Difference between revisions of "Part:BBa K5237015"
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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">
 
       <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
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         <i><b>Figure 1: Fluorescence western blot scan showing expression of myc-tagged intimin by <i>E.coli</i> 10-beta after IPTG induction.</b> <i>E.coli</i> 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the <i>E.coli</i> lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced <i>E.coli</i> lysate.</i>
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          (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
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          after IPTG induction (0.05 mM) and normalized to cell count (OD<sub>600</sub>).
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          Statistical significance was determined with Ordinary two-way ANOVA withŠidák's multiple comparison test, with
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          a single pooled variance. *p &lt; 0.05, ****p &lt; 0.001. Data is depicted as mean (n=3) ± SD</i>
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Revision as of 09:24, 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 1: Fluorescence western blot scan showing expression of myc-tagged intimin by E.coli 10-beta after IPTG induction. E.coli 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the E.coli lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced E.coli lysate.


Sequence and Features No part name specified with partinfo tag.