Materials
Strains
| Strain | Source |
|---|---|
| EcN | Gift from Changtao Jiang |
| EcN △asd | Manufactured in this project |
| DH5alpha | Mei5bio |
| BL21(DE3) | Solarbio |
| BL21(AI) | Angyubio |
Plasmids
| Plasmid | Source |
|---|---|
| pEcCas | Gift from Changtao Jiang |
| pTarget | Gift from Changtao Jiang |
| pet28a-eGFP | Gift from Qingsong Wang |
| pJUMP41-2A | iGEM Kit(Asimov) |
| pJUMP49-2C | iGEM Kit(Asimov) |
Primers
asd knock-out
| Primer | Sequence |
|---|---|
| asd-F | atgaaaaatgttggttttatcggctgg |
| asd-R | ttacgccagttgacgaagcatc |
| asd-up-F | catcacaaggagagcaataagcaa |
| asd-up-R | agctggtcgcccacggtaaattgcatgagaacggagccgac |
| asd-down-F | tttaccgtgggcgaccagct |
| asd-down-R | aaaatggcctgcaattagcgcg |
| PTarget-F | agtcctaggtataatactagtggaatcaggctacccgccaggttttagagctagaaatag |
| PTarget-R | actagtattatacctaggactgag |
| PTarget-N20-F | ggaatcaggctacccgccag |
| PTarget-N20-R | aatggtgacttctacagcgc |
ARG1 plasmid construction
| Primer | Sequence |
|---|---|
| ARG1-T5-F1 | tttgtgagcggataacaattataatatttcacacaggaaacccagtcgagctcatgactcatattta |
| ARG1-T5-F2 | gcgaagcgagcgaatcaaacg |
| ARG1-T5-R1 | tgattcgctcgcttcgcttg |
| ARG1-T5-R2 | attgttatccgctcacaaagcaaataaattttttatgatgcgcgggatcgagatctcgatcctc |
| ARG-T5-Fragment-F | cgcaaggaatggtgcatgccatgcaaggaga |
| ARG-T5-Fragment-R | acaagaattctctggtctcaagagatagttca |
pPepT series construction
| Primer | Seque |
|---|---|
| JUMP-FF+20-R1-F | tgatattaacagctattaaagaggagaaacatatggtagcccgtaaaggcgaa |
| JUMP-FF+20-R1-R | catttgatgtacatcaaatttacctgatcctctagaagcggccgcgaatt |
| JUMP-FF+20-R2-F | tcgcccctcactcctgccatcattctgatattaacagctattaaag |
| JUMP-FF+20-R2-R | atggcaggagtgaggggcgatccatttgatgtacatcaaatt |
| JUMP-empty-F | ttcgcggccgcttctagagcttctgactggttgcacgct |
| JUMP-empty-R | ctctagaagcggccgcgaattc |
| JUMP-ecnpept-B0034-F | gcttcttaataatgttgtcatctagagaaagaggagaaatactagatggtagcccgtaaaggcgaaga |
| JUMP-ecnpept-B0034-R | tgacaacattattaagaagcaacaaaagacaa |
| JUMP-B0034-R1-F | ataatgttgtcatctagagaaagaggagaaatactagatggtagcccgtaaaggcgaagagc |
| JUMP-J61101-R1-F | ataatgttgtcatctagagaaagacaggacccactagtatggtagcccgtaaaggcgaagagc |
| JUMP-R1-R | ttttatccctctagaagcggccgcgaa |
| JUMP-HIP-1-R2-F | tatttgtcttttcttgcttaataatgttgtcatctagagaaag |
| JUMP-HIP-1-R2-R | aagcaagaaaagacaaatattgcgtcaggtcacttttatccctctagaagcgg |
| JUMP-HIP-1-TATA-R2-F | tatttgtcttttcttgctttataatgttgtcatctagagaaag |
| JUMP-HIP-1-FNR-R2-R | aagcaagaaaagacaaatattgattcagatcaattttatccctctagaagcgg |
LldR overexpression construction
| Primer | Sequence |
|---|---|
| lldP-F | atgaatctctggcaacaaaactacg |
| lldR-F | atgattgttttacccagacgcct |
| lldR-R | tcatgcgtttttctccctcga |
| pet28a-F | ggctagctcagccctaggtattatgctagcggaattgtgagcggataaca |
| pet28a-R(lldPR) | tttgttgccagagattcatggtatatctccttctta |
| pet28a-R(lldR) | gtctgggtaaaacaatcatggtatatctccttctta |
Kfic knock-out
| Primer | Sequence |
|---|---|
| kfic-F | caaactattgttcaattattcctgataca |
| kfic-R | gtgaatgtatgaacgcagaatatata |
| kfic-up-F | accacgtacatctttatcacttac |
| kfic-up-R | aataataaggctgtaatctgtatggc |
| kfic-down-F | agattacagccttattattgcacacgagaatttcaatatt |
| kfic-down-R | atggatataaatatttagcatttgataatcaag |
| pTarget-kfic-F | agtcctaggtataatactagtgagctaataaaaaaagcacagttttagagctagaaatag |
| pTarget-kfic-R | actagtattatacctaggactgagctagctg |
Hybrid Promoter Construction
| Primer | Sequence |
|---|---|
| O12-J23117-F | ctagctcagtcctagggattgtgctagctcattatccctacacaa |
| O12-J23117-R | aatccctaggactgagctagctgtcaacattga |
| O12-pept(l1-s1)-F | tatttgtcttttcttgcttwataatgttgtcatcattatccctacacaacacaattgg |
| O12-pept(s2)-F | tatttgtcttttcttgcttwataatgttgtcactacacaacacaattggcagtg |
| O12-pept(s3)-F | tatttgtcttttcttgcttwataatgttgtcaccctacacaacacaattggc |
| O12-peptf(l1)-R | aagcaagaaaagacaaatattgattcagatcaattttatcctactcagatccattgatgtgagttttaattgggaaattg |
| O12-peptf(l2)-R | aagcaagaaaagacaaatattgattcagatcaattttatcctactcagatctgggaaattgtgaaccagaggt |
| O12-peptf(ns)-R | aagcaagaaaagacaaatattgattcagatcaattttatcccattgatgtgagttttaattgggaaattg |
| O12-peptf(s1)-R | aagcaagaaaagacaaatattgattcagatcaattttatcctgggaaattgtgaaccagaggt |
| O12-peptf(s2)-R | aagcaagaaaagacaaatattgattcagatcaattttatccagttttaattgggaaattgtgaaccag |
| O12-peptf(s3)-R | aagcaagaaaagacaaatattgattcagatcaattttatccggaaattgtgaaccagaggtcag |
| O12-pept(l1-s1)-F | tatttgtcttttcttgcttwataatgttgtcatcattatccctacacaacacaattgg |
| O12-pept(s2)-F | tatttgtcttttcttgcttwataatgttgtcactacacaacacaattggcagtg |
| O12-pept(s3)-F | tatttgtcttttcttgcttwataatgttgtcaccctacacaacacaattggc |
| O12-peptw(l1)-R1 | tgcgtcaggtcacttttatcctactcagatccattgatgtgagttttaattgggaaattg |
| O12-peptw(l2)-R1 | tgcgtcaggtcacttttatcctactcagatctgggaaattgtgaaccagaggt |
| O12-peptw(ns)-R1 | tgcgtcaggtcacttttatcccattgatgtgagttttaattgggaaattg |
| O12-peptw(s1)-R1 | tgcgtcaggtcacttttatcctgggaaattgtgaaccagaggt |
| O12-peptw(s2)-R1 | tgcgtcaggtcacttttatccagttttaattgggaaattgtgaaccag |
| O12-peptw(s3)-R1 | tgcgtcaggtcacttttatccggaaattgtgaaccagaggtcag |
| O12-peptw-F2 | tatttgtcttttcttgcttwataatgttgtca |
| O12-peptw-R2 | aagcaagaaaagacaaatattgcgtcaggtcacttttatcc |
Peking 2023 is grateful for all the assistance received in the field of engineering!