Team:Paris Saclay/Notebook/July/29

From 2014.igem.org

Revision as of 10:06, 1 August 2014 by Maschi (Talk | contribs)

Contents

Tuesday 29th July

Lab Work

A - The frame coli Odor free

PCR

by Romain

Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 27.25μl
Green GoTaq buffer 5X 10μl
dNTPs 1μl
FTR-Apra-F 2μl
FTR-Apra-R 2μl
DMSO 1.5μl
MgCl2 4μl
Bacterial culture 2μl
Green GoTaq enzyme 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

95°C

5 min

1

Denaturation 95°C 30 s 30
Annealing 60°C 30 s 30
Extension 72°C 2 min 30
Final extension 72°C 5 min 1
Final extension 12°C hold 1

The different bacterial cultures in each tubes:

  1. Tube 1: MG1655Z1 10I
  2. Tube 2: MG1655 100II
  3. Tube 3: MG1655 50I
  4. Tube 4: MG1655 100I
  5. Tube 5: MG1655Z1 10II
  6. Tube 6: MG1655 50II
  7. Tube 7: MG1655Z1 10I positive control with plasmid

Results of the PCR: The electrophoresis revealed nothing

New PCR with different enyme.

Strains used: MG1655 and MG1655Z1, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 37.25μl
DreamTaq buffer 10X 5μl
dNTPs 1μl
FTR-Apra-F 1μl
FTR-Apra-R 1μl
DMSO 2.5μl
Bacterial culture 2μl
DreamTaq enzyme 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

95°C

5 min

1

Denaturation 95°C 30 s 30
Annealing 60°C 30 s 30
Extension 72°C 2 min 30
Final extension 72°C 5 min 1
Final extension 12°C hold 1
  1. Tube 1: MG1655Z1 10I
  2. Tube 2: MG1655 100II
  3. Tube 3: MG1655 50I
  4. Tube 4: MG1655 100I
  5. Tube 5: MG1655Z1 10II
  6. Tube 6: MG1655 50II
  7. Tube 7: MG1655Z1 10I positive control with plasmid

Results of the PCR: The electrophoresis revealed successfully the DNA!

C - Salicylate Inducible Suppressing System

Digestion

by Fabio

After the experiments done the 28th July, we are sure to manipulate the right plasmid from both BioBricks and also that we have a good concentration of them. Now it's time to start the [http://parts.igem.org/Help:Assembly/3A_Assembly Assembly procedure] in order to concatenate both BioBricks.

  • BioBrick BBa_J61051 (Salicylate promoter + NahR) as Part A
  • BioBrick BBa_K228001 (RNA suppressor) as Part B

TODO: Illustration of the process

BioBrick Assembly - Digest Reaction Protocol

Electrophoresis

by Fabio

'Process A'

  1. BBa_J61051

Results:

First process to verify the digestion PHOTO I: LU000107

we are sure that we have the right BioBricks fragments and in a good concentration

Second one to separate both parts (only with J61051). PHOTO II:

[Electrophoresis Protocol]

D - Lemon scent

Transformation of electrocompetent cells

by Arnaud & Terry

Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)

Make 2 électroporations in cold electroporation cuvettes:

  • A control cuvette(without DNA): 50µl of DY330.
  • A second cuvette: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each cuvette and transfer in 2 tubes to incubate during 1 to 2h at 30°C.

Spread on 4 dishes LB + Cm:

  • 100µl of control (without plasmid)
  • 50µl of transformed DY330 with pJBEI-6409
  • 100µl of transformed DY330 with pJBEI-6409
  • The rest of transformed DY330 with pJBEI-6409 (concentrate in approximately 150µl)

Incubate for the weekend at 30°C.

PCR of BBa_K762100

by Sean

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 35.5μl
Phusion buffer 5X 10μl
dNTPs 1μl
iPS66 1μl
iPS67 1μl
BBa_K762100 1μl
Phusion DNA polymerase 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

98°C

1 min

1

Denaturation 98°C 15 s 25 - 30
Annealing 55°C variable 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1

Members there:

  • Instructors and advisors: Alice, Solenne and Sylvie.
  • Students: Arnaud, Fabio, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/July/29"