Team:Paris/August 27

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=Cloning of EnvZ* in pSB1A2=
=Cloning of EnvZ* in pSB1A2=

Latest revision as of 19:16, 9 September 2008

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Contents

Construction of pFlhB - mRFP Tripart (LVA+)

Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.

Digestion

Measurement of the concentration of D187 purified

Protocol (it's same that for Miniprep) 

=> the experiments of Gel Extraction  have failed, so we need to repeat the step of digestion.

Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D187 MP168.1 RBS - mRFP - term (FV) 9.00 15.7 EcoRI and XbaI

Gel Verification

Protocol

Gel Verification of D187 digestion
Well 1 2 3 4 5 6
Sample 100 pb ladder MP168.1 no sample D187 no sample
Expected size (pb) 2 955 2 940
Measured size (pb) 2 900 2 900


Cloning of EnvZ* in pSB1A2

Transformation results

control insert / vector mass ratio
transformation with pUC19 transformation without plasmid ligation without insert 1,8 / 1 2,4 / 1 3,1 / 1
number of colonies many 0 0 9 6 2
number of clones picked up for screening 4 2 2
number of colonies picked up for screening bis 4 4

PCR screening

  • screening programm
  • elongation time: 2 min
  • number of cycle: 24
  • total volume reaction: 25 µL
  • primers used: O18 and O19
  • positive control: S158 (pSB3K3)
  • negative control: no template

Electrophoresis

Screening of the cloning of EnvZ* - clones 1 to 8
well n° 1 2 3 4 5 6 7 8 9 10 11 12
sample 1 kb DNA ladder positive
control 		negative
control 		L165.1 L165.2 L165.3 L165.4 L165.5 L165.6 L165.7 L165.8 100 bp DNA ladder
expected size 1659 bp
measured size 0,3 kb


No correct clone
The 8 other clones were also screened.

Screening of the cloning of EnvZ* - clones 9 to 16

PCR elongation time: 2 min 30
Electrophoresis

well n° 1 2 3 4 5 6 7 8 9 10 11 12
sample 1 kb DNA ladder positive
control 		negative
control 		L165.9 L165.10 L165.11 L165.12 L165.13 L165.14 L165.15 L165.16 100 bp DNA ladder
expected size 1659 bp
measured size 0,3 kb 


Results: All the clones analysed were not correct.


Cloning of OmpR*

Digestion

Determination of the concentration of DNA

We used the biophotometer

  • 5 µL of template DNA or 5 µL of EB buffer for th blank
  • 55 µL of pure water
Template DNA Concentration of DNA
PCR 147 150 µg/mL
PCR 148 101 µg/mL
MP 101.2 353 µg/mL

Name of the digestions

Name of the digestion Template DNA What's in? Enzymes used
D 188 PCR 148 OmpR* XbaI-PstI
D 189 MP 101.2 pSB1A2 XbaI-PstI

Protocol of digestion

  • D 188 : 3 µL of PCR 148
  • D 189 : 3 µL of MP 101.2
  • 3µL Buffer (n°2) 10X
  • 0.3µL BSA 100X
  • 22.7 µL of pure Water
  • 1 µL of each enzyme
  • Incubate during about 2h30 at 37°C
  • 20 minutes at 65°C

Cleaning of the digestion products

Standard protocol.

Ligation

Determination of the concentration of DNA

We used the biophotometer

  • 10 µL of template DNA or 10 µL of EB buffer for th blank
  • 50 µL of pure water
Template DNA Concentration of DNA
D 188 9 µg/mL
D 189 30 µg/mL

Protocol of ligation L171

  • 2 µL Ligase Buffer 10X
  • 1.5 µL D 189 (vector)
  • 5 µL D 188 (insert)
  • 11.5 µL pure Water (qsp 20 µL)
  • 1 µL T4 DNA ligase at 400 000 U/mL concentration
  • O/N at 16°C


Checking mutagenesis FliA

EcorI/PstI digestion of mutated FliA

For this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels.

Results : No digestion for the mutated sequence --> successful mission !
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