Week 2: DNA Read, Write, and Edit Week 2: DNA Read, Write, and Edit Part 1: Benchling & In-silico Gel Art 1.1 Import Lambda DNA Simulate Restriction Enzyme Digestion Virtual Gel Part 2: Gel Art I have chosen to create a gel art of a person doing a jumping jack through randomization method.
Part 3: DNA Sequence Design 3.1 Protein Selection I have chosen IL23 as I am interested in autoimmune disease such as psoriasis. This protein is related to inflammation and I am curious to learn more about biologics in general.
3.2 Reverse Translation Reverse translation of sp|Q5VWK5|IL23R_HUMAN Interleukin-23 receptor OS=Homo sapiens OX=9606 GN=IL23R PE=1 SV=3 to a 1887 base sequence of most likely codons.
atgaaccaggtgaccattcagtgggatgcggtgattgcgctgtatattctgtttagctgg
tgccatggcggcattaccaacattaactgcagcggccatatttgggtggaaccggcgacc
atttttaaaatgggcatgaacattagcatttattgccaggcggcgattaaaaactgccag
ccgcgcaaactgcatttttataaaaacggcattaaagaacgctttcagattacccgcatt
aacaaaaccaccgcgcgcctgtggtataaaaactttctggaaccgcatgcgagcatgtat
tgcaccgcggaatgcccgaaacattttcaggaaaccctgatttgcggcaaagatattagc
agcggctatccgccggatattccggatgaagtgacctgcgtgatttatgaatatagcggc
aacatgacctgcacctggaacgcgggcaaactgacctatattgataccaaatatgtggtg
catgtgaaaagcctggaaaccgaagaagaacagcagtatctgaccagcagctatattaac
attagcaccgatagcctgcagggcggcaaaaaatatctggtgtgggtgcaggcggcgaac
gcgctgggcatggaagaaagcaaacagctgcagattcatctggatgatattgtgattccg
agcgcggcggtgattagccgcgcggaaaccattaacgcgaccgtgccgaaaaccattatt
tattgggatagccagaccaccattgaaaaagtgagctgcgaaatgcgctataaagcgacc
accaaccagacctggaacgtgaaagaatttgataccaactttacctatgtgcagcagagc
gaattttatctggaaccgaacattaaatatgtgtttcaggtgcgctgccaggaaaccggc
aaacgctattggcagccgtggagcagcctgttttttcataaaaccccggaaaccgtgccg
caggtgaccagcaaagcgtttcagcatgatacctggaacagcggcctgaccgtggcgagc
attagcaccggccatctgaccagcgataaccgcggcgatattggcctgctgctgggcatg
attgtgtttgcggtgatgctgagcattctgagcctgattggcatttttaaccgcagcttt
cgcaccggcattaaacgccgcattctgctgctgattccgaaatggctgtatgaagatatt
ccgaacatgaaaaacagcaacgtggtgaaaatgctgcaggaaaacagcgaactgatgaac
aacaacagcagcgaacaggtgctgtatgtggatccgatgattaccgaaattaaagaaatt
tttattccggaacataaaccgaccgattataaaaaagaaaacaccggcccgctggaaacc
cgcgattatccgcagaacagcctgtttgataacaccaccgtggtgtatattccggatctg
aacaccggctataaaccgcagattagcaactttctgccggaaggcagccatctgagcaac
aacaacgaaattaccagcctgaccctgaaaccgccggtggatagcctggatagcggcaac
aacccgcgcctgcagaaacatccgaactttgcgtttagcgtgagcagcgtgaacagcctg
agcaacaccatttttctgggcgaactgagcctgattctgaaccagggcgaatgcagcagc
ccggatattcagaacagcgtggaagaagaaaccaccatgctgctggaaaacgatagcccg
agcgaaaccattccggaacagaccctgctgccggatgaatttgtgagctgcctgggcatt
gtgaacgaagaactgccgagcattaacacctattttccgcagaacattctggaaagccat
tttaaccgcattagcctgctggaaaaaReverse translation of sp|Q5VWK5|IL23R_HUMAN Interleukin-23 receptor OS=Homo sapiens OX=9606 GN=IL23R PE=1 SV=3 to a 1887 base sequence of consensus codons.
atgaaycargtnacnathcartgggaygcngtnathgcnytntayathytnttywsntgg
tgycayggnggnathacnaayathaaytgywsnggncayathtgggtngarccngcnacn
athttyaaratgggnatgaayathwsnathtaytgycargcngcnathaaraaytgycar
...3.3 Codon Optimization Original Sequence GC Content : 49.34%CAI : 0.83ATGAACCAGGTGACCATTCAGTGGGATGCGGTGATTGCGCTGTATATTCTGTTTAGCTGGTGCCATGGCGGCATTACCAACATTAACTGCAGCGGCCATATTTGGGTGGAACCGGCGACCATTTTTAAAATGGGCATGAACATTAGCATTTATTGCCAGGCGGCGATTAAAAACTGCCAGCCGCGCAAACTGCATTTTTATAAAAACGGCATTAAAGAACGCTTTCAGATTACCCGCATTAACAAAACCACCGCGCGCCTGTGGTATAAAAACTTTCTGGAACCGCATGCGAGCATGTATTGCACCGCGGAATGCCCGAAACATTTTCAGGAAACCCTGATTTGCGGCAAAGATATTAGCAGCGGCTATCCGCCGGATATTCCGGATGAAGTGACCTGCGTGATTTATGAATATAGCGGCAACATGACCTGCACCTGGAACGCGGGCAAACTGACCTATATTGATACCAAATATGTGGTGCATGTGAAAAGCCTGGAAACCGAAGAAGAACAGCAGTATCTGACCAGCAGCTATATTAACATTAGCACCGATAGCCTGCAGGGCGGCAAAAAATATCTGGTGTGGGTGCAGGCGGCGAACGCGCTGGGCATGGAAGAAAGCAAACAGCTGCAGATTCATCTGGATGATATTGTGATTCCGAGCGCGGCGGTGATTAGCCGCGCGGAAACCATTAACGCGACCGTGCCGAAAACCATTATTTATTGGGATAGCCAGACCACCATTGAAAAAGTGAGCTGCGAAATGCGCTATAAAGCGACCACCAACCAGACCTGGAACGTGAAAGAATTTGATACCAACTTTACCTATGTGCAGCAGAGCGAATTTTATCTGGAACCGAACATTAAATATGTGTTTCAGGTGCGCTGCCAGGAAACCGGCAAACGCTATTGGCAGCCGTGGAGCAGCCTGTTTTTTCATAAAACCCCGGAAACCGTGCCGCAGGTGACCAGCAAAGCGTTTCAGCATGATACCTGGAACAGCGGCCTGACCGTGGCGAGCATTAGCACCGGCCATCTGACCAGCGATAACCGCGGCGATATTGGCCTGCTGCTGGGCATGATTGTGTTTGCGGTGATGCTGAGCATTCTGAGCCTGATTGGCATTTTTAACCGCAGCTTTCGCACCGGCATTAAACGCCGCATTCTGCTGCTGATTCCGAAATGGCTGTATGAAGATATTCCGAACATGAAAAACAGCAACGTGGTGAAAATGCTGCAGGAAAACAGCGAACTGATGAACAACAACAGCAGCGAACAGGTGCTGTATGTGGATCCGATGATTACCGAAATTAAAGAAATTTTTATTCCGGAACATAAACCGACCGATTATAAAAAAGAAAACACCGGCCCGCTGGAAACCCGCGATTATCCGCAGAACAGCCTGTTTGATAACACCACCGTGGTGTATATTCCGGATCTGAACACCGGCTATAAACCGCAGATTAGCAACTTTCTGCCGGAAGGCAGCCATCTGAGCAACAACAACGAAATTACCAGCCTGACCCTGAAACCGCCGGTGGATAGCCTGGATAGCGGCAACAACCCGCGCCTGCAGAAACATCCGAACTTTGCGTTTAGCGTGAGCAGCGTGAACAGCCTGAGCAACACCATTTTTCTGGGCGAACTGAGCCTGATTCTGAACCAGGGCGAATGCAGCAGCCCGGATATTCAGAACAGCGTGGAAGAAGAAACCACCATGCTGCTGGAAAACGATAGCCCGAGCGAAACCATTCCGGAACAGACCCTGCTGCCGGATGAATTTGTGAGCTGCCTGGGCATTGTGAACGAAGAACTGCCGAGCATTAACACCTATTTTCCGCAGAACATTCTGGAAAGCCATTTTAACCGCATTAGCCTGCTGGAAAAAImproved DNA Sequence GC Content : 51.56%CAI : 0.91ATGAACCAGGTGACTATCCAGTGGGACGCCGTTATCGCACTGTATATCCTGTTCAGCTGGTGCCACGGGGGCATTACCAACATAAACTGTAGCGGGCACATCTGGGTGGAACCTGCGACCATCTTCAAGATGGGCATGAATATCTCTATCTACTGTCAGGCCGCCATTAAGAACTGCCAGCCCAGGAAGCTGCATTTCTATAAGAATGGGATCAAGGAAAGGTTCCAGATCACCCGGATCAATAAGACCACAGCCCGCCTGTGGTACAAGAATTTTCTCGAGCCTCATGCCTCTATGTACTGTACAGCAGAGTGTCCTAAGCACTTCCAGGAGACTCTGATCTGCGGCAAAGATATTAGCTCCGGGTACCCCCCCGACATCCCCGACGAAGTGACCTGCGTGATCTATGAGTACTCCGGGAATATGACCTGCACCTGGAATGCCGGCAAGCTGACTTACATTGATACAAAGTACGTGGTGCATGTGAAGAGTCTGGAAACTGAGGAGGAACAGCAGTACCTGACAAGCTCCTATATCAATATTTCTACCGACTCTCTGCAGGGCGGCAAGAAGTACCTGGTGTGGGTGCAGGCCGCCAACGCTCTGGGCATGGAAGAGTCTAAGCAGCTGCAGATTCACCTAGATGATATTGTGATCCCATCCGCCGCCGTGATCAGCCGTGCAGAGACAATCAACGCCACCGTGCCTAAAACCATCATCTACTGGGACTCCCAAACCACCATTGAAAAGGTGAGTTGCGAAATGAGGTATAAGGCCACCACCAATCAGACCTGGAACGTGAAGGAATTCGACACAAACTTTACATATGTGCAGCAGAGCGAGTTTTATCTGGAGCCTAATATCAAGTACGTGTTCCAGGTCAGGTGTCAGGAGACAGGGAAGCGCTACTGGCAGCCCTGGAGTTCCCTGTTCTTTCACAAAACCCCAGAAACCGTGCCTCAGGTGACCTCCAAGGCCTTTCAGCATGACACCTGGAATTCCGGCCTGACTGTGGCCTCAATCTCAACTGGACATCTGACCAGCGATAATAGAGGAGACATAGGCCTGCTGCTGGGCATGATCGTGTTCGCAGTGATGCTGAGCATCCTGTCCCTGATCGGGATCTTCAATAGGTCTTTCCGCACCGGCATCAAGAGGAGGATCCTGCTGCTGATCCCCAAGTGGCTGTATGAGGATATCCCCAACATGAAGAACTCAAATGTGGTGAAGATGCTGCAGGAGAATTCCGAACTGATGAACAACAACAGCTCTGAGCAGGTGCTGTATGTGGACCCCATGATTACCGAGATCAAGGAAATCTTCATACCTGAGCACAAGCCCACAGACTACAAAAAAGAGAACACCGGACCACTGGAGACAAGGGATTATCCACAGAATAGCCTTTTCGATAATACAACCGTGGTGTACATCCCCGATCTGAACACCGGCTACAAACCCCAGATCTCTAACTTCCTGCCTGAGGGCTCCCACCTGTCCAATAACAACGAGATCACCAGCCTGACCCTGAAGCCCCCAGTGGACTCCCTGGACTCCGGCAATAATCCCAGACTGCAAAAACACCCTAACTTCGCGTTTTCCGTGTCAAGCGTGAATTCCCTGAGCAACACCATTTTCCTGGGCGAGCTGTCACTGATCCTGAACCAGGGCGAGTGCTCAAGCCCAGACATCCAGAACTCTGTCGAGGAGGAGACTACGATGCTGCTGGAGAATGATAGTCCCTCCGAAACAATCCCAGAGCAGACCCTGCTGCCTGATGAGTTTGTCAGCTGCCTGGGCATCGTGAACGAGGAGCTGCCCTCCATAAATACCTATTTCCCCCAGAATATCCTGGAATCCCACTTCAACAGAATTAGCCTGCTGGAGAAGAvoid Cleavage Sites Why Codon Optimization is Important Codon optimization is important because there is codon usage bias, which means humans and other organisms like E. coli might prefer different codons for the same amino acid. Expressing human gene like IL23 might be difficult because codons natural to human cells are rare in E. coli. If bacterium has low levels of corresponding tRNAs, then it will be slowed down during translation. There will be low protein yield as a result.
The codon optimization here increased GC content so there will be more mRNA stability. Codon adaptation index has also gone up.
3.4 Protein Expression Now, we will use this optimized DNA sequence to create IL23 protein. First we clone the codon optimized sequence into expression vector, and we transform a plasmid into E. coli cells. Bacteria will be shocked by heat to start making protein. The cell’s RNA polymerase will read the DNA and makes mRNA copy. Once the transcription is read, it will begin to build protein using tRNAs in the translation process.
Once this is done, there is a chromatography technique which separates protein from everything else in the cell.
Part 4: IL23 Sequence Analysis Summary Download IL23 Plasmid Map (PDF)
