Difference between revisions of "Part:BBa K4765110"

(→‎Aggregation Assay)
(→‎Characterization)
Line 20: Line 20:
  
 
|}
 
|}
 +
====Successful Protein Expression====
 +
{|
 +
| <html><img style="width:200px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/intimin-lca.png" alt="contributed by Fudan iGEM 2023"></html>
 +
|-
 +
| '''Figure 2. SDS-PAGE electrophoresis of intimin-LCA'''
 +
We constructed intimin-LCA into the pET28a plasmid and transformed it into ''E. coli'' BL21 DE3. Lane 1 to 2 represent intimin-LCA and intimin-LCA + IPTG.  As indicated by the red arrow, we successfully expressed intimin-LCA.
 +
 
====Aggregation Assay====
 
====Aggregation Assay====
 
We conducted sedimentation experiments to validate intimin-LCA's role in mediating the binding of ''E.coli'' and ''Synechococcus elongatus''. 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 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.
 
We conducted sedimentation experiments to validate intimin-LCA's role in mediating the binding of ''E.coli'' and ''Synechococcus elongatus''. 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 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.

Revision as of 14:37, 12 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. 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.

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
Figure 1. Sequencing map of LCA

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

Successful Protein Expression

contributed by Fudan iGEM 2023
Figure 2. SDS-PAGE electrophoresis of intimin-LCA

We constructed intimin-LCA into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represent intimin-LCA and intimin-LCA + IPTG. As indicated by the red arrow, we successfully expressed intimin-LCA.

Aggregation Assay

We conducted sedimentation experiments to validate intimin-LCA's role in mediating the binding of E.coli and Synechococcus elongatus. 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 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.

As shown in Figure 2, at 6 hours, in the aTc-induced E. coli / Synechococcus elongatus samples, the 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 suggest that intimin-LCA can facilitate the binding between the two entities and promote biofilm formation.

contributed by Fudan iGEM 2023
Figure 2. LCA aggregation result

To make results more apparent, we added red fluorescent protein mScarlet after intimin-LCA. After mixing E. coli expressing intimin-LCA with 'Synechococcus elongatus for 6 hours, significant aggregation can be observed, as indicated by the red arrow in the image.

contributed by Fudan iGEM 2023
Figure 3. Bacteria Percentage Remaining in the Supernatant at 6 Hours

The bacterial quantity in the supernatant is quantified by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles).

contributed by Fudan iGEM 2023
Figure 4. Bacteria Remaining in the Supernatant at 0,2,6,24 Hours

The bacterial quantity in the supernatant is quantified by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles).

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