Difference between revisions of "Part:BBa K811004"

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<partinfo>BBa_K811004 short</partinfo>
 
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See BBa_K811003 has a HA tag for purification experiments, does not affect membrane localization.
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===Usage and Biology===
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<b>Are you looking for INPNC-MCS? See <a href="https://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K811005">BBa_K811005<a></b>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K811004 SequenceAndFeatures</partinfo>
 
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=== Characterization ===
 
=== Characterization ===
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INPNC was fused to a red florescent protein, mCherry, and cloned into the pET-26b(+) vector. The construct was then expressed in BL21 cells after induction with 1mM IPTG. The cells were incubated with anti-HA antibody conjugated to Alex Fluor 647 dye from Cell Signalling Technologies and imaged with a confocal microscope (Figure 1).
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INPNC (BBa_K81103) was tagged with an HA tag on the C terminus to verify surface expression.  This construct was cloned into the pET-26b(+) vector and then expressed in BL21 cells after induction with 1mM IPTG. The cells were incubated with anti-HA antibody conjugated to Alex Fluor 647 dye from Cell Signalling Technologies and imaged with a confocal microscope (Figure 1).
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[[Image:DARPinfigure.PNG|400px|thumb|center|Figure 1: mCherry remains in the cell membrane pellet after centrifugation when compared to soluble intein-mCherry.]]
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[[Image:DARPinfigure.PNG|400px|thumb|center|Figure 1: BL21 expressing DARPin, a HER2 binding protein (A), a INPNC-DARPin fusion without antibody (B), and a INPNC-DARPin fusion, induced with IPTG.]]
  
  
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<partinfo>BBa_K811004 parameters</partinfo>
 
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Latest revision as of 22:49, 13 October 2012

INPNC-HA

Are you looking for INPNC-MCS? See BBa_K811005 Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
    Illegal AgeI site found at 823
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Ice nucleation protein (INP) is a protein found in Xanthomonas campestris pc. campestris BCRC 12846. It functions as, as its namesake suggests, causing ice nucleation and formation. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurance in prokaryotes.

The INP protein is composed of a N-terminal region that appears to interact with the phospholipid membrane, a C-terminus hydrophillic region that is exposed to the outside membrane, as well as a central 8, 16, or 48 amino acid motif that is responsible for INP's ice nucleation properties. However, this central amino acid motif is not necessary for INP's surface display properties. Therefore, scientists truncated the protein, retaining only the N (179 aa) and C termini (49 aa) to produce INPNC.

This truncated protein retains INP's membrane display abilities, and also contains a C-terminus HA tag suitable for detection via antibodies.

Characterization

INPNC (BBa_K81103) was tagged with an HA tag on the C terminus to verify surface expression. This construct was cloned into the pET-26b(+) vector and then expressed in BL21 cells after induction with 1mM IPTG. The cells were incubated with anti-HA antibody conjugated to Alex Fluor 647 dye from Cell Signalling Technologies and imaged with a confocal microscope (Figure 1).

Figure 1: BL21 expressing DARPin, a HER2 binding protein (A), a INPNC-DARPin fusion without antibody (B), and a INPNC-DARPin fusion, induced with IPTG.


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