Difference between revisions of "Part:BBa K4765110"

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====Aggregation Assay====
 
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To validate the role of intimin-LCA in mediating the binding of ''E.coli'' and ''Synechococcus elongatus'', we conducted sedimentation experiments. Specifically，bacterial solutions of aTc-induced/not-induced intimin-LCA ''E.coli'' + ''Synechococcus elongatus'', were mixed in a 1:1 ratio (600μL per strain per tube, independent experiment repeat 3 times) and allowed to settle. Sampling was performed at 0, 2, 6,and 24 hours by collecting 100μL aliquots from the upper 25% of each mixture (supernatant) in each tube at each time point. These samples were subsequently transferred to EP tubes and stored at 4℃ until the final sampling. Afterward, they were resuspended and transferred to a 96-well assay plate for OD~600~ and OD~685~ measurement. The percentage of bacteria remaining in the supernatant at 6 hours was determined by dividing the bacterial count at 6 hours (as determined by the OD~600~ and OD~685~ measurement) by the bacterial count at 0 hours.
 
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As shown in Figure 2, at 6 hours, in the aTc-induced ''E. coli'' / ''Synechococcus elongatus'' samples,  bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. As shown in Figure 3, For aTc-induced intimin-LCA ''E.coli'' / ''Synechococcus elongatus'' mixed samples, the bacterial count at 6 hours and 24 hours was significantly lower than the uninduced type. These results suggests that intimin-LCA can facilitate the connection between the two entities and promote biofilm formation.
 
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/yzm/lca-percentage.jpg" alt="contributed by Fudan iGEM 2023"></html>
 
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| '''Figure 2: Bacteria Percentage Remaining in the Supernatant at 6 Hours'''
 
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The bacterial quantity in the supernatant is reflected by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles).
 
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/yzm/lca1.jpg" alt="contributed by Fudan iGEM 2023"></html>
 
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| '''Figure 3: Bacteria Remaining in the Supernatant at 0,2,6,24 Hours'''
 
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The bacterial quantity in the supernatant is reflected by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles).
 
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==Reference==
 
==Reference==

Revision as of 10:20, 10 October 2023

Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop

contributed by Fudan iGEM 2023

Introduction

We’ve developed an E. coli-cyanobacteria adhesion module by transfecting intimin-LCA fusion. Intimin-LCA fusion is composed of intimin and LCA. is a lectin from Lentils that can recognize α-linked mannose residues. It is a common lectin that can specifically bind to the LPS on the surface of S. elongatus PCC7942. Intimin includes a short N-terminal signal peptide to direct its trafficking to the periplasm, a LysM domain for peptidoglycan binding, and a beta-barrel for transmembrane insertion[1] , possesses the outer membrane anchoring of LCA. We’ve constructed this fusion protein into our ribozyme-assisted polycistronic co-expression system：pRAP.

Usage and Biology

This biological component delivers LCA to the surface of E. coli, facilitating adhesion between E. coli, and S. elongatus PCC7942. We envision that the adhesion between cyanobacteria and E. coli can promote the exchange of substances within the biofilm, enhancing the biofilm's survivability.

Characterization

Sequencing map

contributed by Fudan iGEM 2023
Figure1 Sequencing map of LCA

Sequencing is performed using the primer：Kan-F: 5-ATTCTCACCGGATTCAGT-3.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1305


Reference

  1. ↑ Piñero-Lambea, C., Bodelón, G., Fernández-Periáñez, R., Cuesta, A. M., Álvarez-Vallina, L., & Fernández, L. Á. (2015). Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins. ACS Synthetic Biology, 4(4), 463–473. https://doi.org/10.1021/sb500252a