Codon Optimization Codon Optimization Codon Usage Bias Variable

Codon Optimization

Codon Optimization �Codon Usage Bias: Variable frequency of synonomous codons. �Codon preferences reflect natural selection in organisms for translational optimization. �Optimal codons allow faster translation rates, thus highly expressed genes are coded by these. �Codon optimization can be used to produce a synthetic version of a foreign gene for more efficient expression.

Codon Usage of HIV vs. Human

Expression Profile of Synthetic vs. Wildtype gp 120

HIV Codons Decrease Efficiency of Thy-1 Expression -Thy-1 is a highly expressed cellsurface protein. -Replacing codons of Thy-1 with HIV envelope codons significantly decreases expression.

Codon Optimization of GFP � A: Control: Empty vector. � B: Native GFP from Aequorea victoria. � C: Mammalian codon optimized GFP. � D: Mammalian codon optimized GFP + S 65 T replacement.

E. Coli vs. Human Codon Usage

Codon Optimization: Human to Bacteria

Polymerase Chain Reaction �Molecular biology technique used to amplify specific nucleotide sequences. �Developed by Kary Mullis in 1983. �Takes advantage of Taq polymerase from thermophile Thermus aquaticus. � Taq is a heat-resistant enzyme that can function in temperatures above 70°C.

Polymerase Chain Reaction

Components of PCR �Water �Taq Buffer containing divalent and monovalent cations – Provides suitable chemical environment for optimal activity of DNA polymerase (p. H, [salt], cofactors, etc. ) �DNA template containing target region to be amplified. �Sense and anti-sense primers. �Deoxynucleotide triphosphates (d. NTPs), the buildingblocks for synthesis of new DNA. �Taq polymerase – has optimal activity around 70°C.

Homework Assignment �NCBI � Search through GENE databases. � Enter the name of the gene of interest. � Search for Homo sapien or E. coli and select the gene. � Scroll down to m. RNA and proteins and select. � Copy the “translation” �Encorbio � Select the organism that the gene will be codon optimized to. � Paste the amino acid sequence into the top box. � Generate the coding sequence.

Example: Insulin-like Growth Factor 1 Receptor (IGF 1 R) � Amino Acid Sequence � MKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVI FEMTNLKDIGLYNLRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCH PECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSA QMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYR MEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPW TQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTEV CGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSCN HEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVF FYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVN EAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFT VKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSF LEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC � Codon Optimized Sequence � ATGAAAAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGA TTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATAT CTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAG ATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATA TTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGC TGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGGCGTGCCG CCATTATTATTATGCGGGCGTGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGA AGGCTTTGTGATTCATGATGGCGAATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAACTGGAAAACTTTAT GGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTT TTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCAT GGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACC GCATTATTATTACCTGGCATCGCTATCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGGCAG CAACAGCTGGAACATGGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCA TGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCTGATTGTG AAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGC AAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAA AAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGC CGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTT ACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTG ACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGT GAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTT TTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAA CAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGG GCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTT CTGAACGAAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAG CTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGAAATTGCGGATGGCGTATCTGAACGCGAACAAAT TTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCC TGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGG GCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCC CGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACTGCCGGAAGAACTGGATCTGGAACCGGAA AACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCGTGCTGCGCGCGAGCTT TGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGCGCGCTGCCGCAGAGCAGCACCTGC

Example: Insulin-like Growth Factor 1 Receptor (IGF 1 R) � Add Eco. RI (GAATTC) at 5’ end and Bam. HI (GGATCC) at 3’ end. � GAATTCATGAAAAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCG AAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTG ATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCT GGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTG GCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTG GATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAA AAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCG AAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGGCGTGCCGCCATTATTATTATGCGGG CGTGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGC AGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTC CGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCA TTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTAT GTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCT TTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCG AAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAA CGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCGGATTA TCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGGCAGCAACAGCTGG AACATGGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGA AAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCC GCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTG CGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTG ATATTGAAGAAGTGACCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGG CGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGAT GCAGGTGGCGAACACCACCATGAGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATAT CCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCA TGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGG GAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGG TGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCAT TCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTA TTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGC GTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCC GCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCG TGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGG CGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAA AACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGAAATTGCGGATGGCGTATCTGAACGCGAACAAATTTG TGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGAT TATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGA GCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGG CCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAA TTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACTGCCGGAAGAACTGGA TCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCCTGCCGGATCGCCATAGCGGCCATAAAGCGGA AAACGGCCCGGGCGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGCGC GCTGCCGCAGAGCAGCACCTGCGGATCC

Designing Amplification Oligos ~5’ End 5’GAATTCATGAAAAGCGGCGGCGGC 3’ 3’CTTAAGTACTTTTCGCCGCCGCCG 5’ ~*You need an available 3’OH, so for this end you make a primer against the antisense strand. 5’GAATTCATGAAAAGCGGCGGCGGC 3’ 5’GAATTCATGAAAAGCGGC 3’OH 3’CTTAAGTACTTTTCGCCGCCGCCG 5’

Designing Amplification Oligos ~3’ End 5’TGCCGCAGAGCAGCACCTGCGGATCC 3 ’ 3’ACGGCGTCTCGTCGTGGACGCCTAGG 5 ’ ~Again, you need a 3’OH so this time make a primer against the sense strand. 5’TGCCGCAGAGCAGCACCTGCGGATCC 3 ’ HO 3’GGCGTCTCGTCGTGGACGCCTAGG 5’

Designing Amplification Oligos �Primers need to be anywhere from 18 -25 nucleotides. �Don’t include the restriction site or Kozak sequence as part of your 18 -25 nucleotides for the primer. �Should contain at least 50% GC composition. �Must flank the entire CODING sequence.