Labwork

Planning

We started the labwork in June. The first step was the research project scope and bibliography to ensure the feasibility of our project and to plan all the work we had to do during this wonderful iGEM summer. In early July we began the experiments, which was completed in the end of September. In order to be effective, we organized ourselves into four groups of summer interns. The groups were formed by all the members of the team, so even students from areas other than biology could cooperate for the project in the laboratory. To ensure the smooth development of the project by all students, the work had a life cycle :

• Learning: A non biologist had to first learn the basics of biology to understand the lab work.
• Practice: The novice starts to do their experiments with the help of another biology student or an adviser.
• Development: The aforementioned student becomes autonomous and takes initiative.
• Transmission of acquired knowledge: The student, initially oblivious to practices in the lab, can transmit this knowledge at the end of their internship to newcomers.

The E. coli odor-free chassis

• Culture of MG1655 and MG1655Z1 strains- here
• P1 phage stock preparation for the transduction of the Delta-tnaA::Kan - here
• P1 phage transduction using the stock prepared on July 2nd to MG1655 and MG1655Z1 strains - here
• Cultures of MG1655 and MG1655Z1 transductants - here
• Transformations test of competent cells MG1655 and MG1655Z1 - here
• Preparation of competent cells E coli, deletion of the antibiotic cassette in the odorless bacteria - here
• Preparation of competent cells E. coli MG1655 and MG1655Z1 transductants - here
• Transformation of supercompetent cells MG1655 and MG1655Z1 transductants with plasmid BT340- here
• Results of the transformation - here
• Preparation and transformation of competent MG1655 and MG1655Z1 transductants - here
• PCR verification of the strains grown - here
• Preparation and Transformation of competent MG1655 and MG1655Z1 with BT340 - here
• PCR of MG1655 and MG1655Z1 with FTR-Apra-F and FTR-Apra-R - here
• Preparation and transformation of electrocompetent MG1655 and MG1655Z1 with plasmid BT340 - here
• Clone isolation by streak of Delta-tnaA MG1655 and MG1655Z1 on LB dishes - here
• Clone isolation by streak of Delta-tnaA MG1655 and MG1655Z1 on LB and kan dishes - here
• PCR verification of the final strains Delta-tnaA MG1655 and MG1655Z1 - here
• Final stock of MG1655 Delta-tnaA - here

The Lemon Scent

Preparation

• Rehydration of BioBricks BBa_J45014, BBa_K517003 - here
• Preparation of electrocompetent DY330 and transformation via pJBEI-6409 - here
• Extraction of p cola plasmid DNA - here
• Extraction and electrophoresis of BBa_K762100 with pSB1C3 - here
• Gel electrophoresis of p cola - here

pPS1

• PCR targeting with DY330 and pJBEI-6409 - here
• Transformation of DY330 with pJBEI-6409 - here
• Liquid culture of DY330 - here
• Transformation of DY330 with pJBEI-6409 - 29th here
• Culture of DY330 + pJBEI-6409 - here
• Transformation of DY330 with pJBEI-6409 - here
• Electroporation of DY330+pJBEI-6409 - here

pPS2-pPS3-pPS4

• PCR of the differents genes or Biobrick Hereand here
• Cloning in Topo vector and transformation of competent E.coli here and here
• Checking of the cloning here and here
• Plasmids extraction here
• Digestion to have the insert here and here
• Ligation of the insert in the differents plasmids here and Here
• Transformation Here and here
• PCR on colony here and here
• Checking of the insert sens here and here and here
• Final stock and extraction of pPS3 and pPS4 here

pPS5

• Preparation of the pPS4 plasmid with SalI digestion here
• Ligation here

PSBIC3

• Preparation of the plasmids here

Salicylate Inducible Suppressing System

• Rehydration of BioBricks BBa_J61051 and BBa_K228001- 23rd July
• Transformation of competent E.coli cells - 24th July
• Bacterial culture - 25th July
• Plasmid DNA Purification - 28th July
• Digestion to check & Electrophoresis - 28th July
• Main Digestion of BBa_J61051 and BBa_K228001 - 29th July
• Segregate Process - 29th July
• DNA purification gel agarose - 30th July
• Ligation - 30th July
• Transformation of competent E.coli cells - 31st July
• Liquid Culture - 1st August
• Plasmid DNA Purification - 4th August
• Digestion to check & Electrophoresis - 4th August
• Final Stock BBa_K1372000 - 4th August
• Liquid Culture - 5th August
• Plasmid DNA Purification - 6th August
• Digestion of BBa_K1372000 and BBa_B0015- 6th August
• Segregate Process - 6th August
• DNA purification gel agarose - 7th August
• Ligation - 7th August
• Transformation of competent E.coli cells - 8th August
• Liquid Culture - 11th August
• Plasmid DNA Purification - 12th August
• Digestion to check & Electrophoresis - 12th August
• Final Stock BBa_K1372001 - 12th August

Construction of the Fusion Color Chromoprotein

• Plasmid DNA extraction and electrophoresis - 24th July
• PCR of the Chromoprotein - 18th August
• PCR Cloning in pGEMT easy - 20th August
• Transformation of DH5α with chromoprotein (in pGEMT easy) - 21st August
• Plasmid extraction - 25th August
• Plasmid extraction - 26th August
• Electrophoresis of pGEMT - 27th August
• Transformation of DH5α by pSB1C3 - 28th August
• Colony replication - 1st September
• Liquid culture - 2nd September
• Plasmid extraction and gel electrophoresis - 3rd September
• PCR - 5th September
• Check sequence of the w6 clone - 8th September
• PCR Transformation for stock of the w6 clone - 15th September
• PCR colony w6 clone, Liquid culture - 16th September

The Lemon Shaping

• Concentration Agar Test - here
• Concentration Agar Test - here
• Agar mold test - here
• Agar mold test - here
• Incubation of E. Coli with plasmid FNR RBS AmylCP in LB here
• Coloration of agar here
• Coloration of agar here
• Preparation of M63 medium here
• Incubation of E. Coli with plasmid FNR RBS AmylCP in M63 here
• Transformation of odor free E. coli with plasmids coding Fluo Protein here
• Results of Fluorescent Protein here