"
Team:Groningen/project AND gate
From 2011.igem.org
AND gate
The engineered riboregulator by Isaacs et al.
In order to integrate the memory units, we designed an AND gate system which is based on the engineered riboregulator presented by [http://www.ncbi.nlm.nih.gov/pubmed/15208640 Isaacs at al.] in 2004.
The design is based on two subparts, a cis-repressed mRNA (crRNA) and a trans-activating RNA. The crRNA is composed of the sequence of the target gene and an upstream short nucleotid sequence which is complementary to the ribosome binding site (RBS). Repression of gene expression is achieved through sequestration of the RBS by formation of a stem loop structure by the 5'-untranslated region of the crRNA. This prevents binding of the RBS by the 30s ribosomal subunit and subsequent translation.
The taRNA exhibits partial complementarity to the cis-repressive sequence. Its expression, which is driven by a second promoter, allows formation of a RNA duplex molecule and is accompanied by a conformational change in the crRNA structure. The stem-loop structure is unfolded, subsequently abolishing RBS obstruction and allowing translation of the target gene. The RBS present in the taRNA sequence is sequestrated by a stem structure. Twenty six nucleotides are involved in the intermolecular interaction, providing 10^15 unique taRNA-crRNA pairs.
The system possesses several advantages over regular RNA interference to control gene expression: First and most importantly, it can repress as well as activate translation. This characteristic is crucial for the functionality of our system, as it will allow reset of our circuit to the initial state. Second, both the modular structure and the functionality with a variety of genes predestine the artificial riboregulator of Isaacs at al. for the use in synthetic biology and, more specifically in our genetic circuit.
The riboregulator in Count coli
We built two BioBricks which represent our artificial riboregulator and will link the two memory units as an AND gate.
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607005 Part:BBa_K607005] is a combination of a cis-repressive sequence with the encoding region of the lasR protein under transcriptional control of the Prm promoter.
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K607000 BBa_K607000] is the complementary taRNA with an upstream hybB promoter.
TODO
To test the functionality of the engineered riboregulator we want to compare expression of a crRNA-GFP reporter construct in absence and presence of taRNA.
