All Parts were ordered with prefix and suffix sequences to allow for TypeIIS assembly into Devices. BioBrick assembly was used to clone two devices together to build a System. DNA was kindly synthesised by either IDT or Twist.
Basic parts
| Registry number | Part Name | Type | Information | Notes |
|---|---|---|---|---|
| J23100 | P_J23100 | Promoter | Constitutive promoter- strong | Inserted into holding plasmid and sequence verified |
| B0034 | B0034 | RBS | Strong RBS | Inserted into holding plasmid and sequence verified |
| B0015 | B0015 | Terminator | Strong terminator | Inserted into holding plasmid and sequence verified |
| Term_T7 | Terminator | From the Novagen pET21 plasmid | Inserted into holding plasmid and sequence verified | |
| K4339000 | CycA_CDS | CDS | An inner membrane permease, from E. coli. Transports in non-polar amino acids, including L-alanine, via a proton symport mechanism[1] | Inserted into holding plasmid and sequence verified |
| K3619001 | TrxA_CDS | CDS | Participates in various redox reactions through the reversible oxidation of the active centre dithiol to a disulfide and catalyses dithiol-disulfide exchange reactions[2]. Can be used as a fusion partner, increasing protein solubility. From E. coli, ordered to test thioredoxin expression[3]. | Inserted into holding plasmid and sequence verified |
| K4339001 | ala_tRNA_synthetase | CDS | An enzyme that catalyses the binding of alanine to the corresponding tRNA, from E. coli[4]. | DNA synthesised |
| K4339002 | MaSp1_K3264003mod_CTEa | CDS | K3264003mod— GreatBay2019 2Rep region[5], composed of 2 repeated poly-glycine and poly-alanine domains CTEa— CT domain from E. australis[6]. |
Inserted into holding plasmid and sequence aligned with region of K3264003 missing |
| K4339003 | K3264003mod_K3264002 | CDS | K3264003mod— GreatBay2019 2Rep region[5] K3264002— GreatBay2019 CT domain from A. ventricosus[5]. |
Inserted into holding plasmid and sequence aligned with Gly-rich region missing |
| K4339004 | K3264003mod | CDS | K3264003mod— GreatBay2019 2Rep region[5] but this sequence has 6 bp mutations to reduce repeats and GC rich regions. | Inserted into holding plasmid but not sequence aligned |
| K4339005 | MaSp1 4Rep_CTEa | CDS | MaSp1 4Rep— Rep region from Construct 1 of the Stark 2007 paper[6], composed of 4 repetitive alanine-rich domains linked by glycine-rich regions CTEa— CT domain from E. australis[6] |
DNA synthesis failed |
| K4339006 | MaSp1 4Rep_K3264002 | CDS | MaSp1 4Rep— Rep region from Construct 1 (as above)[6] K3264002— GreatBay2019 CT domain[5] |
DNA synthesis failed |
| K4339007 | MaSp1 4Rep | CDS | MaSp1 4Rep— Rep region from Construct 1 (as above)[6] | Inserted into holding plasmid but large region of CDS missing from sequence |
| K4339008 | MaSp2 6Rep_CT | CDS | MaSp2 6Rep— Cobbled together rep region using domains from L. hesperus[7] [8] CT—CT domain from L. hesperus[8] |
Inserted into holding plasmid and sequenced aligned with repetitive regions missing |
| K4339009 | MaSp2 6Rep | CDS | MaSp2 6Rep— Cobbled together rep region using domains from L. hesperus[7] [8] | Inserted into holding plasmid but large region of CDS missing from sequence |
Composite parts
| Part Name | Type | Information | Notes |
|---|---|---|---|
| P_T7_RBS | Promoter + RBS | Inducible promoter with inbuilt RBS. From the Novagen pET21 plasmid but with XbaI removed. | Inserted into holding plasmid and sequence verified |
| P_T7_RBS_His | Promoter + RBS + His-tag | Inducible promoter with inbuilt RBS linked to a start codon and His-tag | Inserted into holding plasmid and sequence verified |
| P_T7_TrxA_His_S_TEV | Promoter + RBS with TrxA and cleavable tag | Inducible promoter driving expression of thioredoxin fused to a His-tag and an S-tag with a TEV cleavage site. From the Novagen pET32 plasmid but with XbaI removed. | Inserted into holding plasmid and sequence verified |
| P_T7_TrxA_His_S_Thrombin | Promoter+BS with TrxA and cleavable tag | Inducible promoter driving expression of thioredoxin fused to a His-tag and an S-tag with a Thrombin cleavage site. From the Novagen pET32 plasmid but with XbaI removed. | Inserted into holding plasmid and sequence verified |
| B0034_His | RBS + His-tag | Strong RBS. Linked to a start codon and His-tag |
Devices
| Purpose | Promoter/RBS | CDS | Term | Plasmid | Results To Date | |
|---|---|---|---|---|---|---|
| Positive control for expression | P_T7-His | mCherry | T7 | Medium copy, AmpR | Previously built by PI. Successfully transformed into BL21 DE3 pLyS and Rosetta (by visual confirmation), expression not confirmed by Western blotting | |
| CycA expression | PJ23100-B0034 | CycA | B0015 | Medium copy, AmpR | Plasmid successfully built. Not sequence verified | |
| His-tagged CycA expression | PJ23100-B0034-His | CycA | B0015 | Medium copy, AmpR | Plasmid successfully built and sequence verified. Transformed into BL21 DE3 pLysS. No expression seen on Western-Blot. Alanine assay showed decrease in net alanine uptake relative to control strain | |
| Thioredoxin expression | P_T7 | TrxA | T7 | Medium copy, AmpR | Plasmid successfully built, poorly sequenced | |
| His-tagged thioredoxin expression | P_T7-His | TrxA | T7 | Medium copy, AmpR | Plasmid successfully built and sequence verified. Transformed into Rosetta. No expression seen on Western-Blot. | |
| Alanine tRNA synthetase expression | P_T7 | ala_tRNA_synthetase | T7 | Medium copy, AmpR | Device not constructed | |
| His-tagged alanine tRNA synthetase expression | P_T7-His | ala_tRNA_synthetase | T7 | Medium copy, AmpR | Device not constructed | |
| His-tagged MaSp1 expression | P_T7-His | K3264003mod_CTEa | T7 | Medium copy, AmpR | Device constructed and sequence verified. Transformed into Rosetta. No expression seen on Western Blot | |
| His-tagged MaSp1 expression | P_T7-His | K3264003mod_K3264002 | T7 | Medium copy, AmpR | Device constructed and sequence verified (with 2 point mutations) | |
| His-tagged MaSp1 expression | P_T7-His | K3264003mod | T7 | Medium copy, AmpR | Device constructed and sequence verified with 1 Gly-rich region missing. Transformed into Rosetta. No expression seen on Western Blot | |
| His-tagged MaSp1 expression | P_T7-His | MaSp1 4Rep_CTEa | T7 | Medium copy, AmpR | Device not constructed | |
| His-tagged MaSp1 expression | P_T7-His | MaSp1 4Rep_K3264002 | T7 | Medium copy, AmpR | Device not constructed | |
| His-tagged MASP1 expression | P_T7-His | MaSp1 4Rep | T7 | Medium copy, AmpR | Device constructed and sequence verified. Transformed into Rosetta. No expression seen on Western Blot | |
| His-tagged MaSp2 expression | P_T7-His | MaSp2 6Rep_CT | T7 | Medium copy, AmpR | Device constructed and sequence verified (with 4 point mutations) | |
| His-tagged MaSp2 expression | P_T7-His | MaSp2 6Rep | T7 | Medium copy, AmpR | Device constructed and sequence verified (with 4 point mutations and 1 base insertion) | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | K3264003mod_CTEa | T7 | Medium copy, AmpR | Device constructed, but only with poor sequence fidelity | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | K3264003mod_K3264002 | T7 | Medium copy, AmpR | Device constructed and sequence verified. Transformed into Rosetta. No expression seen on Western Blot | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | K3264003mod | T7 | Medium copy, AmpR | Device constructed and sequence verified (with 1 Ala-rich region missing). Transformed into Rosetta. No expression seen on Western Blot | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | MaSp1 4Rep_CTEa | T7 | Medium copy, AmpR | Device not constructed | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | MaSp1 4Rep_K3264002 | T7 | Medium copy, AmpR | Device not constructed | |
| TrxA fused His-tagged MaSp1 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | MaSp1 4Rep | T7 | Medium copy, AmpR | Device constructed, but only with poor sequence alignment (repetitive regions missing) | |
| TrxA fused His-tagged MaSp2 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | MaSp2 6Rep_CT | T7 | Medium copy, AmpR | Device constructed and sequence verified. Transformed into Rosetta. No expression seen on Western Blot | |
| TrxA fused His-tagged MaSp2 expression (with thrombin cleavage site) | P_T7_TrxA_His_S_Thrombin | MaSp2 6Rep | T7 | Medium copy, AmpR | Device constructed and sequence verified. Transformed into Rosetta. No expression seen on Western Blot | |
| tRNA expression | P_J23100-B0034 | E. coli_tRNA_GGC | B0015 | Medium copy, AmpR | Plasmid successfully built. Transformed into Rosetta and sequence verified. Whole RNA extracted, but initial assay failed to show expression of the tRNA. | |
| tRNA expression | P_J23100-B0034 | E. coli_tRNA_TGC | B0015 | Medium copy, AmpR | Plasmid successfully built. Transformed into Rosetta and sequence verified. Whole RNA extracted, but initial assay failed to show expression of the tRNA | |
| tRNA expression | P_J23100-B0034 | H. sapiens_tRNA_AGC | B0015 | Medium copy, AmpR | Plasmid successfully built. Transformed into Rosetta and sequence verified. Whole RNA extracted, but initial assay failed to show expression of the tRNA. | |
| tRNA expression | P_J23100-B0034 | H. sapiens_tRNA_CGC | B0015 | Medium copy, AmpR | Plasmid successfully built. Transformed into Rosetta and sequence verified. Whole RNA extracted, but initial assay failed to show expression of the tRNA. |
Systems
| Purpose | Device 1 | Device 2 | Plasmid | Results To Date |
|---|---|---|---|---|
| Co-expression of CycA and tRNA | P_J23100-B0034-E. coli_tRNA_GGC-B0015 | PJ23100-B0034-His-CycA-B0015 | Medium copy, compatible, KanR | Plasmid successfully built. No transformation attempted. |
| Co-expression of CycA and tRNA | P_J23100-B0034-E. coli_tRNA_TGC-B0015 | PJ23100-B0034-His-CycA-B0015 | Medium copy, compatible, KanR | Plasmid successfully built. No transformation attempted. |
| Co-expression of CycA and tRNA | P_J23100-B0034-H. sapiens_tRNA_AGC-B0015 | PJ23100-B0034-His-CycA-B0015 | Medium copy, compatible, KanR | Plasmid successfully built. No transformation attempted. |
| Co-expression of CycA and tRNA | P_J23100-B0034-H. sapiens_tRNA_CGC-B0015 | PJ23100-B0034-His-CycA-B0015 | Medium copy, compatible, KanR | Plasmid successfully built. No transformation attempted. |
References
[1] - Hook, C. et al. The Escherichia coli amino acid uptake protein CycA: Regulation of its synthesis and practical application in L-isoleucine production. Microorganisms. 2022;10(3): 647. doi: 10.3390/microorganisms10030647.
[2] - Watson W et al. Thioredoxin and its role in toxicology. Toxicol Sci. 2004;78(1): 3-14. doi: 10.1093/toxsci/kfh050
[3] - LaVallie E et al. Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods in Enzymology. 2000;326: 322-340. doi: 10.1016/S0076-6879(00)26063-1
[4] - Putney S et al. Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits. J Biol Chem. 1981;256(1): 198-204. doi: 10.1016/s0021-9258(19)70119-7
[5] - Andersson, Marlene, et al. Biomimetic Spinning of Artificial Spider Silk from a Chimeric Minispidroin. Nature Chemical Biology. 2017;13(3): 262–264. doi: 10.1038/nchembio.2269
[6] - Stark, M. et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules. 2007;8(5): 1695–1701. doi: 10.1021/bm070049y
[7] - Ayoub, N et al. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes. PLoS ONE. 2007;2(6). doi: 10.1371/journal.pone.0000514
[8] - You, Z. et al. Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin. Scientific Reports. 2018;8(1): 1–14. doi: 10.1038/s41598-018-34150-y