Improve
# Overview
B.HOME is built on the foundation of existing iGEM parts. With support from our Parthub 2, we can quickly identify suitable parts from previous iGEM teams, use directly or modify according to the functionalities we aim to achieve. Please visit our Software page for more details.
In this year's project, we focused on three major improvements:
- Enhance the ribozyme-assisted polycistronic co-expression (pRAP) system[1] by introducing synthetic stem-loops.
- Improve the existing parts for biofilm formation by presenting the Ag-Nb pairs and lectin on the surface of E. coli, enabling binding between E. coli-E. coli and E. coli-cyanobacteria.
- Use multiple methods to characterize the adhesive capabilities of exopolysaccharides within our EPS module.
Furthermore, we made minor improvements to various other parts.
# Improve pRAP
Last year, we only used the hammerhead ribozyme (opens new window), which could cleave ploycistronic mRNA into several monocistronic mRNA, allowing for efficient expression of proteins for metabolic engineering. We also demonstrated by changing RBS strength (opens new window) we could control protein expression levels, ultimately successfully expressing enzymes related to synthetic β-carotene production.
However, in this year's experiments, we observed degradation of monocistronic mRNA after ribozyme cleavage, as reported in the original pRAP paper[1:1], affecting our functional characterization. Therefore, we added the stem-loop (liu2023) at the 3' end of the CDS to prevent mRNA degradation. We also design synthetic stem-loops using our software, with different strength, and experimental tested their anti-degradation capabilities. Please visit our Part stem-loop test BBa_K4765129 (opens new window) for more details..
Simultaneously, we also constructed various ribozymes into our Part ribozyme test: leaky expression BBa_K4765120 (opens new window).
# Improve Biofilm Formation
Taking inspiration from GreatBay_SCIE iGEM 2022 (opens new window) BBa_K4275026 (Neae-Nb3) (opens new window), we used the previous described antigen-nanobody (Ag-Nb) pairs[2][3] presented by intimin to facilitate the binding between E. coli. To build a programmable biofilm, We required Ag-Nb pairs with varying binding strengths, and we tested three different pairs. Subsequently, We successfully constructed a biofilm using the most effective pair, as described in BBa_K4765106 (opens new window).
We also explored lectin-mediated binding between cyanobacteria and E. coli. To find the appropriate surface display system, we compared intimin (opens new window) from GreatBay_SCIE iGEM 2022 (opens new window) with INPNC (opens new window) from XMU iGEM 2022 (opens new window), and chose the more effective one - intimin. We added LCA and MVN to the C-terminus of intimin, resulting in Part BBa_K4765109 (Twister P1 + T7_RBS + intimin-MVN fusion + stem-loop) (opens new window) and Part BBa_K4765110 (Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop) (opens new window). E. coli expressing these parts demonstrated significant binding capabilities with Microcystis aeruginosa PCC7806 and S. elongatus PCC7942 respectively.
# Improve EPS Characterization
Both XJTU iGEM 2020 (opens new window) and 2022 (opens new window) teams have overexpressed galU and pgmA to increase exopolysaccharide (EPS) production. We adopted their methods for EPS production while focusing on the adhesion capabilities.
We incorporated red fluorescent protein mScarlet after galU and pgmA, creating Part BBa_K4765121 (ribozyme connected: galU + pgmA + mScarlet) (opens new window). We found EPS expressing bacteria is "heavier", precipitate faster, likely due to more "sticky". Before forcely pepitting, these EPS expressing bacteria form cluster in liquid culture. Under a fluorescence microscope, by increasing the flow speed of culture media, we observed that E. coli with red fluorescence (simultaneously expressing galU and pgmA) were the very last ones being washed away, confirming superior adhesion capability.
# Other Improvements
In addition, we did the following:
- Codon-optimized the enzymes for synthesize MAA (from BBa_K4765010 (opens new window) to BBa_K4765011 (opens new window))
- Co-expressed the anti-desiccation protein SAHS 33020 with H. ex mtSSB to test their anti-desiccation capabilities
- Analyzed factors affecting SacC (opens new window) functionality, and improve culture media for bacteria
# Improved Parts
# References
Liu Y, Wu Z, Wu D, Gao N, Lin J (2023). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synth Biol, 12(1), 136-143. https://doi.org/10.1021/acssynbio.2c00416 ↩︎ ↩︎
Glass DS, Riedel-Kruse IH (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell, 174(3), 649-658.e16. https://doi.org/10.1016/j.cell.2018.06.041 ↩︎
Kim H, Skinner DJ, Glass DS, Hamby AE, Stuart BAR, Dunkel J, Riedel-Kruse IH (2022 Aug). 4-bit adhesion logic enables universal multicellular interface patterning. Nature, 608(7922): 324-329. https://doi.org/10.1038/s41586-022-04944-2 ↩︎
