Revision as of 21:19, 20 October 2020

knt-gfp entangled CDSs for aminoglycoside resistance and GFP

Generation of the biobrick sequence with CAMEOS software We used a software named Cameos which can produce a sequence with entangled genes, i.e. overlapping coding sequences where the entire coding sequence of gene A is comprised within the sequence of gene B but read in another reading frame. We chose to entangle genes encoding phenotypes that would be relatively easy to test, like the ability to survive in the presence of antibiotics and the production of a fluorescent protein. Here we chose to entangle a gene conferring resistance to an aminoglycoside antibiotic with a gene encoding GFP. In order to perform entanglement of the genes, the software CAMEOS needs alignments of the gene products with many homologs. These alignements give information to the software about regions that are more or less conserved and domains of the protein that might interact although they are not in direct proximity in the primary structure. CAMEOS identifies regions where it can introduce changes in the sequence that are less likely to change the protein structure and thus its function. At the end, Cameos suggests a list of entangled genes that we call HuGenesS, since one coding sequence is longer than the second one and sort of embraces it, as if in a loving hug.

Knt Sequence (113 amino acids + stop codon) :

MIEQDGLHAGSPAAWVERLFGYEWAKETNYCSDSYQFSLSYQGRLTDIVSAYLAKALETLQEESS RLSWLAQQGVYQSAVLDSSQRSGRDWLALGEVPTTDLLTSSLNPANQKDTYKNALRRLRTLDPTK CELKSSSKETLSRTRSNLEASTVKKTDLYSEHEGNTTTTLTTRISARMPKKTDLKVTSRDASTSK IMVYNSRTTTNNDCLSETVQYSYQTITIYPRNLNSELTGTSADRILYYYGSSQPQASRIYFYRLLDEFF

GFP Sequence (113 amino acids + stop codon) :

MGKGDELLLGLVPILVELSGQVNGHRFSVSGKGTGDATRGKLTLKLACTTGSLPVSSPGLVTTLGAGLAC FGGSPDHRPAHKFFKSSKPEGYVQERTASAKDSGSYKMRAEVKFEGDTLANKIELRGIDSKEDGPILGTR REYNYNSHNAYISADAQKNGLKSNFKRRINIEDNGVQLADHYQQRLPIGDGPVLLPDNHYLSTQSQLGAD GNERRSHLVLLRFVTAAGITHILLPLIG

To characterize the knt-gfp entangled genes, the DNA fragment encoding the two fully overlapping CDS in different reading frames has been inserted in the plasmid pBAD24-MoClo. The entangled genes are under the control of a promoter inducible with arabinose.

Knt DNA Sequence:

5' - atgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggatatgaATGG GCAAAGGAGACGAACTACTGCTCGGACTCGTACCAATTCTCGTTGAGCTATCAGGGCAGGTTAACGGACATC GTTTCAGCGTATCTGGCAAAGGCACTGGAGACGCTACAAGAGGAAAGCTCACGCTTAAGCTGGCTTGCACAA CAGGGAGTCTACCAGTCAGCAGTCCTGGACTCGTCACAACGCTCGGGGCGGGACTGGCTTGCTTTGGGGGAA GTCCCGACCACCGACCTGCTCACAAGTTCTTTAAATCCAGCAAACCAGAAGGATACGTACAAGAACGCACTG CGTCGGCTAAGGACTCTGGATCCTACAAAATGCGAGCTGAAGTCAAGTTCGAAGGAGACACTCTCGCGAACA AGATCGAACTTAGAGGCATCGACAGTAAAGAAGACGGACCTATACTCGGAACACGAAGGGAATACAACTACA ACTCTCACAACGCGTATATCAGCGCGGATGCCCAAAAAAACGGACTTAAAAGTAACTTCAAGAGACGCATCA ACATCGAAGATAATGGTGTACAACTCGCGGACCACTACCAACAACGACTGCCTATCGGAGACGGTCCAGTAC TCCTACCAGACAATCACTATCTATCCACGCAATCTCAACTCGGAGCTGACGGGAACGAGCGCAGATCGCATC TTGTACTACTACGGTTCGTCACAGCCGCAGGCATCACGCATATACTTCTACCGCTTATTGGATGAAttcttctaa - 3'

GFP DNA Sequence + BsaI sites :

5’ - ATGGGCAAAGGAGACGAACTACTGCTCGGACTCGTACCAATTCTCGTTGAGCTATCAGGGCAGGTTAACGGACATCGT TTCAGCGTATCTGGCAAAGGCACTGGAGACGCTACAAGAGGAAAGCTCACGCTTAAGCTGGCTTGCACAACAGGGAGTCTACCA GTCAGCAGTCCTGGACTCGTCACAACGCTCGGGGCGGGACTGGCTTGCTTTGGGGGAAGTCCCGACCACCGACCTGCTCACAAG TTCTTTAAATCCAGCAAACCAGAAGGATACGTACAAGAACGCACTGCGTCGGCTAAGGACTCTGGATCCTACAAAATGCGAGCT GAAGTCAAGTTCGAAGGAGACACTCTCGCGAACAAGATCGAACTTAGAGGCATCGACAGTAAAGAAGACGGACCTATACTCGGA ACACGAAGGGAATACAACTACAACTCTCACAACGCGTATATCAGCGCGGATGCCCAAAAAAACGGACTTAAAAGTAACTTCAAG AGACGCATCAACATCGAAGATAATGGTGTACAACTCGCGGACCACTACCAACAACGACTGCCTATCGGAGACGGTCCAGTACTC CTACCAGACAATCACTATCTATCCACGCAATCTCAACTCGGAGCTGACGGGAACGAGCGCAGATCGCATCTTGTACTACTACGG TTCGTCACAGCCGCAGGCATCACGCATATACTTCTACCGCTTATTGGATGA - 3’