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From 2013.igem.org

Safety

Genetic Modifications

The project “Thermocoli” consists in a circuit of transcription factors and reporter genes, some of which are under the post-transcriptional regulation of thermoregulable RNA elements, also known as RNA thermometers. These genes are arrange in a circuit constructed in such a way that three distinguishable states should emerge, characterized by the expression and repression of two different reporter fluorescent proteins.

In this work, we intend to regulate the expression not only reporter proteins, but also the expression of at least one transcription factor using RNA thermometers. If proved possible, the RNA-thermoregulation of transcription factors will widen the spectrum of genetic circuit topologies that can be used for a number of purposes, most remarkably, the research of basic cellular processes and the replacement of chemical inducers for industrial-scale processes. Our system can be subdivided in five different modules that can be characterized separately. Figure 1 shows those submodules labeled from A to E. Here we describe each one of them:

A. This synthesized construction has a gene that codes for the green fluorescent protein (GFP) variant that has an LVA degradation tag; the gene is under the regulation of a pLac promoter and a thermolabile ribosome binding site (RBS) that should allow for translation only at temperatures above 32°C. At the 3’ end of the gene dubbed “GFP-LVA” are two consecutive transcription termination regions.

The GFP sequence is similar to the one found in part BBa_K145015 and is a mutant derived from the green fluorescent protein found in jellyfish (Andersen et al., 1998). The LVA tag that is in frame with the coding sequence of the TetR gene, is actually a derivation of the C-terminal AANDENYALAA tagging sequence, which makes proteins susceptible to degradation by the ClpX and ClpA proteases (McGiness, Baker and Sauer, 2006).

After testing different variants for the last three amino acid residues in the C-terminal part of the peptide, it was found that AANDENYALAA variants show different degradation rates. We chose the LVA because is the most commonly employed in iGEM projects and we attached it to the coding sequence of GFP using a spacer (or scar) similar to the one present in part BBa_J04450. The transcription termination sequences employed are two: part BBa_B0010 (derived from T1 of E. coli rrnB) and part BBa_B0012 (derived from TE of bacteriophage T7). Both sequences are stem loops that hinder the processivity of E. coli RNA polymerase.

B. A construction that has a gene coding for the LacI transcription factor, which also has a LVA degradation tag. The LacI gene is transcribed through a constitutive promoter and a thermolabile RBS with an optimal translation temperature of 37°C. Two termination sites were added at the 3’ end of the gene.

The constitutive promoter corresponds to the sequence of part BBa_J23119, the most potent member of a family of promoter variants obtained from a combinatorial library. Transcription factor LacI is the same one found in E. coli lac operon and the sequence employed is the same as the one from part BBa_C0012, which is the same as the one employed in the Elowitz and Leibler 2000 study as stated in the “design” section of the registry entry for part BBa_C0012, this LacI variant differs from the wild-type in that the GTG start present in nature was changed to an ATG start.