Difference between revisions of "Part:BBa K4765109"

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===Introduction===
 
===Introduction===
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We’ve developed an ''E. coli''-cyanobacteria adhesion module by transfecting intimin-MVN fusion. Intimin-MVN fusion is composed of intimin and MVN. MVN is a lectin isolated from the cyanobacteria ''Microcystis aeruginosa'' PCC7806 and it was tested by iGEM14_Peking. Intimin which 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 MVN.
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We’ve developed an ''E. coli''-cyanobacteria adhesion module by transfecting intimin-MVN fusion. Intimin-MVN fusion is composed of intimin and MVN. MVN is a lectin isolated from the cyanobacteria ''Microcystis aeruginosa'' PCC7806 and it was tested by [https://2014.igem.org/Team:Peking iGEM14_Peking]. Intimin which 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 MVN.
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We’ve constructed this fusion protein into our ribozyme-assisted polycistronic co-expression system：pRAP.
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4765109 SequenceAndFeatures</partinfo>
 
  
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<!-- Uncomment this to enable Functional Parameter display
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====Successful Protein Expression====
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===Functional Parameters===
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{|
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<partinfo>BBa_K4765109 parameters</partinfo>
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| <html><img style="width:200px" src="https://static.igem.wiki/teams/4765/wiki/zsl/protein-gel/intimin-mvn.png" alt="contributed by Fudan iGEM 2023"></html>
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|-
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| '''Figure 2. SDS-PAGE electrophoresis of intimin-MVN'''
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We constructed intimin-MVN into the pDSG plasmid and transformed it into ''E. coli'' DH5α. Lane 1 to 2 represent intimin-MVN and intimin-MVN + aTc.  As indicated by the red arrow, we successfully expressed intimin-MVN.
  
 
====Aggregation Assay====
 
====Aggregation Assay====
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To validate the role of intimin-MVN in mediating the binding of ''E.coli'' and ''Microcystis aeruginosa'', we conducted sedimentation experiments. Specifically，bacterial solutions of aTc-induced/not-induced intimin-MVN ''E.coli'' + ''Microcystis aeruginosa'', 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.
+
To validate the role of intimin-MVN in mediating the binding of ''E.coli'' and ''Microcystis aeruginosa'', we conducted sedimentation experiments. Specifically，bacterial solutions of aTc-induced/not-induced intimin-MVN ''E.coli'' + ''Microcystis aeruginosa'', 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 OD600 and OD685 measurement. The percentage of bacteria remaining in the supernatant at 6 hours was determined by dividing the bacterial count at 6 hours (as quantified by the OD600 and OD685 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'' / ''Microcystis aeruginosa'' samples,  bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. As shown in Figure 3, For aTc-induced intimin-MVN ''E.coli'' / ''Microcystis aeruginosa'' mixed samples, the bacterial count at 2 hours and 6 hours was significantly lower than the uninduced type. These results suggests that intimin-MVN can facilitate the connection between the two entities and promote biofilm formation within a relatively short time.
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As shown in Figure 3, at 6 hours, in the aTc-induced ''E. coli'' / ''Microcystis aeruginosa'' samples,  bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. As shown in Figure 4, For aTc-induced intimin-MVN ''E.coli'' / ''Microcystis aeruginosa'' mixed samples, the bacterial count at 2 hours and 6 hours was significantly lower than the uninduced sample. These results suggests that intimin-MVN can facilitate the connection between the two entities and promote biofilm formation within a relatively short time.
  
 
{|
 
{|
 
| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/yzm/mvn-percentage.jpg" alt="contributed by Fudan iGEM 2023"></html>
 
| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/yzm/mvn-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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| '''Figure 3. 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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The bacterial quantity in the supernatant is quantified by measuring the OD600 (1 OD600 corresponds to 10^8 bacterial particles).  
 
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| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/yzm/mvn1.jpg" alt="contributed by Fudan iGEM 2023"></html>
 
| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/yzm/mvn1.jpg" alt="contributed by Fudan iGEM 2023"></html>
 
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|-
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| '''Figure 3: Bacteria Remaining in the Supernatant at 0,2,6,24 Hours'''
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| '''Figure 4. 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).  
+
The bacterial quantity in the supernatant is quantified by measuring the OD600 (1 OD600 corresponds to 10^8 bacterial particles).  
 
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|}
  
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==Reference==
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<!-- -->
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===Sequence and Features===
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<partinfo>BBa_K4765109 SequenceAndFeatures</partinfo>
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 +
<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
 +
<partinfo>BBa_K4765109 parameters</partinfo>
 +
<!-- -->
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 +
===Reference===

Latest revision as of 15:47, 12 October 2023

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

contributed by Fudan iGEM 2023

Introduction

We’ve developed an E. coli-cyanobacteria adhesion module by transfecting intimin-MVN fusion. Intimin-MVN fusion is composed of intimin and MVN. MVN is a lectin isolated from the cyanobacteria Microcystis aeruginosa PCC7806 and it was tested by iGEM14_Peking. Intimin which 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 MVN.

Usage and Biology

This biological component delivers MVN to the surface of E. coli, facilitating adhesion between E. coli and Microcystis aeruginosa PCC7806. 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 MVN

Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.


Successful Protein Expression

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

We constructed intimin-MVN into the pDSG plasmid and transformed it into E. coli DH5α. Lane 1 to 2 represent intimin-MVN and intimin-MVN + aTc. As indicated by the red arrow, we successfully expressed intimin-MVN.

Aggregation Assay

To validate the role of intimin-MVN in mediating the binding of E.coli and Microcystis aeruginosa, we conducted sedimentation experiments. Specifically，bacterial solutions of aTc-induced/not-induced intimin-MVN E.coli + Microcystis aeruginosa, 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 OD600 and OD685 measurement. The percentage of bacteria remaining in the supernatant at 6 hours was determined by dividing the bacterial count at 6 hours (as quantified by the OD600 and OD685 measurement) by the bacterial count at 0 hours.

As shown in Figure 3, at 6 hours, in the aTc-induced E. coli / Microcystis aeruginosa samples, bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. As shown in Figure 4, For aTc-induced intimin-MVN E.coli / Microcystis aeruginosa mixed samples, the bacterial count at 2 hours and 6 hours was significantly lower than the uninduced sample. These results suggests that intimin-MVN can facilitate the connection between the two entities and promote biofilm formation within a relatively short time.

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 OD600 (1 OD600 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 OD600 (1 OD600 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
    Illegal SapI site found at 2058


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