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 1.1 Tools used 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 Sites

Preliminaries

Tools used

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] These were done via known domain databases from GenBank, with a ClustalW Realignment option in the Web Services menu of JalView

• Clustal Color Scheme
• Hydrophobicity Color Scheme
• Helix Propensity Color Scheme
• Turn Propensity Color Scheme

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

Transmembrane Prediction - [http://www.sbc.su.se/~miklos/DAS/| DAS Transmembrane Prediction]

JalView File of Seq. Alignments - doesn't work ATM

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 get picture

SopII Analysis

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

• SopII Transmembrane

This shows consistent TM helicies throughout the whole sequence

HtrII Analysis

Methyl-accepting chemotaxis proteins (MCP) are hypothesized to be modular in structure (ref needed). This modularity and homology between most MCPs is evidence by the clear separation of HAMP domains from methyl-accepting helical domains and the sensor kinases. Below is shown evidence for this in HtrII.

• Hypothesized MCP structure

This hisitidine kinase also has a clear TM region, from residues 0-80

• HtrII Transmembrane

Genbank mentions the HAMP domain (Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases)

This is the key to the propagation of the excitation signal according to (ref needed)

Evidence for the HAMP domain is contained in the following sequence alignment with known HAMP domains from GenBank

• HtrII HAMP

evidence for HAMP domain. Alignment suggest homogoly from residue 65 to 134. This conforms with the GenBank analysis shown belown

evidence for methyl-accepting chemo-taxis like domain (these are the likely helices (hydrophobic residue every 7 AA, after the HAMP domain)

• HtrII MCPa

• HtrII MCPb

• HtrII MCPc

The above clearly suggest that HtrII follows the modular structure. The helical domains are evidence by the hydrophobic (blue) residues, that occur every 7-8 residues. This begins at residues 210 in the sequence (EVMDR), which is simliar to the GenBank Analysis

This is further shown by looking at the hydrophicity traces below. Although the consistency of the 7-8 repeat of hydrophobic residue depends on helix turn propensity

• HtrII Methyl-accepting helicies

GenBank Analysis

COG5000 Location:99–249 Blast Score:90 NtrY; Signal transduction histidine kinase involved in nitrogen fixation and metabolism regulation [Signal transduction mechanisms]

smart00283 Location:239–446 Blast Score:277 MA; Methyl-accepting chemotaxis-like domains (chemotaxis sensory transducer). Thought to undergo reversible methylation in response to attractants or repellants during bacterial chemotaxis.

pfam00672 Location:99–133 Blast Score:85 HAMP; HAMP domain.

imply that the design with tsr worked as this was also a MCP (probably don't need to give evidence for this)

evidence for the kinase

ComP Analysis

evidence for HAMP domain

• ComP HAMP

transmembrane prediction conferes with this as follows

• ComP TM

same alignment as for HtrII above, it is likely that the gaps are loops. We will account for this in the fusion variants.

evidence for kinase

• ComP Kinase

confers with GenBank analysis below

evidence for methylated (or accepting) helices... analysis based on region between HAMP domain and kinase (between residues KNTILD| and |MFAEIK)

• ComP Helix Propensity

• ComP Turn Propensity

• ComP Hydrophobicity

also refer to direct aligment between comP and HtrII helical regions

• ComP / HtrII Comparison of helical regions

GenBank Analysis cd00075 Location:681–767 Blast Score:129 HATPase_c; Histidine kinase-like ATPases; This family includes several ATP-binding proteins for example: histidine kinase, DNA gyrase B, topoisomerases, heat shock protein HSP90, phytochrome-like ATPases and DNA mismatch repair proteins pfam07730 Location:568–638 Blast Score:138 HisKA_3; Histidine kinase. This is the dimerisation and phosphoacceptor domain of a sub-family of histidine kinases. It shares sequence similarity with pfam00512 and pfam07536.

Chosen Fusion Sites

from the paper. they chose the were roughly at the start, middle, end of the HAMP domain ...

so along the those lines, since primer synthesis has gotten cheaper, we have chosen the following 7 sites. However, these are located immediate after the HAMP domain, so as not to disrupt the signaling. Note the Bold residue is overlap and is lost.

The next 5 are reference to after the HAMP domain finishes in both HtrII and ComP.

Construct 1 - position 0 HtrII (Q S V R T S || ComP (VL L V S K A)

Construct 2 – position 1 HtrII (S V R T S L|| ComP (LL V S K A Y)

construct 3 – position 2 HtrII (V R T S L E|| ComP (LV S K A Y T)

construct 4 – position 3 HtrII (R T S L E D|| ComP (VS K A Y T F)

construct 5 – position 4 htrII (EDAEA|| ComP (VSMEN)

construct 6 – position 5 HtrII (ERFGE|| ComP (VTSEI)

construct 7 – position 96/95 htrII (EDAEA|| ComPp (VSMEN)

File:Seq align.jpg Media:Example.ogg