"
Team:EPF-Lausanne
From 2011.igem.org
(→Project summary) |
(→Project summary) |
||
| Line 10: | Line 10: | ||
Our project goal was to set up a high-throughput method for generating and characterizing new transcription factors (TFs) that would recognize different promoter sequences. We mutated the TetR transcription factor and obtained ten interesting new mutants. For each of these mutants, we characterized their affinity to the consensus sequence (the Ptet promoter) as well as their position-weight matrices. This characterization was done <i>in vitro</i> on a microfluidic chip using a process called MITOMI, which allowed us to test a single mutant against hundreds of Ptet sequences. | Our project goal was to set up a high-throughput method for generating and characterizing new transcription factors (TFs) that would recognize different promoter sequences. We mutated the TetR transcription factor and obtained ten interesting new mutants. For each of these mutants, we characterized their affinity to the consensus sequence (the Ptet promoter) as well as their position-weight matrices. This characterization was done <i>in vitro</i> on a microfluidic chip using a process called MITOMI, which allowed us to test a single mutant against hundreds of Ptet sequences. | ||
| - | As a complement to the <i> in vitro </i> approach, the next step in characterizing these TF mutants was to produce an <i>in vivo</i> readout system. | + | As a complement to the <i> in vitro </i> approach, the next step in characterizing these TF mutants was to produce an <i>in vivo</i> readout system. To that end, we created and tested two different reporter systems based on RFP expression, with one producing a positive and the other a negative selection of the TetR-Ptet interaction. Because we also wanted a high-throughput method, we decided to use a lysis cassette as a reporter gene. The idea was to transform cells with one plasmid containing a TetR mutant and another plasmid containing a Ptet sequence (either the consensus or a mutated one), and then to have automatic lysis of the cells in which the TetR mutant recognizes the Ptet sequence, the goal being to recover their DNA. This approach could then be coupled to microfluidic chemostat in which hundreds of cell colonies can be grown all at the same time. To enhance the modularity of this readout system, we also created T7 promoter variants which can be coupled to the RFP or lysis genes. Lysis and DNA recovery experiments using this readout system have been efficiently conducted, demonstrating the feasibility of our project design. |
Revision as of 09:01, 19 September 2011
Welcome
It's on! After some warmup experiments during the semester, our team is now fully operational, and already having trouble getting away from the lab. Follow our adventures on twitter: IGEM_EPFL
Team, remember the Todo List
Project summary
Our project goal was to set up a high-throughput method for generating and characterizing new transcription factors (TFs) that would recognize different promoter sequences. We mutated the TetR transcription factor and obtained ten interesting new mutants. For each of these mutants, we characterized their affinity to the consensus sequence (the Ptet promoter) as well as their position-weight matrices. This characterization was done in vitro on a microfluidic chip using a process called MITOMI, which allowed us to test a single mutant against hundreds of Ptet sequences.
As a complement to the in vitro approach, the next step in characterizing these TF mutants was to produce an in vivo readout system. To that end, we created and tested two different reporter systems based on RFP expression, with one producing a positive and the other a negative selection of the TetR-Ptet interaction. Because we also wanted a high-throughput method, we decided to use a lysis cassette as a reporter gene. The idea was to transform cells with one plasmid containing a TetR mutant and another plasmid containing a Ptet sequence (either the consensus or a mutated one), and then to have automatic lysis of the cells in which the TetR mutant recognizes the Ptet sequence, the goal being to recover their DNA. This approach could then be coupled to microfluidic chemostat in which hundreds of cell colonies can be grown all at the same time. To enhance the modularity of this readout system, we also created T7 promoter variants which can be coupled to the RFP or lysis genes. Lysis and DNA recovery experiments using this readout system have been efficiently conducted, demonstrating the feasibility of our project design.
Results of the different experiments can be found on the data page.
For more details on the assembly and mutagenesis strategies, please refer to the following pages: TetR mutants, T7 promoter variants, Reporter plasmids.
