Melbourne/Blue Photosensor Background

From 2007.igem.org

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insert TM analysis here. reference the three papers cited in background
insert TM analysis here. reference the three papers cited in background
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* [[Melbourne/Blue Photosensor Background/sopII_TM| SopII Transmembrane ]]
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* [[Melbourne/Blue Photosensor Background/sopII_TM| SopII Transmembrane prediction ]]
This shows consistent TM helicies throughout the whole sequence
This shows consistent TM helicies throughout the whole sequence

Revision as of 13:27, 26 October 2007

<return to top of background> <return to home page>

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

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

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

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.

HtrII BLAST analysis


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


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

This alignment suggest homogoly from residue 65 to 134 in the HtrII sequence. This conforms with the GenBank analysis shown below.


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


The above clearly suggest that HtrII follows the modular structure. The presence of helical domains are suggested by the hydrophobic (blue) residues, that occur every 7-8 residues. This begins at residues 210 in the sequence (EVMDR), which is similar 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



Analysis of ComP motifs

evidence for HAMP domain


ComP BLAST analysis

This is aligned to the same pfam00672 family as HtrII is above. It is likely that the gaps in the ComP alignment are loops. We will account for this in the fusion variants. transmembrane prediction confers with this as follows


evidence for kinase


confers with GenBank analysis below


Evidence for methylated (or accepting) helices: analysis based on region between HAMP and Kinase domains (between residues KNTILD| and |MFAEIK)


also refer to direct aligment between comP and HtrII helical regions



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 overlaps and is lost :)

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

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

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

Construct 3 – position 2 HtrII (V R T S L E || ComP (L V S K A Y T) >> ...VRTSLEVSKAYT...

Construct 4 – position 3 HtrII (R T S L E D || ComP (V S K A Y T F) >> ...RTSLEDSKAYTF...

Construct 5 – position 5 htrII (S L E D A K || ComP (K A Y T F E V) >> ...SLEDAKAYTFEV...

Construct 6 – position 16 HtrII (A E Q A Q K || ComP (K V I F L D K) >> ...AEQAQKVIFLDK...

Construct 7 – position 24 htrII (E E I N T E || ComP (E V G P D W N) >> ...EEINTEVGPDWN...

Future Directions

Will try deleting residues between fusion point so as to rotate the kinase domain relative to the HAMP domain to see what effect this has on the chimera function.


File:Seq align.jpg Media:Example.ogg