Difference between revisions of "Part:BBa K5237015"
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<partinfo>BBa_K5237327 short</partinfo>
 
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This part can be used for the display of nanobodies on the surface of <i>E.coli</i>. Salema, V. et al, (2013) showed efficient presentation of nanobodies on the surface of <i>E.coli</i> K-12 cells by fusing them to the &#946; domain of Intimin. The part comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a &#946;-barrel that inserts into the outer membrane. The C-terminus is exposed to the extracellular milieu and contains a myc tag (EQKLISEED) that can be used for purification or detection of full-length protein.
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This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on <i>E.coli</i>.
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Salema et al., (2013) showed efficient presentation of nanobodies on the surface of <i>E.coli</i> K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane.  
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The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.
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       <img alt="FRET_tetR-Oct1" src="https://static.igem.wiki/teams/5237/wetlab-results/sist-fret-final.svg" style="width:99%;" class="thumbimage">
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       <img alt="FRET_tetR-Oct1" src="https://static.igem.wiki/teams/5237/wetlab-results/anti-myc-wb-intimin-myc.png" style="width:99%;" class="thumbimage">
 
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         <i><b>Figure 3: Fluorescence measurement of mNeonGreen, mScarlet-I and FRET.</b> Fluorescence intensity of mNeonGreen
 
         <i><b>Figure 3: Fluorescence measurement of mNeonGreen, mScarlet-I and FRET.</b> Fluorescence intensity of mNeonGreen

Revision as of 08:52, 30 September 2024


No part name specified with partinfo tag.

This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on E.coli. Salema et al., (2013) showed efficient presentation of nanobodies on the surface of E.coli K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.


FRET_tetR-Oct1
Figure 3: Fluorescence measurement of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 hours after IPTG induction (0.05 mM) and normalized to cell count (OD600). Statistical significance was determined with Ordinary two-way ANOVA withŠidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Data is depicted as mean (n=3) ± SD


Sequence and Features No part name specified with partinfo tag.