Team:EPF-Lausanne/Our Project/T7 promoter variants

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{{:Team:EPF-Lausanne/Templates/Header|title=T7 promoter variants}}
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{{:Team:EPF-Lausanne/Templates/T7lysisHeader|title=Intro}}
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Our goal is to make two families of T7 promoter variants. One family has mutations on the T7 promoter consensus sequence while the other has the same set of mutations on the consensus sequence but also has a lac operator downstream of the T7 promoter. In each family, we produce six designed variants with different predicted promoter strengths compared to the wildtype as well as three sets of randomer variants which we want to use to check the overall range of promoter strengths.  
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One major challenge in designing new regulatory parts is to determine which combinations of transcription factors and binding sequences match.
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From previous research and our own MITOMI experiments, we know which DNA sequences TetR binds to, and which residues of TetR participate in binding, but we do not know how changing these residues will affect either binding affinity or specificity.
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Molecular dynamics simulations and other theoretical approaches have not come any closer to answering these questions.
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In short, we know too little about protein-DNA interaction to intelligently design transcription factors.
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To make up for this lack of knowledge, we present an experimental system to select valid binding pairs from many random tetR and pTet mutants, based on an inducible lysis gene.
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=== The Making Of A Variant ===
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[[File:EPFL-Solange-Lysis.jpg|700px]]
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To produce these T7 promoter variants, we use a two-step PCR process. The first PCR, which we call "gene-specific" PCR, is a typical PCR that adds a ribosome-binding site (rbs) in front of either RFP or the lysis operon and adds a terminator downstream.  
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The system – in a way a "survival of the weakest" – is related to directed evolution.
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A lysis system based on the K112808 lysis device is indirectly activated by tetR.
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Therefore, if in a given cell the tetR variant present can bind to the tetR promoter, the cell lyses and releases its DNA into the culture media.
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From there, DNA can be recovered and amplified, tranformed, or directly sequenced.
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By design, this DNA codes for a combination of TF and promoter with high mutual affinity, and therefore almost directly yields a valid regulatory part.
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In this light, it is a useful component of our transcription factor development pipeline.
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[[File:rbs_rfp_term.png]]
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This is a direct and practical way of solving the problem of selecting high affinity pairs among the millions of possible combinations of transcription factors and promoters.
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It can be seen as a form of DNA-based information processing, and is therefore also a neat example of a problem more efficiently solved by non-conventional computation.
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[[File:rbs_lys_term.png]]
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To develop the system, we began with a [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/lysis|simple experiment]] to check that a lysis cassette in a plasmid could lyse with greater efficiency as a function of IPTG concentration. The next step towards our goal was to demonstrate that [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/recovery|plasmid DNA can be adequately recovered]] and repackaged (PCR amplified, transformed into a different strain, etc...) as a result of lysing. In a [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/selection|more elaborate experiment]], we were able to show that not only did the lysing efficiently release plasmids from the cells, but that it could be made to do so selectively in a large culture containing a variety of strains. Finally, cognizant of the fact that a good lysis selection method ought to be flexible with regards to the larger reporter system, we manufactured [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/t7prom|twelve different T7 promoter variants]] that exhibit a wide range of strengths and induction efficiencies. The latter will play a crucial role in being able to accomodate the activation time-scales of fragile and complex selection systems.  
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With this PCR product now serving as the DNA template, we start a second PCR which we call the "extension" PCR. It extends the product by adding the T7 promoter, with or without a lac operator downstream. In these illustrations, we have substituted the lysis operon for the RFP gene but the same process is done for RFP.
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[[File:t7_rbs_lysis_term.png]]
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[[File:t7_lac_rbs_lysis_term.png]]
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In the last stage, we run another PCR with a set of primers that will add Gibson overhangs for the [https://2011.igem.org/Team:EPF-Lausanne/Tools/Gibson_assembly Gibson assembly] that will add this promoter construct into the desired plasmid.
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[[File:gibson_t7_lac_lysis_gibson.png]]
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=== The different variants ===
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Vdog, please explain the different variants here :) They should only be listed on the data page, and it's trange to talk about them here without really showing them
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=== Characterization with RFP ===
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''LacI repression''
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With T7 and T7-lac promoter variants in hand, we want to characterize their relative strengths. To obtain the right set-up, we transform the promoter variant plasmids into a different strain of E. coli called BL21. These cells have a mutation in the promoter for the lacI gene. As a result of this mutation, LacI protein is overproduced and is found abundantly in these cells.  
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[[File:basic_bl21.png]]
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In this same strain, the gene for the T7 RNA polymerase is preceded by a lac operator. Since LacI is a repressor and is strongly present in BL21 cells, the production of T7 RNA polymerases is severely repressed.
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[[File:bl21_w_t7rnap_repression.png]]
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With very few T7 RNA polymerases available, there is very little recognition and binding of T7 promoters and consequently very little expression of the gene driven by the T7 promoter. Moreover, the expression of a gene driven by a T7-lac promoter would be even less, since the presence of LacI would block any action of the T7 RNA polymerase.  
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[[File:bl21_w_t7lacreporter.png|left|700px]]
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''IPTG induction''
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With such a system in place, the transformation of our T7 and T7-lac promoter variant plasmids into BL21 cells would produce little to no reporter expression. Now to induce promoter activity, we rely on the fact that the chemical IPTG blocks the repressor action of LacI.
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[[File:bl21_induction_start.png|700 px]]
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Without LacI inhibition, the cell can resume production of T7 RNA polymerases. These in turn can bind to the T7 and T7-lac promoters to express the reporter gene without interruption. So the adding of IPTG to a BL21 cell culture containing these T7 and T7-lac promoter variant plasmids ought to produce high levels of RFP or Lysis expression. Since lysis is a rather binary process (either the cell is lysed or it is not), we use RFP fluorescence as a gauge of promoter strength and efficiency.
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[[File:bl21_induction_complete.png|700px]]
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=== DNA Recovery with Lysis ===
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Having a lysis cassette driven by a T7 promoter is an important step towards being able to recover the DNA sequences of transcription factor and promoter mutants that have strong mutual affinities. To verify that basic lysing can be
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induced, we use the same IPTG experiment as for RFP, using both large samples for visual, qualitative confirmation and small samples in a platereader for quantitative, numerical confirmation.
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Once the basic mechanism of cell lysing is confirmed, the next step is to show that DNA can be recovered from the supernatant. We grow two large cultures of cells. One contains cells that will lyse and release plasmids into the supernatant while the other has non-lysing, "normal" cells.
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[[File:broth_noiptg.png]]
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Adding IPTG to both flasks induces lysis in one set of cells but not in the others.
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[[File:broth_iptg_induction.png]]
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Thanks to qPCR, the supernatant harvested from the lysing culture reveals increased numbers of plasmids while the non-lysing culture exhibits significantly lower numbers of plasmids. A similar test involves transforming the supernatant (whose plasmid content is not known a priori) into cells and counting the number of resulting colonies. The plates containing transformations from the lysing supernatant have vastly superior number of colonies compared to the non-lysing supernatant transformations.
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[[File:iptg_platecount_text.png|500px]]
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To round out the experiments for DNA recovery, it is essential that we show that the recovered DNA is in fact plasmid DNA from the relevant lysed cells.  
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All the components of the selection machine have been separately tested experimentally and found to work. Therefore we are convinced the system can work, but it would still require a full-circle experiment to demonstrate its usefulness.
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 03:03, 22 September 2011

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