Virtual restriction enzyme on enterobacteria phage lambda (48k bp) with 7 different restriction enzymes, simulated in Benchling:
Some gels we made at Genspace, 18 Feb 2025. (None of these lanes were mine, since I hadn’t joined the node yet when they designed the digests in the previous meeting.)
In 2016, they discovered a PETase in bacteria, an enzyme that can digest PET plastic: https://en.wikipedia.org/wiki/PETase. The enzyme is a single protein, so it’s something a beginner might be able to synthesize.
The protein sequence is 290 amino acids long: https://www.uniprot.org/uniprotkb/A0A0K8P6T7/entry
>sp|A0A0K8P6T7|PETH_PISS1 Poly(ethylene terephthalate) hydrolase OS=Piscinibacter sakaiensis OX=1547922 GN=ISF6_4831 PE=1 SV=1
MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRP
SGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQP
SSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAA
PQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCA
NSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS
The gene is the section labeled “ISF6_4831,” whose sequence as submitted by the discoverers is here: https://www.ncbi.nlm.nih.gov/nuccore/BBYR01000074.1?report=genbank&from=1351&to=2223
1 atgaactttc cccgcgcttc ccgcctgatg caggccgccg ttctcggcgg gctgatggcc
61 gtgtcggccg ccgccaccgc ccagaccaac ccctacgccc gcggcccgaa cccgacagcc
121 gcctcactcg aagccagcgc cggcccgttc accgtgcgct cgttcaccgt gagccgcccg
181 agcggctacg gcgccggcac cgtgtactac cccaccaacg ccggcggcac cgtgggcgcc
241 atcgccatcg tgccgggcta caccgcgcgc cagtcgagca tcaaatggtg gggcccgcgc
301 ctggcctcgc acggcttcgt ggtcatcacc atcgacacca actccacgct cgaccagccg
361 tccagccgct cgtcgcagca gatggccgcg ctgcgccagg tggcctcgct caacggcacc
421 agcagcagcc cgatctacgg caaggtcgac accgcccgca tgggcgtgat gggctggtcg
481 atgggcggtg gcggctcgct gatctcggcg gccaacaacc cgtcgctgaa agccgcggcg
541 ccgcaggccc cgtgggacag ctcgaccaac ttctcgtcgg tcaccgtgcc cacgctgatc
601 ttcgcctgcg agaacgacag catcgccccg gtcaactcgt ccgccctgcc gatctacgac
661 agcatgtcgc gcaatgcgaa gcagttcctc gagatcaacg gtggctcgca ctcctgcgcc
721 aacagcggca acagcaacca ggcgctgatc ggcaagaagg gcgtggcctg gatgaagcgc
781 ttcatggaca acgacacgcg ctactccacc ttcgcctgcg agaacccgaa cagcacccgc
841 gtgtcggact tccgcaccgc gaactgcagc tga
I used the codon optimization tool in Benchling to optimize the sequence for E. coli. If I were actually synthesizing this enzyme the lab, E. coli is easier to come by than the original bacteria (Ideonella sakaiensis) that this PETase was found in.
Codon optimization is useful because the abundance of tRNA in E. coli might not match what’s in the original organism, so we want to pick different codons that are more common in E. coli (or avoid depleting a certain tRNA if its codon sequence appears a lot in the original sequence). We could also change the codons to avoid sequences that will be targeted by restriction enzymes we might want to use later, but since I don’t know what restriction enzymes I’ll be using (if any), I did not optimize for that.
Benchling produced this optimized sequence:
atgaactttccacgtgcttcccgtctgatgcaggcagctgttttaggtgggctgatggccgtgagtgcagccgcgaccgcccagaccaacccctatgcacgcggcccgaatccgacagcggcctcacttgaagctagcgcaggtccgttcaccgtccgctcatttactgtaagccggccgagcggctatggcgcgggaactgtgtactatccaaccaacgcaggcgggaccgtgggcgctattgctattgtaccggggtacaccgcgcgtcaatctagcataaaatggtggggaccacggctggcatctcatggtttcgttgtcatcacaattgatacgaattccacgctagatcagccttccagccgctcgtcgcaacagatggcggcgctgcgccaggtggcgtcgttgaacggtaccagcagtagtccgatttacggtaaggtagacaccgcacgtatgggcgttatgggctggtcgatgggaggtggcggctcgttgatctcagcggccaataatccttcactgaaagcagcggcgccgcaggcaccgtgggatagttctacgaatttctcttcagtcaccgttcccacgctgatctttgcctgtgaaaacgacagcattgctccggtcaactcttccgccctgcctatttatgacagcatgtcgcgtaatgcgaaacaatttctcgaaatcaatggtggtagccactcctgcgccaacagcggtaacagcaatcaggcgttaatcgggaaaaaaggcgtggcctggatgaagcgctttatggataacgatacgagatattccacgtttgcctgtgagaatccaaacagtacacgagtgtctgatttccgcactgcgaactgcagttaa