Melbourne/Blue Photosensor Background
From 2007.igem.org
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==== HtrII Analysis ==== | ==== 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 ==== | ==== 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 ... | ||
[[Image:seq_align.jpg]] | [[Image:seq_align.jpg]] | ||
[[Media:Example.ogg]] | [[Media:Example.ogg]] | ||
Revision as of 12:09, 6 August 2007
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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 |
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
