Difference between revisions of "Part:BBa K4765110"
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===Introduction=== | ===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<ref>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 </ref> , possesses the outer membrane anchoring of LCA. | 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<ref>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 </ref> , possesses the outer membrane anchoring of LCA. | ||
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===Usage and Biology=== | ===Usage and Biology=== |
Revision as of 11:37, 11 October 2023
Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop
Contents
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.
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
Figure1 Sequencing map of LCA
Sequencing is performed using the primer:Kan-F: 5-ATTCTCACCGGATTCAGT-3. |
Aggregation Assay
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.
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.
Figure 2: Bacteria Percentage Remaining in the Supernatant at 6 Hours
The bacterial quantity in the supernatant is reflected by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles). |
Figure 3: Bacteria Remaining in the Supernatant at 0,2,6,24 Hours
The bacterial quantity in the supernatant is reflected by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles). |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1305
Reference
- ↑ 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