Revision as of 01:20, 22 September 2011

Introduction
Our Project
Tools
- Gibson Assembly
- MITOMI
Notebook
- Protocols
- May
- June
- July
- August
- September
- October
Considerations
- Human Practices
- Safety
Acknowledgements
- Sponsors
- Partners

Data

team, please check this page: [1]

New parts

we must have 3 favourite, then the others come in a "other biobrick" section

Variants without additional lac operator (constitutive):

[http://partsregistry.org/Part:BBa_K613001 K613001]

[http://partsregistry.org/Part:BBa_K613002 K613002]

[http://partsregistry.org/Part:BBa_K613003 K613003]

[http://partsregistry.org/Part:BBa_K613004 K613004]

[http://partsregistry.org/Part:BBa_K613005 K613005]

[http://partsregistry.org/Part:BBa_K613006 K613006]

Variants with additional lac operator (LacI repressed):

[http://partsregistry.org/Part:BBa_K613007 K613007]

[http://partsregistry.org/Part:BBa_K613008 K613008]

[http://partsregistry.org/Part:BBa_K613009 K613009]

[http://partsregistry.org/Part:BBa_K613010 K613010]

[http://partsregistry.org/Part:BBa_K613011 K613011]

[http://partsregistry.org/Part:BBa_K613012 K613012]

Medium-strength Plac

[http://partsregistry.org/Part:BBa_K613000 K613000]

blablabla

TetR Mutants

TetR variants	Mutagenesis	Promoter	Cterminal	DNA form	Expression	MITOMI vs 1-off	Biobrick	Sequenced	Sent to registry

E37AW43ST141A	PCR-induced	T7	His-tag	linear	ITT tested	v	BBa_K613015	v	v


P39K	PCR-induced	T7	His-tag	template	ITT tested, worked	v	BBa_K613016	v	v

Y42F	PCR-induced	T7	His-tag	linear	ITT tested	v	BBa_K613017	v	v
Y42FK108E	PCR-induced	T7	His-tag	linear	ITT tested		BBa_K613018	v	v

T7 promoter variants

Pre-existing parts

add something about the lysis cassette, and add also on its page on the partsregistry!

In vivo characterization - Readout systems

We created two different readout systems to characterize TetR mutants in vivo. This is the last step of our whole method, after having selected the interesting TetR-Ptet mutant pairs with the lysis device and having characterized them in vitro.

TetR - RFP

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. For more details about them and the experimental results, please refer to the Reporter systems page.

TetR - LacI - RFP

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. The experimental results are available on the Reporter systems page.

@@ Line 81: / Line 81: @@
   add something about the lysis cassette, and add also on its page on the partsregistry!
-== T7 Promoter Variants ==
-===Non-random T7 promoter variants===
-=== Characterization and DNA Recovery with Lysis ===
-Since a major component of our scheme for selecting promoters and transcription factors with strong binding affinities required the ability to lyse cells, we also wanted to test a T7-driven lysis cassette.
-[[File:lysis_dynamics.png|700px]]
-Induction with various concentrations of IPTG reveals a steady increase in the amount of lysis that can be obtained. Here 500 uM yields the most substantial amount of lysis, and that concentration was used in all further experiments dealing with lysis. The negative controls were two-fold: one is an unsuccessful attempt at inserting the lysis cassette downstream of a T7 promoter in the psB3K1 plasmid, while the other is a T7 promoter upstream of an RFP gene. Neither should express lysis.
-[[File:dose_response.png|700px]]
 == In vivo characterization - Readout systems ==

Team:EPF-Lausanne/Our Project/Data

From 2011.igem.org