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Data

TetR Mutants

• V36F
• P39K
• Y42F
• P39Q-Y42M

T7 Promoter Plasmids

• pSB3K1-T7-const-RFP

Description: The T7 promoter upstream of the RFP gene, set in the pSB3K1 backbone

Sequence:

Plasmid Map:

Saturation Curves:

Dose-Response Curves:

• pSB3K1-T7-lac-RFP

• pSB3K1-T7-lac2-RFP

• pSB3K1-T7-const-Lysis

• pSB3K1-T7-lac-Lysis

T7 Promoter Variants

Reporter Plasmids

J61002 Ptet-RFP

TetR plasmids

pSB3K1 Pconst-TetR

Description Parts assembled:

• Plasmid backbone: pSB3K1 from ETHZ 2007 [http://partsregistry.org/wiki/index.php?title=Part:pSB3K1 "pSB3K1"] (taken from the delivery plate)
• Pconst: J23116 from Berkeley 2006 [http://partsregistry.org/Part:BBa_J23116 "j23116"] (sequence copied into our primers)
• RBS and spacer: (sequence copied into our primers)
• TetR: "TetR sequence" (623 bp) The sequence lacks a stop codon, we added TAA with our primers

Sequence

Sequenced data compared to the sequence of Pconst,RBS+spacer and TetR gene:"pSb3K1_TetR_seq" All these parts are correct. Plasmid map

ATC induction The platereader experiment was run for 12h, using 0-0.1-5-25-200-300-600-800-1000-1200-1500 ng/ul final concentrations of ATC. The cells were cotransformed with pSB3K1 Pconst-TetR and J61002 Ptet-RFP, to use the fluorescent protein as a readout. All the concentrations were tested on 4 different cultures and the results were averaged.

File:EPFL timevsRFP comp.jpg

Dose-response

pSB3K1 Pconst-TetR Ptet-LacI

Parts assembled

• Plasmid backbone containing Pconst and TetR: see precedent section
• Terminator: B0014 from the Registry [http://partsregistry.org/Part:BBa_B0014 "B0014"] (sequence copied into our primers)
• Ptet: R0040 from Registry [http://partsregistry.org/Part:BBa_R0040 "R0040"] (sequence copied into our primers)
• LacI: amplified from Repressilator plasmid "LacI sequence" The sequence lacks a stop codon, we added TAA with our primers.

Sequencing results: