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From 2014.igem.org

Contents 1 Thursday 24th July 1.1 Lab work 1.1.1 B - Construction of the fusion protein (color) 1.1.1.1 Plasmid DNA Extraction and Electrophoresis 1.1.2 C - Lemon scent 1.1.2.1 Plasmid DNA Extraction & Electrophoresis 1.1.2.2 Gel electrophoresis of p cola 1.1.2.3 Results of the PCR Targeting 1.1.3 E - Salicylate Inducible Suppressing System 1.1.3.1 Plasmid DNA Extraction & Electrophoresis 1.1.3.2 Transformation of competent E.coli cells

Thursday 24th July

Lab work

B - Construction of the fusion protein (color)

Plasmid DNA Extraction and Electrophoresis

by Terry

Biobricks used:

• 1. BBa_K1033927 with pSB1C3 (pink chromoprotein)
• 2. BBa_K1033910 with pSB1C3 (yellow chromoprotein)
• 3. BBa_K1033921 with pSB1C3 (red chromoprotein)
• 4. BBa_K1033922 with pSB1C3 (red chromoprotein)
• 5. BBa_K1033902 with pSB1C3 (blue chromoprotein)
• 6. BBa_K731201 with pSB1C3 (Arabinose inductible araC-pBAD promoter)
• 8. BBa_K1033925 with pSB1C3 (pink chromoprotein)
• 9. BBa_K1033905 with pSB1C3 (purple chromoprotein)
• 10. BBa_K1033913 with pSB1C3 (orange chromoprotein)
• 11. BBa_K1033929 with pSB1C3 (blue chromoprotein)

Protocol

C - Lemon scent

Plasmid DNA Extraction & Electrophoresis

by Terry

Biobricks used:

• 7. BBa_K762100 with pSB1C3 (Limonen synthase1 with RBS)

Protocol

Gel electrophoresis of p cola

by Sean

p cola used: sample left overnight yesterday resuspended in 30μl of H₂O milliQ.

Protocol

Results

Results of the PCR Targeting

by Romain

PCR targeting made the July 22nd. Strains used: DY330 (clone I and II). Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)

Results:

• Nothing has grown on the control dishes.
• Failure, nothing has grown on the clone I dishes.
• Failure, other bacteria have grown on the clone II dishes.

We have to make another PCR. Liquid culture of DY330 made in 30ml LB.

E - Salicylate Inducible Suppressing System

Plasmid DNA Extraction & Electrophoresis

by Terry

Biobricks used:

• 12 & 13. BBa_J45017 with pSB1C3 with pSB1AK3 (pchBA synthase (converts chorismate to salicylate))

Protocol

Transformation of competent E.coli cells

by Fabio

We transformed in DH5α both BioBricks rehydrated yesterday (BBa_J61051 and BBa_K228001).

Samples:

1. BBa_J61051 Concentrated
2. BBa_J61051 Undiluted
3. BBa_K228001 Concentrated
4. BBa_K228001 Undiluted
5. DH5α Control

Protocol

Members present:

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

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