Team:Paris/August 20

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==PCR Screening==
==PCR Screening==
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==>[[Team:Paris/Notebook/Protocols#PCR Screening| Protocol]]
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[[Team:Paris/Notebook/Protocols#PCR Screening| Protocol]]
[[Image:J23100-J23101+D165.jpg |thumb| Ligations results  J23100+D165 and J23101+D165]]
[[Image:J23100-J23101+D165.jpg |thumb| Ligations results  J23100+D165 and J23101+D165]]

Revision as of 17:37, 5 September 2008

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Contents

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 number Miniprep number Biobricks Description
S163.1 MP163.1 B0032 Icon rbs.png
RBS
S163.2 MP163.2
S164.1 MP164.1 E0422 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP+ LVA+ term
S164.2 MP164.2
S165.1 MP165.1 E0430 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP+ LVA- term
S165.2 MP165.2
S166.1 MP166.1 E0432 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP LVA+ term
S166.2 MP166.2
S167.1 MP167.1 E0420 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP LVA- term
S167.2 MP167.2
S168.1 MP168.1 I732078 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
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) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

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 Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png J23100 - rbs-LasR-Double terminator
MP169.2 L153.2
MP170.1 L154.1 Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png J23101 - rbs-LasR - Double terminator
MP170.2 L154.2

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 20-08-08.png 20-08-08-coupe.png

D184 D185 20-08-08bis.png 20-08-08bis-coupe.png


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 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

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.

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