Difference between revisions of "Part:BBa K2591013"

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LoxP-BGH polyA-LoxP

Usage and Biology

In the field of molecular biology and cell biology, the basic concepts of transcription termination in eukaryotes is widely applied to molecular cloning. For the DNA polymerase II, the transcription termination is always mediated by the process called polyadenylation, which is initiated by poly A signal in the 3’ downstream region of coding sequence. BGH poly A signal is a commonly used terminator in molecular cloning to build a normal expression construct initiated by Pol II. Cre-loxP recombination system is a widely used system to generate deletions, translocations and inversions at specific site of DNA. It consists of two directional 34bp loxP sequence, when these two loxP sites possess the same direction, the sequence between them will be excised by Cre recombinase, when these two loxP sites possess the opposite direction, the sequence between them will be inverted. This method is implemented both in eukaryotic and prokaryotic cells, particularly, Cre-loxP system is used in transgenic mice as well as neurobiology. Nevertheless, it should have great potential in synthetic biology as a switch. This system is relatively simple compared to other mechanic switch, it only need two LoxP sites and one single Cre enzyme, and then, inversion or deletion happens.

</center>Figure1. Deletion event mediated by Cre recombinase when two LoxP sequence posses the same direction.</center>

In the case that BGH polyA signal is flanked by two LoxP site, this construct is leaded by a promoter and is followed by a functional gene, and there is no Cre recombinase, transcription happens but terminate before coding sequence, producing a shorter mRNA, which goes to abortion.(Fig2) But when we introduce Cre recombinase, recombination happens, BGH polyA signal sequence is deleted. This whole sequence can be transcribed to a much longer mRNA, which contains the coding sequence. Finally, the functional protein is expressed.(Fig3)

Figure2. Shorter mRNA terminated by BGH polyA before coding sequence and the abortion of mRNA

Figure3. Longer mRNA terminated by BGH polyA after coding sequence and expression of functional protein

Characterization

Application

LSL can be insert in front of any functional gene or some switches in synthetic biology. It can mediate time and stage-specific expression of some essential genes. For example, scientists had use this strategy to activate some oncogenes in specific stage. Also, for the following iGEMers, when they have constructed a circuit and there is an essential signal, LSL can act as a global turn-on button.

Reference

Jackson EL; Willis N; Mercer K; Bronson RT; Crowley D; Montoya R; Jacks T; Tuveson DA. 2001. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev  15(24):3243-8PubMed: 11751630MGI: J:73445.

Sequence and Features