Melbourne/Blue Photosensor Background

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== Preliminaries ==
== Preliminaries ==
 +
=== Tools used===
Restriction Site Analysis - [http://tools.neb.com/NEBcutter2/index.php| NEB cutter]
Restriction Site Analysis - [http://tools.neb.com/NEBcutter2/index.php| NEB cutter]
Line 21: Line 22:
Transmembrane Prediction - [http://www.sbc.su.se/~miklos/DAS/| DAS Transmembrane Prediction]
Transmembrane Prediction - [http://www.sbc.su.se/~miklos/DAS/| DAS Transmembrane Prediction]
-
[[Media:htrII comP HAMP and MCP comparison.jar| JalView File of Seq. Alignments ]]
+
[[Media:comparison.jar| JalView File of Seq. Alignments ]] - doesn't work ATM
==Sequences and Restriction Sites==
==Sequences and Restriction Sites==
-
==== SopII ====
+
==== SopII (sensory rhodopsin II)====  
-
sensory rhodopsin II
+
[[Melbourne/Blue Photosensor Background/SopII/DNA| DNA]],
[[Melbourne/Blue Photosensor Background/SopII/DNA| DNA]],
Line 35: Line 35:
-
 
+
==== HtrII (sensory rhodopsin II transducer)====  
-
==== HtrII ====
+
-
sensory rhodopsin II transducer
+
-
 
+
[[Melbourne/Blue Photosensor Background/HtrIIA| DNA]],
[[Melbourne/Blue Photosensor Background/HtrIIA| DNA]],
[[Melbourne/Blue Photosensor Background/HtrII/AA| AA]],
[[Melbourne/Blue Photosensor Background/HtrII/AA| AA]],
Line 44: Line 42:
* No IGEM restriction sites
* No IGEM restriction sites
* No useful restriction site
* No useful restriction site
-
 
==== SopII/HtrII fusion ====
==== SopII/HtrII fusion ====
Line 56: Line 53:
* Use site from SopII (this is the one that will be used)
* Use site from SopII (this is the one that will be used)
-
 
+
==== ComP (two-component sensor histidine kinase)====
-
 
+
-
==== ComP ====
+
-
two-component sensor histidine kinase
+
[[Melbourne/Blue Photosensor Background/ComP/DNA| DNA]] (includes IGEM forward and reverse primers (VF2 + VR) and IGEM prefix and suffix,
[[Melbourne/Blue Photosensor Background/ComP/DNA| DNA]] (includes IGEM forward and reverse primers (VF2 + VR) and IGEM prefix and suffix,
Line 68: Line 62:
[[Melbourne/primary_Restriction_enzymes| HaeII]] (@1902) inserted
[[Melbourne/primary_Restriction_enzymes| HaeII]] (@1902) inserted
-
 
+
==== ComA (sensory rhodopsin II transducer)====
-
 
+
-
==== ComA ====
+
-
sensory rhodopsin II transducer
+
[[Melbourne/Blue Photosensor Background/ComA/DNA| DNA]],
[[Melbourne/Blue Photosensor Background/ComA/DNA| DNA]],
Line 85: Line 76:
==== Overview ====
==== Overview ====
-
Refer to 4 PCR primer method here, 2001 paper
+
Chimera will be constructed using a PCR based stitching approach as described by Spudich, [[Melb:Blue_Photosensor#References|(refer to reference)]]
-
get picture
+
-
 
+
==== Analysis of SopII motifs ====
-
==== SopII Analysis ====
+
transmembrane photoreceptor, requires all-trans retinal as substrate... linked to HtrII
transmembrane photoreceptor, requires all-trans retinal as substrate... linked to HtrII
insert TM analysis here. reference the three papers cited in background
insert TM analysis here. reference the three papers cited in background
-
 
+
* [[Melbourne/Blue Photosensor Background/sopII_TM| SopII Transmembrane prediction ]]
-
[[Image:sopII_TM.jpg| SopII Transmembrane]]  
+
This shows consistent TM helicies throughout the whole sequence
This shows consistent TM helicies throughout the whole sequence
 +
==== Analysis of HtrII motifs ====
 +
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 Analysis ====
+
[[Melbourne/Blue Photosensor Background/HtrII_BLAST| '''HtrII BLAST 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.
+
* [[Melbourne/Blue Photosensor Background/MCP_structure| Hypothesized MCP structure ]]
-
 
-
[[Image:MCP_structure.jpg| Hypothesized MCP structure]]
 
This hisitidine kinase also has a clear TM region, from residues 0-80
This hisitidine kinase also has a clear TM region, from residues 0-80
-
[[Image:htrII_TM.jpg| HtrII Transmembrane]]
+
* [[Melbourne/Blue Photosensor Background/HtrII Transmembrane| HtrII TM finder plot ]]
 +
 
Genbank mentions the HAMP domain (Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases)
Genbank mentions the HAMP domain (Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases)
Line 118: Line 107:
Evidence for the HAMP domain is contained in the following sequence alignment with known HAMP domains from GenBank
Evidence for the HAMP domain is contained in the following sequence alignment with known HAMP domains from GenBank
 +
* [[Melbourne/Blue Photosensor Background/HtrII HAMP| HtrII-pfam00672 Jalview alignment]]
 +
This alignment suggest homogoly from residue 65 to 134 in the HtrII sequence. This conforms with the GenBank analysis shown below.
-
[[Image:htrII_HAMP.jpg| HtrII HAMP alignment]]
 
-
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))
 +
* [[Melbourne/Blue Photosensor Background/HtrII MCPa| HtrII-Methyl-accepting chemotaxis proteins(MCP)sequence alignment residues 390-490 ]]
-
evidence for methyl-accepting chemo-taxis like domain (these are the likely helices (hydrophobic residue every 7 AA, after the HAMP domain)
+
* [[Melbourne/Blue Photosensor Background/HtrII MCPb| residues 490-590 ]]
-
[[Image:htrII_MCPa.jpg| HtrII MCPa]]
+
* [[Melbourne/Blue Photosensor Background/HtrII MCPc| residues 590-700 ]]
-
[[Image:htrII_MCPb.jpg| HtrII MCPb]]
+
-
[[Image:htrII_MCPc.jpg| 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
+
 
 +
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  
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  
-
[[Image:htrII_helicies.jpg| HtrII Methyl-accepting helicies]]
+
* [[Melbourne/Blue Photosensor Background/HtrII Methyl-accepting helicies| HtrII Methyl-accepting helicies ]]
 +
==== Analysis of ComP motifs ====
-
* GenBank Analysis
+
evidence for HAMP domain
-
COG5000
+
-
Location:99–249
+
-
Blast Score:90
+
-
    NtrY; Signal transduction histidine kinase involved in nitrogen fixation and metabolism regulation [Signal transduction mechanisms]
+
-
smart00283
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[[Melbourne/Blue Photosensor Background/ComP_BLAST| '''ComP BLAST analysis''' ]]
-
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
+
* [[Melbourne/Blue Photosensor Background/ComP HAMP| ComP-HAMP sequence alignment ]]
-
Location:99–133
+
-
Blast Score:85
+
-
    HAMP; HAMP domain.
+
 +
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
-
imply that the design with tsr worked as this was also a MCP (probably don't need to give evidence for this)
+
* [[Melbourne/Blue Photosensor Background/ComP TM| ComP TM finder plot ]]
 +
Evidence for existence of kinase domain:
 +
* [[Melbourne/Blue Photosensor Background/ComP Kinase| ComP-Kinase alignment ]]
-
evidence for the kinase
 
-
==== ComP Analysis ====
+
Evidence for methylated (or accepting) helices:
 +
analysis based on region between HAMP and Kinase domains (between residues KNTILD| and |MFAEIK)
-
evidence for HAMP domain
+
* [[Melbourne/Blue Photosensor Background/ComP Helix Propensity| ComP Helix Propensity alignment]]
-
[[Image:comP_HAMP.jpg| ComP HAMP]]  
+
-
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
+
* [[Melbourne/Blue Photosensor Background/ComP Turn Propensity| ComP Turn Propensity alignment]]
-
[[Image:comP_kinase.jpg| ComP Kinase]]  
+
-
confers with GenBank analysis below
+
* [[Melbourne/Blue Photosensor Background/ComP Hydrophobicity| ComP Hydrophobicity alignment]]
 +
Also refer to direct aligment between comP and HtrII helical regions
 +
* [[Melbourne/Blue Photosensor Background/ comparision| ComP / HtrII Comparison of helical regions ]]
-
evidence for methylated (or accepting) helices...
 
-
analysis based on region between HAMP domain and kinase (between residues KNTILD| and |MFAEIK)
 
-
[[Image:comP_helix-prop.jpg| ComP Helix Turn Propensity]]
+
==== Chosen Fusion Sites ====
-
[[Image:comP_turn-prop.jpg| ComP Turn Propensity]]
+
-
[[Image:comP_hydrophob.jpg| ComP Hydrophobicity]]
+
-
also refer to direct aligment between comP and HtrII helical regions
+
Spudich chose fusion sites that were roughly at the start, middle, end of the HAMP domain ...
-
[[Image:comP_HtrII_MCP-comp.jpg| ComP / HtrII Comparison of helical regions]]
+
 +
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 :)
-
GenBank Analysis
+
The next 5 are referenced to after the HAMP domain finishes in both HtrII and ComP.
-
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.
+
 +
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...
-
==== Chosen Fusion Sites ====
+
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.
-
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]]

Latest revision as of 13:40, 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

Chimera will be constructed using a PCR based stitching approach as described by Spudich, (refer to reference)

Analysis of SopII motifs

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

Analysis of HtrII motifs

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 existence of kinase domain:


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

Spudich chose fusion sites that 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

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