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From 2010.igem.org

Phototaxis

Progress report: Today the decision to atempt to make our backteria synthesise retinal endogenously was made. We will be using old material from the Cambridge 2009 project (Part:BBa_K274210) to make our bacteria synthesise beta-carotene, and we have designed a new biobrick around the ninaB gene from D. melanogaster, that has been shown to produce a beta-carotene 15,15'-monooxygenase [http://www.jbc.org/content/275/16/11915.long][http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14720/?tool=pubmed]. An enzyme that catalyses cleavage beta-carotene into two retinal molecules. This protein has been shown to function in E. coli[http://www.jbc.org/content/275/16/11915.long[1]], along with a synthetic operon closely matching the one from the cambridge project.

We have already isolated the gene sequence, and suggested biobricks for the coding region (BBa_K343001) and a combined part (BBa_K343002) that will work as a protein generator. The relevant cDNA is available for purchase, and should be easy to acquire.

On top of this we deduced the coding sequence for the SopII-HtrII-Tsr fusion, chimera protein from Spudich et al. [http://jb.asm.org/cgi/content/full/183/21/6365] From the information given in the materials and methods section of the article, we could deduce the exact sequence. The amount of amino acid residues were given and the gene sequences known. From there we could find out where and how the gene sequences were stitched together. By that we could already sandbox the planned biobrick for the bluelight receptor sensory rhodopsin II coupled to the chemotaxis pathway of E.Coli via the HtrII and Tsr fusion. The next part is obtaining the physical DNA, for which we will have to contact Spudich lab and ask for a plasmid containing the chimera-protein.

BioBrick Design: Today we have proposed four biobricks:

BBa_K343000 -

BBa_K343001 - Sandboxed coding sequence from ninaB gene

BBa_K343002 - Sandboxed coding sequence from ninaB gene on one of the weaker Anderson promoters, with rbs and dual terminator.

BBa_K343003 - Sandboxed coding sequence for the SopII-HtrII-Tsr fusion protein.

The sequence for the D. melanogaster gene is taken from the cDNA sequence from flybase.org, and matched by length to the b-diox protein. Choice of promoter was made on the consideration that much of this enzyme might not be needed to supply sufficient retinal. Rbs and terminators are entirely standard.

The Cambridge part needed is already on one of our distribution plates: Spring 2010 Distribution 2010 Kit Plate 3, Well 6N, pSB1A2

--CKurtzhals 17:11, 2 July 2010 (UTC)

References:

1. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal.,von Lintig J, Vogt K., J Biol Chem. 2000 Apr 21;275(16):11915-20.

2. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo, Johannes von Lintig,* Armin Dreher, Cornelia Kiefer, Mathias F. Wernet, and Klaus Vogt, Proc Natl Acad Sci U S A. 2001 January 30; 98(3): 1130–1135.

3. "An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli", Jung K-H, Spudich EN, Trivedi VD and Spudich JL ; Journal of Bacteriology, Nov. 2001, p. 6365–6371.

Flagella

Biobrick design: We want to make a constitutive active FlhDC operon. For this we will use a sigma 70 promotor. Sigma 70 is a constitutive active housekeeping gene. We have ordered FlhDC and are waiting for it to arrive. Also we need a terminator and a ribosomal binding sequence (RBS). The promotor (BBa_J23100), the terminator (BBa_B0015) and the RBS (BBa_J61100) are all in the 2010 spring kit.

We have created this as a part on Partsregistry.org