Difference between revisions of "Part:BBa K811003"
 
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=== Characterization ===
 
=== Characterization ===
 
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 lysed, and spun for 1 hour at 20000g to separate the membrane fragments from the lysate. The results were compared to Intein-mCherry, a soluble version of mCherry (Figure 1).
 
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 lysed, and spun for 1 hour at 20000g to separate the membrane fragments from the lysate. The results were compared to Intein-mCherry, a soluble version of mCherry (Figure 1).
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[[Image:MCherry-vs-INPNC-mCherry.jpg|frame|center|Figure 1: mCherry remains in the cell membrane pellet after centrifugation when compared to soluble intein-mCherry.]]
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[[Image:MCherry-vs-INPNC-mCherry.jpg|400px|thumb|center|Figure 1: mCherry remains in the cell membrane pellet after centrifugation when compared to soluble intein-mCherry.]]
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The UGent Belgium 2016 iGEM team codon optimised this part for <i> E. coli </i> and used the sequence in [[Part:BBa_K1896013|BBa_K1896013]].
  
 
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<partinfo>BBa_K811003 parameters</partinfo>
 
<partinfo>BBa_K811003 parameters</partinfo>
 
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===Parts improvement by ELTE team (2022)===
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Team ELTE from 2022 improved this part integrated into their project.
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The improvment involved codon optimalisation for protein expression in E. coli. (Codon optimalised INPNC for Surface Display <partinfo>BBa_K4375007</partinfo>)
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Moreover, they added novel features to it, and they designed upon it a composite part, called MiniNano. This construct is compact and integrates the tumor targeting and detection features into one component. It visualizes tumor tissues with a near-IR emitting fluorescence protein called miRFP670 nano, so it is more applicable since light at this wavelength range penetrates tissues deeper. (To know more please refer to <partinfo>BBa_K4375018</partinfo>)
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[[Image:BBa_K4375018_MiniNano.png|450px|thumb|center|'''Figure 2:''' Use of '''INPNC''' by the ELTE team.]]

Latest revision as of 14:46, 10 October 2022

INPNC 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]

Ice Nucleation Protein (N, C termini only). Surface display protein.


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.

Characterization

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 lysed, and spun for 1 hour at 20000g to separate the membrane fragments from the lysate. The results were compared to Intein-mCherry, a soluble version of mCherry (Figure 1).

Figure 1: mCherry remains in the cell membrane pellet after centrifugation when compared to soluble intein-mCherry.


The UGent Belgium 2016 iGEM team codon optimised this part for E. coli and used the sequence in BBa_K1896013.


Parts improvement by ELTE team (2022)

Team ELTE from 2022 improved this part integrated into their project.

The improvment involved codon optimalisation for protein expression in E. coli. (Codon optimalised INPNC for Surface Display BBa_K4375007)

Moreover, they added novel features to it, and they designed upon it a composite part, called MiniNano. This construct is compact and integrates the tumor targeting and detection features into one component. It visualizes tumor tissues with a near-IR emitting fluorescence protein called miRFP670 nano, so it is more applicable since light at this wavelength range penetrates tissues deeper. (To know more please refer to BBa_K4375018)

Figure 2: Use of INPNC by the ELTE team.