Employing homologous ends to stitch together fragments into full length products
There are many strategies for assembling synthon or gene-length DNAs into larger molecules. In PCR-based methods, DNAs are stitched together into full length products through polymerase-based reactions. Section divider: Homology-based Assembly Methods. Employing homologous ends to stitch together fragments into full length products.(aka cassette mutagenesis) (aka megaprimer mutagenesis)
# PCR 2 fragments PCR targAf targAr p20N5 pcrA PCR targBf targBr pTargetF pcrB # Gibson Assemble them Gibson pcrA pcrB pTarg2 # Transform them Transform pTarg2 Mach1 Amp # Sequences oligo targAf GAGTTCATGTGCAGCTCCATAAGCTGAAATTCTGCCTCGTGATAC oligo targAr GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC oligo targBf GAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC oligo targBr GTATCACGAGGCAGAATTTCAGCTTATGGAGCTGCACATGAACTC>p20N5 attaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcctgcaaCTCGAGcgatcagatACTAGTaaccaatataccaaataaagagttgaggacgtcaaggATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAGGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGATTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAGGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAAGAATTCccctAGAGACCCCTCCTCcagaaatcatccttagcgaaagctaaggattttttttatctgaaattctgcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggCtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgt >pTargetF catgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatgctggatccttgacagctagctcagtcctaggtataatactagtcatcgccgcagcggtttcaggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattctctagagtcgacctgcagaagcttagatctattaccctgttatccctactcgagttcatgtgcagctccataagcaaaaggggatgataagtttatcaccaccgactatttgcaacagtgccgttgatcgtgctatgatcgactgatgtcatcagcggtggagtgcaatgtcatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataagatgccgctcgccagtcgattggctgagctcataagttcctattccgaagttccgcgaacgcgtaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca >pTarg2 attaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatgctggatccttgacagctagctcagtcctaggtataatactagtcatcgccgcagcggtttcaggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattctctagagtcgacctgcagaagcttagatctattaccctgttatccctactcGAGTTCATGTGCAGCTCCATAAGCTGAAATTCTGCCTCGTGATACgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggCtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgt A Gibson construction file, set as code in four commented blocks. First two PCRs, one off p20N5 and one off pTargetF, giving pcrA and pcrB. Then a single Gibson line assembling those two products into pTarg2. Then a transformation of pTarg2 into Mach1 selecting on ampicillin. Then the sequences block, listing the four oligos targAf, targAr, targBf and targBr that put the shared ends on the two PCR products.
Replace the RFP cds in pSB1A2-I13521 with amilGFP retaining its native regulation using Gibson assembly
amilGFP ORF:
ATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGC AGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGG AAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTT GACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGAC ATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTA TTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTC ATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAG AAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGA GGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCAC TCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAA ATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACAT GCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAATo design this, start by constructing the sequence of the product keeping note of the junctions between the vector and the inserted amilGFP sequence. Every oligo gets a ~20bp annealing region on its own template. Then the 20bp overlap is added once per junction, as a 5’ tail on one of the two oligos that meet there: here both tails go on the insert oligos, so the vector is amplified with plain primers. That is the step to say out loud, because tailing both oligos at a junction is the obvious thing to do and it gives you a 40bp overlap, not a 20bp one. # PCR amplification of fragments PCR o1 o2 amilGFP amil PCR o3 o4 pSB1A2-I13521 vec # Gibson Assembly of PCR products Gibson amil vec pdt # Transformation of assembly product Transform pdt Mach1 # Sequences oligo o1 agaaagaggagaaatactagATGTCTTATTCAAAGCATGGC oligo o2 ctacactagcactatcagcgTTATTTAACCTTCAAAGGGTTAAC oligo o3 cgctgatagtgctagtgtag oligo o4 ctagtatttctcctctttctc >pdt gaattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaagaggagaaatactagATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGCAGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGGAAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTTGACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGACATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTATTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTCATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAGAAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGAGGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCACTCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAAATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACATGCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAAcgctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagttgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctg A question for the room, marked with the deck's thinking figure. Replace the RFP coding sequence in pSB1A2-I13521 with amilGFP, keeping its native regulation, using Gibson assembly. The amilGFP open reading frame is printed below in twelve lines of sequence. The answer is not on the slide.
