Part:BBa_K4765109

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

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 , 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

Figure1 Sequencing map of MVN Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.

Successful Protein Expression

Figure 2. SDS-PAGE electrophoresis of intimin-MVN We constructed intimin-MVN into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represent intimin-MVN and intimin-MVN + IPTG. 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 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 / 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. 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 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