Part Collection
Our favourite part is BBa_K4765129 (stem-loop test) (opens new window).
# Ribozyme-Assisted Polycistronic co-expression
We've improved previously published Ribozyme-Assisted Polycistronic co-expression (pRAP) system[1]. This improvement allows for the simultaneous expression of multiple proteins by inserting all B.HOME CDS sequences into one (opens new window). Our Software RAP could: 1) design synthetic stem-loops with different strengths to control avaiable mRNA; 2) leverages enzyme
A pRAP module consists of four components arranged from the 5' to the 3' end: a Twister ribozyme, RBS, CDS, and a stem-loop structure.
The Twister ribozyme, an RNA molecule capable of self-cleavage at specific sites[2][3], is strategically incorporated between coding CDSs within a polycistron. The chosen ribozyme efficiently cleaves the polycistronic mRNA transcript, transforming it into individual mono-cistrons. This process eliminates inter-cistron interactions, ensuring comparable translation initiation rates for each mono-cistron.
To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of the CDSs[1:1].
Figure 1: Model for pRAP system.
Twistwe-ribozyme, RBS, CDS, and stem-loop form a basic pRAP component.
Subsequently, we developed an assessment setup to evaluate pRAP performance. This validation system comprises two genes, stayGold (BBa_K4162001 (opens new window)) and mScarlet (BBa_K4765022 (opens new window)). The green fluorescent protein stayGold is positioned at the upstream, while the red fluorescent protein mScarlet is positioned at the downstream. Subsequently, we examined the fluorescence intensity per OD600 and the ratio of red to green fluorescence of the bacteria. This not only allowed us to confirm that both genes can be expressed from the pRAP system. Furthermore, the fluorescence ratio provides additional insights into the efficiency of ribozyme cleavage, as well as the protection of mRNA from degradation by the stem-loop structure.
Figure 2: Stem-loop test.
This module can be used to evaluate stem-loop efficiency.
Table 1: Parts for pRAP system
| Part Name | Type | Description |
|---|---|---|
| BBa_K4765020 (opens new window) | RNA | Twister P1 ribozyme, from 10.1080/15476286.2022.2123640 |
| BBa_K4765021 (opens new window) | RNA | stem-loop, from 10.1021/acssynbio.2c00416 |
| BBa_K4765022 (opens new window) | Coding | mScarlet |
| BBa_K4765119 (opens new window) | DNA | ribozyme test: constitutive expression |
| BBa_K4765120 (opens new window) | DNA | ribozyme test: T7 leaky expression |
| BBa_K4765129 (opens new window) | DNA | stem-loop test |
| BBa_K4765130 (opens new window) | DNA | shuttle test |
We found the protein expression driven by T7 promoter without adding IPTG (BBa_K4765120 (opens new window)) in BL21(DE3) cells, is higher than driven by a constitutive promoter (BBa_K4765120 (opens new window)). Thus, we use T7 leaky setting for most of our funcitonal characterization.
Finally, we integrated CDS sequences from the Survival System, Biofilm Formation System, and Terraforming System into pRAP, with help from RAP (more details on our Software page).
# Survival System
The environment on Mars is extremely harsh, which is why we need to equip the biofilm with robust survival capabilities. To achieve this, we have introduced three modules: Anti-freeze, Anti-UV, and Anti-desiccation.
To enhance the biofilm's resistance to freezing, we have chosen a protein called AnAFP (BBa_K4765015 (opens new window)). Following a recent publication, we have test a new protein called H. ex mtSSB, derived from tardigrades[4]. In comparison to separately introduced the anti-desiccation protein SAHS (BBa_K2306003 (opens new window)), it possesses both anti-desiccation and anti-UV capabilities, with significantly lower metabolic stress on E. coli compared to producing MAA (BBa_K4765118 (opens new window)).
Following another publication[5], we also tested the anti-UV capability of Rv Dusp, FEN1, and XRCC1.
Figure 3: Survival system.
With specific genes inserted into pRAP system, this module confers bacteria the ability to survive harsh conditions.
Table 2: Parts for survival system
| Part Name | Type | Description |
|---|---|---|
| BBa_K4765010 (opens new window) | Coding | MysA codon optimized |
| BBa_K4765011 (opens new window) | Coding | MysB codon optimized |
| BBa_K4765012 (opens new window) | Coding | MysC codon optimized |
| BBa_K4765013 (opens new window) | Coding | MysD codon optimized |
| BBa_K4765014 (opens new window) | Coding | MysH codon optimized |
| BBa_K4765015 (opens new window) | Coding | AnAFP |
| BBa_K4765016 (opens new window) | Coding | H.exemplaris mitochondrial single-stranded DNA binding protein (H. ex mtSSB) |
| BBa_K4765018 (opens new window) | Coding | FEN1 |
| BBa_K4765019 (opens new window) | Coding | XRCC1 |
| BBa_K4765111 (opens new window) | DNA | Twister P1 + T7_RBS + AnAFP + stem-loop |
| BBa_K4765112 (opens new window) | DNA | Twister P1 + T7_RBS + SAHS 33020 + stem-loop |
| BBa_K4765113 (opens new window) | DNA | Twister P1 + T7_RBS + H. ex mtSSB + stem-loop |
| BBa_K4765117 (opens new window) | DNA | ribozyme connected: H. ex mtSSB + SAHS 33020 |
| BBa_K4765118 (opens new window) | DNA | ribozyme connected: MysABCDH |
| BBa_K4765126 (opens new window) | DNA | ribozyme connected: H. ex mtSSB + SAHS 33020 + AnAFP |
| BBa_K4765127 (opens new window) | DNA | ribozyme connected: H. ex mtSSB + AnAFP + SAHS 33020 |
| BBa_K4765128 (opens new window) | DNA | ribozyme connected: AnAFP + SAHS 33020 + H. ex mtSSB |
| BBa_K4765140 (opens new window) | DNA | B.HOME v1 |
# Symbiotic System
To ensure nutrient supply, we need to establish a symbiotic system that allows cyanobacteria to produce and secrete sugars, and then enable E. coli to efficiently utilize them. We are immensely grateful for the plasmids containing CscB (BBa_K4115045 (opens new window)) and SacC (BBa_K4115017 (opens new window)) provided by ShanghaiTech-China iGEM 2023 (opens new window), which enable us to complete the circulation of nutrients for both cyanobacteria and E. coli.
# Biofilm Formation System
Drawing inspiration from lichens, our B.Home project aims to establish biofilm involving cyanobacteria and E. coli.
We need to create physical connections between bacteria to form a self-assembling, robust, and scalable biofilm. For this purpose, we introduce connections between E. coli and E. coli, as well as between E. coli and cyanobacteria. For the former, we have adopted the LAMBA (Living Assembled Material by Bacteria Adhesion) approach, which has been reported as a cell-cell adhesion toolbox[6] or a self-healing material with programmable physical structures[7]. We have integrated it into the pRAP system. This approach produced two strains of E. coli to display antigens (Ag, BBa_K4765006 (opens new window)) or nanobodies (Nb, BBa_K4765106 (opens new window)) on bacteria outer surface, allowing the antigen-expressing E. coli to bind to the antibody-expressing E. coli, forming a biofilm. We have chosen intimin (BBa_K4765001 (opens new window)) as the surface-presenting protein because it offers superior presentation efficacy compared to INPNC (BBa_K4765008 (opens new window)), another commonly used surface-presenting protein, and inserted a short spacer between intimin and Ag/Nb to avoid steric hindrance.
As for the connection between E. coli and cyanobacteria, we have selected Synechococcus elongates PCC7942 due to their ease of genetic modification. We intend to have E. coli display LCA (BBa_K4765009 (opens new window)), a kind of lectin, on their membrane surface and establish a physical connection with the lipopolysaccharides on the surface of S. elongates. Again, we have chosen intimin as the surface-presenting protein for bacteria.
Figure 4: Biofilm Formation System
Bacteria form cell-cell adhesion through this module, thus forming biofilm.
Table 3: Parts for biofilm formation system
| Part Name | Type | Description |
|---|---|---|
| BBa_K4765001 (opens new window) | Coding | intimin |
| BBa_K4765002 (opens new window) | Coding | Ag1, Akt3PH from 10.1016/j.cell.2018.06.041 |
| BBa_K4765003 (opens new window) | Coding | Nb1, 3AKH13 from 10.1016/j.cell.2018.06.041 |
| BBa_K4765004 (opens new window) | Coding | Ag2, EPEA from 10.1016/j.cell.2018.06.041 |
| BBa_K4765005 (opens new window) | Coding | Nb2, antiEPEA from 10.1016/j.cell.2018.06.041 |
| BBa_K4765006 (opens new window) | Coding | Ag3, P53TA from 10.1016/j.cell.2018.06.041 |
| BBa_K4765007 (opens new window) | Coding | Nb3, R4P43 from 10.1016/j.cell.2018.06.041 |
| BBa_K4765008 (opens new window) | Coding | INPNC |
| BBa_K4765009 (opens new window) | Coding | LCA |
| BBa_K4765017 (opens new window) | DNA | linker after INPNC |
| BBa_K4765101 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Ag1 fusion + stem-loop |
| BBa_K4765102 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Nb1 fusion + stem-loop |
| BBa_K4765103 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Ag2 fusion + stem-loop |
| BBa_K4765104 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Nb2 fusion + stem-loop |
| BBa_K4765105 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Ag3 fusion + stem-loop |
| BBa_K4765106 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-Nb3 fusion + stem-loop |
| BBa_K4765107 (opens new window) | DNA | Twister P1 + T7_RBS + INPNC-Ag3 fusion + stem-loop |
| BBa_K4765108 (opens new window) | DNA | Twister P1 + T7_RBS + INPNC-Nb3 fusion + stem-loop |
| BBa_K4765109 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-MVN fusion + stem-loop |
| BBa_K4765110 (opens new window) | DNA | Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop |
| BBa_K4765131 (opens new window) | DNA | ribozyme connected: intimin-Nb1 + mScarlet |
| BBa_K4765132 (opens new window) | DNA | ribozyme connected: intimin-Nb2 + mScarlet |
| BBa_K4765133 (opens new window) | DNA | ribozyme connected: intimin-Nb3 + mScarlet |
# Terraforming System
Our objective is to modify the biofilm to firmly attach to the surfaces of Martian rocks and facilitate the process of rock erosion. To achieve this, we have introduced the oxalic acid module and the extracellular polysaccharide (EPS) module.
As for the oxalic acid module, following multiple rounds of discussions with CAU-China_2022, we have decided to adopt their composite part (BBa_K4192120 (opens new window)) to produce oxalic acid.
Regarding the EPS module, inspired by XJTU iGEM 2020 (opens new window), we have overexpressed the key enzymes, PgmA and GalU, both from E. coli involved in EPS synthesis, to increase the amount of EPS on bacteria. To facilitate EPS functional characterization, we have also incorporated these enzymes into the pRAP system (BBa_K4765121 (opens new window)), connected with a red fluorescent protein.
Figure 5: EPS module.
This module assists bacteria to attach to Martian rocks while facilitating rock weathering.
Table 4: Parts for terraforming system
| Part Name | Type | Description |
|---|---|---|
| BBa_K4765121 (opens new window) | DNA | ribozyme connected: galU + pgmA + mScarlet |
| BBa_K4765122 (opens new window) | DNA | ribozyme connected: pgmA + galU + mScarlet |
In summary, our Survival System, Biofilm Formation System and Terraforming System were all sucessfully built and test functionally, proving our concept of using synthetic biology to promote terraforming. Later, we functional characterized B.HOME v1 (intimin-MVN, mtSSB, AnAFP, SAHS, pgmA, galU, mScarlet), all modules in one bacteria and function as designed.
# References
Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143. https://doi.org/10.1021/acssynbio.2c00416 ↩︎ ↩︎
Eiler, D., Wang, J., & Steitz, T. A. (2014). Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proceedings of the National Academy of Sciences, 111(36), 13028–13033. https://doi.org/10.1073/pnas.1414571111 ↩︎
Chen, Y., Cheng, Y., & Lin, J. (2022). A scalable system for the fast production of RNA with homogeneous terminal ends. RNA Biology, 19:1, 1077-1084. https://doi.org/10.1080/15476286.2022.2123640 ↩︎
Hibshman, J. D., Clark-Hachtel, C. M., Bloom, K. S., & Goldstein, B. (2023). A bacterial expression cloning screen reveals tardigrade single-stranded DNA-binding proteins as potent desicco-protectants. bioRxiv, 2023.08.21.554171. https://doi.org/10.1101/2023.08.21.554171 ↩︎
Clark-Hachtel CM, Hibshman JD, Buysscher TD, Goldstein B (2023). Tardigrades dramatically upregulate DNA repair pathway genes in response to ionizing radiation. bioRxiv, 2023.09.07.556677. https://doi.org/10.1101/2023.09.07.556677 ↩︎
Glass, D. S., & Riedel-Kruse, I. H. (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, D. J., Glass, D. S., Hamby, A. E., Stuart, B. A. R., Dunkel, J., & Riedel-Kruse, I. H. (2022). 4-bit adhesion logic enables universal multicellular interface patterning. Nature, 608(7922), 324–329. https://doi.org/10.1038/s41586-022-04944-2 ↩︎
