Melbourne/Blue Photosensor Background

From 2007.igem.org

(Difference between revisions)
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==== HtrII Analysis ====
==== HtrII Analysis ====
-
Yeh!!!! sequence analysis.
+
evidence for HAMP domain
 +
 
 +
evidence for methyl-accepting chemo-taxis like domain (these are the likely helices (hydrophobic residue every 7 AA, after the HAMP domain)
 +
 
 +
link to RICE power point and rip picture
 +
give general structure of MAPK
 +
imply that the design with tsr worked as this was also a MAPK (probably don't need to give evidence for this)
 +
 
 +
evidence for the kinase
==== ComP Analysis ====
==== ComP Analysis ====
-
==== Chosen Fusion ====
+
evidence for HAMP domain
 +
 
 +
evidence for methylated (or accepting) helices...
 +
 
 +
evidence for the kinase
 +
 
 +
 
 +
 
 +
 
 +
 
 +
==== Chosen Fusion Sites ====
 +
from the paper we chose the were roughly at the start, middle, end of the HAMP domain ...
[[Image:seq_align.jpg]]
[[Image:seq_align.jpg]]
[[Media:Example.ogg]]
[[Media:Example.ogg]]

Revision as of 12:09, 6 August 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

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

evidence for HAMP domain

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

link to RICE power point and rip picture give general structure of MAPK imply that the design with tsr worked as this was also a MAPK (probably don't need to give evidence for this)

evidence for the kinase

ComP Analysis

evidence for HAMP domain

evidence for methylated (or accepting) helices...

evidence for the kinase




Chosen Fusion Sites

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

File:Seq align.jpg Media:Example.ogg