Melbourne/Blue Photosensor Background

From 2007.igem.org

Latest revision as of 13:40, 26 October 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

Preliminaries

Tools used

Restriction Site Analysis - NEB cutter

Sequence Translation - Translate

Sequence Aligment - 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 - NetPrimer

Transmembrane Prediction - DAS Transmembrane Prediction

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

Sequences and Restriction Sites

SopII (sensory rhodopsin II)

DNA, AA, genbank

• No IGEM restriction sites
• HaeII (@168) present

HtrII (sensory rhodopsin II transducer)

DNA, AA, 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,

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, genbank

SpeI (@927) deleted HaeII (@1902) inserted

ComA (sensory rhodopsin II transducer)

DNA, AA, 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

• SopII Transmembrane prediction

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

• Hypothesized MCP structure

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

• HtrII TM finder plot

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-pfam00672 Jalview alignment

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))

• HtrII-Methyl-accepting chemotaxis proteins(MCP)sequence alignment residues 390-490

• residues 490-590

• residues 590-700

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

• HtrII Methyl-accepting helicies

Analysis of ComP motifs

evidence for HAMP domain

ComP BLAST analysis

• ComP-HAMP sequence alignment

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

• ComP TM finder plot

Evidence for existence of kinase domain:

• ComP-Kinase alignment

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

• ComP Helix Propensity alignment

• ComP Turn Propensity alignment

• ComP Hydrophobicity alignment

Also refer to direct aligment between comP and HtrII helical regions

• ComP / HtrII Comparison of 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