Team:EPF-Lausanne/Our Project/Data

From 2011.igem.org

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=== Readout system - TetR and RFP ===
=== Readout system - TetR and RFP ===
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The first readout system is composed of TetR with a constitutive promoter, followed by RFP with a pTet promoter. If TetR '''binds''' to pTet, then RFP is ''' repressed'''. The plasmids used here are pSB3K1 pConst-TetR and J61002 Ptet-RFP. This readout system is convenient for fluorescence detection experiments, but it would not be suited for using the lysis cassette as the reporter gene is being repressed upon TetR-pTet interaction.
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The first readout system is composed of TetR with a constitutive promoter, followed by RFP with a pTet promoter. If TetR '''binds''' to pTet, then RFP is ''' repressed'''. The plasmids used here are pSB3K1 pConst-TetR and J61002 Ptet-RFP. This readout system is convenient for fluorescence detection experiments, but it would not be suited for using the lysis cassette as the reporter gene is being repressed upon TetR-pTet interaction. With the lysis device, the interesting cells (where TetR binds to pTet) would survive and we would recover only the useless TetR mutants.
[[File:EPFL_Summary_without_LacI.jpg]]
[[File:EPFL_Summary_without_LacI.jpg]]
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''Sequence''
 
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''Plasmid map''
 
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This plasmid contains a p15A replication origin as well as a Kanamycin resistance marker.
 
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[[File:EPFL_Tetr_plasmid.jpg‎|300px]]
 
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''Dose-response''
''Dose-response''
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=== pSB3K1 Pconst-TetR Ptet-LacI ===
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J61002 only
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''Description''
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This is the final TetR plasmid, containing the TetR gene as well as the LacI inverter with a Ptet promoter. Here we have wild-type TetR and Ptet sequences, but this plasmid is also intended to be used with mutant TetR genes and mutant Ptet binding sequences.
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Parts assembled
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* Plasmid backbone containing Pconst and TetR: see precedent section
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* Terminator: B0014 from the Registry [http://partsregistry.org/Part:BBa_B0014 "B0014"] (sequence copied into our primers)
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* Ptet: R0040 from Registry [http://partsregistry.org/Part:BBa_R0040 "R0040"] (sequence copied into our primers)
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* LacI: amplified from Repressilator plasmid [https://static.igem.org/mediawiki/2011/7/7f/EPFL_LacI_sequence.txt "LacI sequence"] The sequence lacks a stop codon, we added TAA with our primers.
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''Sequence''
 
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Add results for TetR sequencing
 
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Add results for LacI asap
 
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=== Readout system - TetR and lysis ===
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''Plasmid map''
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This second readout system is composed of 3 genes: TetR under pConst control, LacI under pTet control (playing the role of an inverter) and finally RFP under pLac control. Here, RFP is '''induced''' when TetR '''binds''' to pTet. Lysis cassette can be put instead of RFP in thy system, having as a consequence that the cells where TetR mutants bind to pTet would lyse.
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This plasmid has the same structure as pSB3K1 Pconst-TetR: p15A replication origin and Kanamycin resistance marker. However, Ptet-LacI has been added after the p15A sequence.
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To get this readout system, we cotransformed pSB3K1 Pconst-TetR Ptet-LacI with J61002 Plac-RFP. Unfortunately, the sequence of Ptet in front of LacI got mutated during the assembly process, resulting in only a partial repression of LacI by TetR. Still, our results do show the effects of TetR and LacI on the whole system.
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[[File:EPFL_TetR_plasmid_with_LacI.jpg‎|300px]]
 
''ATC induction''
''ATC induction''

Revision as of 18:08, 17 September 2011

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