Revision as of 22:01, 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 DNA Sequence (with GFP entangled DNA sequence in capital letters):

5’ - atgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggatatgaATGGGCAAAGGAGACGAA CTACTGCTCGGACTCGTACCAATTCTCGTTGAGCTATCAGGGCAGGTTAACGGACATCGTTTCAGCGTATCTGGCAAAGGCACTGG AGACGCTACAAGAGGAAAGCTCACGCTTAAGCTGGCTTGCACAACAGGGAGTCTACCAGTCAGCAGTCCTGGACTCGTCACAACGC TCGGGGCGGGACTGGCTTGCTTTGGGGGAAGTCCCGACCACCGACCTGCTCACAAGTTCTTTAAATCCAGCAAACCAGAAGGATAC GTACAAGAACGCACTGCGTCGGCTAAGGACTCTGGATCCTACAAAATGCGAGCTGAAGTCAAGTTCGAAGGAGACACTCTCGCGAA CAAGATCGAACTTAGAGGCATCGACAGTAAAGAAGACGGACCTATACTCGGAACACGAAGGGAATACAACTACAACTCTCACAACG CGTATATCAGCGCGGATGCCCAAAAAAACGGACTTAAAAGTAACTTCAAGAGACGCATCAACATCGAAGATAATGGTGTACAACTC GCGGACCACTACCAACAACGACTGCCTATCGGAGACGGTCCAGTACTCCTACCAGACAATCACTATCTATCCACGCAATCTCAACT CGGAGCTGACGGGAACGAGCGCAGATCGCATCTTGTACTACTACGGTTCGTCACAGCCGCAGGCATCACGCATATACTTCTACCGC TTATTGGATGAattcttctaa - 3’

Knt protein sequence resulting from the entanglement :

MIEQDGLHAGSPAAWVERLFGYEWAKETNYCSDSYQFSLSYQGRLTDIVSAYLAKALETLQEESSRLSWLAQQGVYQ SAVLDSSQRSGRDWLALGEVPTTDLLTSSLNPANQKDTYKNALRRLRTLDPTKCELKSSSKETLSRTRSNLEASTVKK TDLYSEHEGNTTTTLTTRISARMPKKTDLKVTSRDASTSKIMVYNSRTTTNNDCLSETVQYSYQTITIYPRNLNSELTGT SADRILYYYGSSQPQASRIYFYRLLDEFF

GFP protein sequence resulting from the entanglement :

MGKGDELLLGLVPILVELSGQVNGHRFSVSGKGTGDATRGKLTLKLACTTGSLPVSSPGLVTTLGAGLACFGGSPDHRPAHKFFKSSKPEGYVQERTASAKDSGSYKMRAEVKFEGDTLANKIELRGIDSKEDGPILGTRREYNYNSHNAYISADAQKNGLKSNFKRRINIEDNGVQLADHYQQRLPIGDGPVLLPDNHYLSTQSQLGADGNERRSHLVLLRFVTAAGITHILLPLIG

Cloning our biobrick with entangled genes knt-gfp

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.

entangled GFP DNA Sequence :

5’ - ATGGGCAAAGGAGACGAACTACTGCTCGGACTCGTACCAATTCTCGTTGAGCTATCAGGGCAGGTTAACGGACATCGTTTCAGCGTATCTGGCAAAGGCACTGGAGACGCTACAAGAGGAAAGCTCACGCTTAAGCTGGCTTGCACAACAGGGAGTCTACCAGTCAGCAGTCCTGGACTCGTCACAACGCTCGGGGCGGGACTGGCTTGCTTTGGGGGAAGTCCCGACCACCGACCTGCTCACAAGTTCTTTAAATCCAGCAAACCAGAAGGATACGTACAAGAACGCACTGCGTCGGCTAAGGACTCTGGATCCTACAAAATGCGAGCTGAAGTCAAGTTCGAAGGAGACACTCTCGCGAACAAGATCGAACTTAGAGGCATCGACAGTAAAGAAGACGGACCTATACTCGGAACACGAAGGGAATACAACTACAACTCTCACAACGCGTATATCAGCGCGGATGCCCAAAAAAACGGACTTAAAAGTAACTTCAAGAGACGCATCAACATCGAAGATAATGGTGTACAACTCGCGGACCACTACCAACAACGACTGCCTATCGGAGACGGTCCAGTACTCCTACCAGACAATCACTATCTATCCACGCAATCTCAACTCGGAGCTGACGGGAACGAGCGCAGATCGCATCTTGTACTACTACGGTTCGTCACAGCCGCAGGCATCACGCATATACTTCTACCGCTTATTGGATGA - 3’