From 2007.igem.org

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The background to the design of the blue photosensor is included here. Specifically the design for the chimeric fusion protein that will be the key link in blue light pathway

Contents 1 Preliminaries 2 Sequences and Restriction Sites 2.1 SopII 2.2 HtrII 2.3 SopII/HtrII fusion 2.4 ComP 2.5 ComA 3 Chimera Design 3.1 Overview 3.2 SopII Analysis 3.3 HtrII Analysis 3.4 ComP Analysis 3.5 Chosen Fusion

Preliminaries

Restriction Site Analysis - [http://tools.neb.com/NEBcutter2/index.php| NEB cutter]

Sequence Translation - [http://au.expasy.org/tools/dna.html| Translate]

Sequence Aligment - [http://www.jalview.org/download.html| JalView]

PCR primer design - [http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html| NetPrimer]

Sequences and Restriction Sites

SopII

sensory rhodopsin II

DNA, AA, [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=sopII genbank]

• No IGEM restriction sites
• HaeII (@168) present

HtrII

sensory rhodopsin II transducer

DNA, AA, [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3702851&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum genbank]

• No IGEM restriction sites
• No useful restriction site

SopII/HtrII fusion

DNA, AA (frame 2 in Translate - only SopII + HtrII + Linker included in link), linker = TSASA SNGASA,

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=sopII genbank]

• No IGEM restriction sites
• Use site from SopII (this is the one that will be used)

ComP

two-component sensor histidine kinase

DNA (includes IGEM forward and reverse primers (VF2 + VR) and IGEM prefix and suffix, AA, [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=sopII genbank]

SpeI (@927) deleted HaeII (@1902) inserted

ComA

sensory rhodopsin II transducer

DNA, AA, [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=sopII genbank]

SpeI (@12) deleted HaeII (@433) inserted

Chimera Design

Overview

Refer to 4 PCR primer method here, 2001 paper

SopII Analysis

transmembrane photoreceptor, requires all-trans retinal as substrate... linked to HtrII insert TM analysis here. reference the three papers cited in background

HtrII Analysis

Yeh!!!! sequence analysis.

ComP Analysis

Chosen Fusion

File:Seq align.jpg Media:Example.ogg