Team:Paris/August 8

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Contents

Minipreps: Plasmid extraction

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

  • List of Oligos :
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


  • 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_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/Analysis

Analysis of PCR product

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
migration 30min at 100V, on a 1% agarose gel

  • Results :
Name Promotor Band Expected size Measured size
PCR_Control - control - 2 0 pb
0 pb
PCR_127 FlhDC gene 3 972 pb
0 pb
PCR_128 OmpR gene 5 762 pb
700 pb
PCR_129 EnvZ gene 4 1421 pb
1400 pb

==> Conclusion : we observed the size expected for the PCR products, but not for FlhDC gene.
We hypothesis that its value of amplification for this gene it's low as we can't visualize it. So we try to continue to experiments to know if there is something inside or not this tube.


  • Quantification of the PCR products purified

Blank : 2µl of buffer EB + 98µl of water
Samples : 2µl of PCR purified + 98µl of water.

  • Results :
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
  • Incubate 2h30 at 37°C


Analysis by electrophoresis

Analysis of PCR product digestion
  • Conditions

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

  • Results :
Name Promotor Band Expected size Measured size
D139 FlhDC gene 4 972 pb
0 pb
D140 OmpR gene 5 762 pb
700 pb
D141 EnvZ gene 6 1421 pb
1000 pb
D142 -vector digested- 7 2057 & 707 pb
2000 & 700 pb
D142 -vector not digested- 8 2764 pb
2100 pb

==> Conclusions : We validate the digestion of the vector and the insert. Now we are sure, that we don't detect anything for FlhDC genes.

The return of the promoters

Yesterday, we could not see anything on the electrophoresis after the digestion. Today, we will try the digestion again.

Protocol

Digestion name Template DNA Enzymes Quantity of DNA used
D132 PCR 124 - pflgA EcoRI-SpeI 10 µL
D133 PCR 125 - pflgB EcoRI-SpeI 10 µL
D134 PCR 16 - pflhB EcoRI-SpeI 10 µL

Results

Results of the digestion of the promoters

To analyze our digestions, we made an electrophoresis.

  • Gel : 1.5% Agar
  • Ladder : 100 bp
  • 4µL DNA + 2µL Loading Dye
Name Promotor Band Expected size Measured size
D132 pflgA 2 250 bp
250 bp
D133 pflgB 3 250 bp
250 bp
D133 pflhB 4 249 bp
250 bp
    Conclusion
    *The bands are not visible on this picture, but with a long exposition time, we managed 
     to see the bands at their right place.
    *The concentration of DNA is low!

We will do the ligation tomorrow.

Transformation with promotors of interest

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 PCR screening


Ligation L128 L129 L130 L131
Name pFlgA pFlgB pFlhB pFlhDC
n° clone 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
fluorescence red red no no no no no no red red red red red no no no red no no no no no no no red red red red red red red no


Protocol of screening PCR

  • Mix

25µl of Quick Load 2X
1µl of forward primer 10µM
1µl of reverse primer 10µM
23µl of water


  • primers used
Ligation Name primers
L128 pFlgA O100 & O101
L129 pFlgB O102 & O 103
L130 pFlhB O108 & O 109
L131 pFlhDC O111 & O 113


  • 50µl of PCR Mix by tube/clone
  • one toothpick of each clone's colony per 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 100V on 1,5% gel

Results of electrophoresis


gel 1 gel 1 gel 2 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 primers which are not specific.


Building of the standard measurement plasmid

This morning we MiniPreped E0240 and pSB3K3 which are the two important parts of the standard measurement plasmid.

Before the digestion, we have to determine the DNA concentration of the MiniPreps

Measurement of DNA concentration

Template Concentration
(µg/µL)
Ratio DO260/DO280
MP 142
pSB3K3
0.04 1.76
MP 143
E0240
0.16 1.66

Digestion

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 137 MP 142 - pSB3K3 EcoRI-SpeI 25 µL
D 138 MP 143 - E0240 EcoRI-SpeI 6.25 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).

Results of the digestion

Result of the digestion to build the measurement plasmid

Electrophoresis settings

  • Gel : 1 % agar
  • 4µL template DNA for D137
  • All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long.
  • 10µL QuickLoad DNA ladder 1 kb


Name Band Expected size Measured size
D 137 2
2727 bp
3000 bp
D 138 3&4
899 bp
~1000 bp

Gel extraction and DNA purification

To extract the biobrick E0240 from the gel, we used the standard protocol number 8.
To purify the DNA we used the standard protocol number 10

The ligation will be done tomorrow.

Building an other measurement plasmid

PCR to create the special E0240

Protocol

PCR Mix for cloning with Taq polymerase

  • 25µL Quick-load Mix
  • 1µL Oligo F (10µM)
  • 1µL Oligo R (10µM)
  • 1µL Template DNA (MP 143)
  • 23µL pure water

We try to build a measurement tool with and without included RBS.

PCR name Template DNA Oligo F Oligo R
RBS + MP 143 O 141 O 140
RBS - MP 143 O 142 O 140
T empty O 142 O 140

PCR Program

    LID 105°C
1. 95°C 5 min 2. 95°C 1 min 3. 60°C 30 sec 4. 72°C 1 min 30 5. go to : 2 rep : 29 6. sound : 1 7. hold : 10°C

Results

Results of the PCR

Electrophoresis settings

  • Gel : 1 % agar
  • 5µL PCR products
  • 10µL QuickLoad DNA ladder 1 kb
Name Band Expected size Measured size
RBS + 2
900 bp
~ 3000 bp
~ 1000 bp (low fluorescence)
RBS - 3
881 bp
~ 3000 bp
~ 1000 bp (strong fluo)
T 4
nothing
nothing
    Conclusion:
    - There is DNA that is around 3,000 bp. Actually, it is the template DNA (MP 143), there were too much DNA.
      We will have to extract on gel the right piece of DNA.
    - RBS - worked better than RBS +.