Wet Lab · Composite Part
Composite Part
Our project sought to advance the synthetic biology of composite parts
Our Composite Part
Our composite part combines existing BioBrick elements, PelB signal peptides, dockerin and cohesin interaction domains, and TEV cleavage sites, with two enzyme domains, ICCG and TfCa, that had not previously been fused to this kind of scaffolding system. The two constructs below, ICCG-DoT and TfCa-DoG, are designed to co-localize on a shared cohesin scaffold so that PET and MHET hydrolysis can happen in sequence rather than as separate, uncoordinated reactions.
Sequence Feature Legend
- T7 / Lac Promoters
- Ribosome Binding Site (RBS)
- Signal Peptides (pelB)
- Enzymes / Domains (ICCG, TfCa)
- Linkers / Spacers
- Interaction Modules (Dockerin / Cohesin)
- Cleavage Sites (TEV) & Purification (Histag)
- Point Mutations / Variants
Constructs
Construct 1: pET28a-pelB-ICCG-DoT
Features: T7 promoter | Lac promoter | Ribosome binding site | PelB signal peptide | ICCG | Linker | Dockerin T | TEV cutting site | 8xHistag
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCC TCTAGAAAGGAGATATACC ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC AGCAATCCGTATCAGCGTGGTCCGAATCCGACCCGTAGCGCACTGACCGCAGATGGTCCGTTTAGCGTTGCAACCTATACCGTTAGCCGTCTGAGCGTTAGCGGTTTTGGTGGTGGTGTTATTTATTATCCGACCGGTACCAGCCTGACCTTTGGTGGTATTGCAATGAGCCCGGGTTATACCGCAGATGCAAGCAGCCTGGCATGGCTGGGTCGTCGTCTGGCAAGCCACGGTTTTGTTGTTCTGGTTATTAATACCAATAGCCGTTTTGATGGTCCGGATAGCCGTGCAAGCCAGCTGAGCGCAGCACTGAATTATCTGCGTACCAGCAGCCCGAGCGCAGTTCGTGCACGTCTGGATGCAAATCGTCTGGCAGTTGCAGGTCATAGCATGGGTGGTGGTGGTACCCTGCGTATTGCAGAACAGAATCCGAGCCTGAAAGCAGCAGTTCCGCTGACCCCGTGGCATACCGATAAAACCTTTAATACCAGCGTTCCGGTTCTGATTGTTGGTGCAGAAGCAGATACCGTTGCACCGGTTAGCCAGCATGCAATTCCGTTTTATCAGAATCTGCCGAGCACCACCCCGAAAGTTTATGTTGAACTGTGTAATGCAAGCCATATTGCACCGAATAGCAATAATGCAGCAATTAGCGTTTATACCATTAGCTGGATGAAACTGTGGGTTGATAATGATACCCGTTATCGTCAGTTTCTGTGTAATGTTAATGATCCGGCACTGTGTGATTTTCGTACCAATAATCGTCATTGTCAG ACTAGTGGAGGAGGTGACGACGGTGGAAGCGGTGGTCCTCAAGGTACC ACATATAAAGTACCTGGTACTCCTTCTACTAAATTATACGGCGACGTCAATGATGACGGAAAAGTTAACTCAACTGACGCTGTAGCATTGAAGAGATATGTTTTGAGATCAGGTATAAGCATCAACACTGACAATGCCGATTTGAATGAAGACGGCAGAGTTAATTCAACTGACTTAGGAATTTTGAAGAGATATATTCTCAAAGAAATAGATACATTGCCGTAC AAGAACGAGCTCGAGAACCTTTACTTCCAAGGTCACCACCATCATCATCACCATCATTAA
Construct 2: pET28a-TfCa-doG (Wild Type)
Features: T7 promoter | Lac promoter | Ribosome binding site | TfCa | Linker | Dockerin G | TEV cutting site | 8xHistag
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCC TCTAGAAAGGAGATATACC ATGGAAATTGTTATCCGGACAGGGAGTGGTGACGTTCGCGGATCAAAGGAGAATGGAATCGCTGTGTTCAGAGGCATCCCGTACGCAGAGCCACCGGTAGGTGCACATAGATTTACGGCCCCTAGACCACCGCGGCCATGGGACGGTGTGCGTGATGCGACGGAGTTCTCTGCTACCGCCCCTAGACCACCATATCCTGAGGCAATAGGCGCCCTTCTGATCGAGCGCTTCATCCCTGGCGACGATTATCTGACACTGAACGTATGGACTCCAGACCCTAATGCGGTGGGTCTGCCTGTCATGGTGTGGATACATGGCGGAGCCTTCACTAACGGATCCGGTTCGGAGCCTGTTTACGACGGTGCGGCATTTGCGAGAGACGGAGTGGTCTTCGTGTCGTTTAATTACCGTTTGGGTATCATTGGCTTCGCGGATTTGCCTGACGCACCTTCGAACAGAGGCCTGCTGGATCAAATTGCTGCACTGGAGTGGGTCCGGGACAACATCGCTCGATTCGGCGGTGATCCGGGTAACGTGACCGTATTCGGGGAGAGTGCCGGCGCTATGAGCGTCTGCACCTTAATGGCCACCCCAAGAGCTCGTGGCTTGTTTAGACGAGCCATTCTTCAGAGTGGTGCAGGGAATATGGCCGTAGCCGCTGAGGACGCGACAACGATAGCAGCAGTTATTGCTCACCGCTTAGGTGTCGAACCAACGGCTGCCGCCCTGGCCCATGTGCCCGTCGCCCAATTGCTGGATGTCCAACAGCAGGTAGCCCAGGAGATACAAGGAGCGCCTGATCCAGCCGTTTGGGGAGAACGGATCGCCGGCGGTTCTGTCTTACTGCCATTTGCTCCTGTCATCGACGGTGAGTTGCTTAGCCAACGTCCCGCCGAGGCCATTGCGGGCGGTGCCGGGCACGATGTTGACCTGCTGTTTGGAACCACGACAGACGAGTATAGATTGTTCTTGGCGCCAACAGGACTTCTGCCATTTATTACCAGTGACTATGTTACTGCACATCTTGCCAAATCCGGGCTGGACGCAGATGCAGCAAAAGCATACACGGCGGAAGGCCGTGGCGAAGAGCCCGGCGATATCCTGGCATCGATTATAACCGACCAAGTATTCAGAATACCTGCCTTGAGAATTGCTGAGTCCCGGGTTGACGCTCCTGCTCGGACGTTTGGGTATGAGTTTGCGTGGCGTACTCCACAACTCGATGGTATTCTTGGAGCGTGCCACGCCGTTGAGCTGCCCTTCGTATTTCGCACCTTAGACCGTGCCGCCAGTCTTGTTGGAACCAACCCACCGGAAGAGTTAGCCGAGACCGTACACAATGCCTGGGT
We are submitting this composite part for consideration under iGEM's Best Composite Part special prize category.