Team:Paris/August 6

Cloning of flagella gene promotors into J61002 plasmid

We have to digest (by EcoRI and SpeI) the PCR products of yesterday (amplification of pFlgA (MP124), pFlgB (MP125), pFlhB (MP126) and pFlhDC (MP127) in order to clone them into the J61002 plasmid (that we must extract and then digest by EcoRI and SpeI).

Plasmid extraction

The J61002 plasmid was extracted from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.

• Number of this plasmid extraction : MP123
• Carried out 3 times (3 tubes)

Quantification of DNA

In order to quantify the DNA contained either in the Miniprep product of MP123 or in the PCR products of MP124, MP125, MP126 and MP127 previously purifed yesterday by the QIAquick Gel Extraction Kit, we carried out :

By electrophoresis

Assay to quantify DNA

• 10µl of 100pb DNA ladder
• 5 µL of the sample + 1 µL of 6X blue loading dye
• Migration in a 1,5% agarose gel; ~30' at 100W.

==>Results : The MP123 plasmid is clearly visible but the PCR products (purified by QIAquick Gel Extraction) aren't. There might be a problem during the purification step using the QIAquick Gel Extraction. Whatever, we still go on with the digestion.

By Spectrophotometer

Besides the MP123 sample (J61002 plasmid) was quantified using a Bio Photometer (Eppendorf).

==> Conclusion :we took the MP123 n°2 to do digestions

Digestion

For each sample (MP123 and PCR products of MP124, MP125, MP126 and MP127):

• 10 µL of DNA (Miniprep eluate or PCR products)
• 12,5 µL water
• 2,5 µL buffer 2
• 0,25 µL BSA
• 1 µL EcoRI

Incubation 1h30 at 37°C
Incubation 1h30 more after adding 1 µL of SpeI

Electrophoresis of the digested J61002 plasmid

Electrophoresis of digested J61002 (D136)

• 10µl of 1 kb DNA ladder
• 30µL of the digestion + 5 µL of 6X blue loading dye
• 3µl of not digested plasmid + 1µl of 6X blue loading dye
• Migration in a 0,5% agarose gel; ~30' at 100W.

==>Conclusion :' we observe a band near 3000 pb, and we excpeted a size of 3002 pb. So we observe the linearise plamsid (only one band at the good size).

Analysis of the purified digested DNA:

gel 2 : D136

Purification of the digested PCR products using the same kit.

• band 1 : ladder 1kb 10µL
• band 2 : D136 4µL + 1µL Loading dye

=> Concentration : +/- 12ng/4µL -> 3ng/µL

Ligation

Protocol

For each sample,

• 1 µl Ligase
• X µl Vector
• Y µl Insert (3x more than vector)
• 2 µl Ligase Buffer 10x
• 20 µl qsp H2O

• Incubated O/N at 4°C (in the fridge)

List of ligations

Cloning of pflhDC

Yesterday the isolation of pflhDC did not work : the PCR product measured more than 1.5 kb. We checked the primers, they are well designed but they contain a sequence similarity with other sequences in E.coli K12. To amplify more precisely the promoter, we decided to do a PCR with gradient.

Protocol

• 10µL Phusion buffer 5X
• 1µL dNTP
• 2.5 µL Oligo F (O111) 10mM
• 2.5 µL Oligo R (O113) 10mM
• 1µL template DNA
• 0.5µL Phusion polymerase

PCR Program

   PROMOTE2
   * LID : 105 °C
   *1. T: 95°C     5min
   *2. T: 95°C     1min
   *3. T: 55°C     30s  ~>G: 5°C
   *4. T: 72°C     1min30
   *5. GO TO: 2 REP: 29
   *6. T: 72°C     5min    
   *7. HOLD: 10°C

Results

PCR with gradient : amplification of pflhDC

On the left side, the temperature was 50°C, in the center 55°C and on the right 60°C.

We can't see anything on this electrophoresis (except the ladder) !

Remarks :

• We forgot the negative control
• The temperature should be 60°C +- 5°C
• For a promoter, 45s of elongation is enough (~1min per kb)
• We could have used a taq polymerase instead of the phusion one
• We will do it again

The return of PCRs for amplification of promoters

As our results were not very encouraging, we started again a PCR to amplify:

• pflhDC ( 12 times with 12 different temperatures between 55°C and 65°C)
• pflgA (60°C)
• pflgB (60°C)
• pflhB (60°C)
• Negative control

Protocol

We used the taq polymerase, following a typical protocol.

Program

The PCR Program was a new version of PROMOTE2 :

   PROMOTE2
   * LID : 105 °C
   *1. T: 95°C     5min
   *2. T: 95°C     1min
   *3. T: 60°C     30s  ~>G: 5°C
   *4. T: 72°C     45 sec
   *5. GO TO: 2 REP: 29
   *6. T: 72°C     5min    
   *7. HOLD: 10°C

Results

We will have the results tomorrow

Culture for glycerol stocks and MiniPreps

• E.coli K12 strain MG1655, to create a glycerol stock of this wonderful bacteria
• pJ61002

Protocol :

• 5 mL LB with the appropriate antibiotic (here 5µL Amp 1000X)
• With a toothpick, we select a single colony
• 37°C 225 rpm, overnight

Isolation of colonies

We isolated colonies of several strains on a petri dish (streaked):

• S119
• S120
• E0240
• pSB3K3

Culture : 37°C overnight