
| pUC19
| Amp
| 36
| 0
|
PCR Screening of Ligation Transformants
Use of 8 clones of Ligation transformants for screening PCR
| Ligation
| Name
| n° clone
| fluorescence
| primers used
|
| L128
| pFlgA
| 1
| red
|
| 2
| red
|
| 3
| no
|
| 4
| no
|
| 5
| no
|
| 6
| no
|
| 7
| no
|
| 8
| no
|
|
| L129
| pFlgB
| 1
| red
|
| 2
| red
|
| 3
| red
|
| 4
| red
|
| 5
| red
|
| 6
| no
|
| 7
| no
|
| 8
| no
|
|
|
|
| L130
| pFlhB
| 1
| red
|
| 2
| no
|
| 3
| no
|
| 4
| no
|
| 5
| no
|
| 6
| no
|
| 7
| no
|
| 8
| no
|
|
|
|
| L131
| pFlgB
| 1
| red
|
| 2
| red
|
| 3
| red
|
| 4
| red
|
| 5
| red
|
| 6
| red
|
| 7
| red
|
| 8
| no
|
|
|
|
Protocol of screening PCR
| 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 100 pb
- 10µl of screening PCR
- migration ~30min at 100W on 1,5% gel
Electrophoresis Purification of PCR
When the PCR cycles were finished,
conditions :
- 10µl of ladder 1 kb (unlike 100 pb)
- 2 x 30µl of PCR products added with 10µl of loading Dye 6x
- migration ~30min at 100W on a 1,5% agarose gel.
Results of electrophoresis
gel 1 
gel 2 
| Name
| Promotor
| Gel
| Band
| Expected size
| Measured size
|
| PCR_124'
| pFlgA
| 1
| 2 to 9
| 261 pb
| 300 pb
|
| PCR_125'
| pFlgB
| 1
| 10 to 17
| 261 pb
| 300 pb
|
| PCR_126'
| pFlhB
| 2
| 2 to 9
| 260 pb
| 300 pb
|
| PCR_127'
| pFlhDC
| 2
| 10 to 17
| 446 pb
| 1,000 pb
|
==> Conclusion:
- PCR of pFlgA, pFlgB and pFlhB have succeed, but we always have a problem with pFlhDC probably because of the oligos wich are not specific.
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