Week 2 – DNA Read, Write, and Edit Laboratory Notebook Entry Field Value Course HTGAA Week 2 Dan Wright Topic DNA Read · Write · Edit
Abstract This laboratory exercise explored the modern molecular biology workflow: restriction digest simulation, wet-lab electrophoresis, gene design, codon optimization, DNA synthesis preparation, and genome read/write/edit technologies. A developmental transcription factor (MSX-1) was selected, reverse-translated, optimized, and engineered into an expression cassette.
1. Restriction Digest Simulation & Gel Art Objective Simulate Lambda DNA restriction digests and generate a gel-art visualization.
Enzymes Used EcoRI · HindIII · BamHI · KpnI · EcoRV · SacI · SalI
Methods Imported Lambda genome into Benchling Performed multi-enzyme restriction digest Visualized predicted fragment sizes Result Figure 1. Simulated restriction digest of Lambda DNA.
2. Wet Lab Restriction Digest Objective Perform physical restriction digestion and electrophoresis following the designed protocol.
3. Gene Design Workflow 3.1 Protein Selection Target Protein: Homo sapiens )
Rationale:
3.2 Reverse Translation Full reverse-translated nucleotide sequence:
atggcccccgccgccgacatgaccagcctgcccctgggcgtgaaggtggaggacagcgcc
ttcggcaagcccgccggcggcggcgccggccaggcccccagcgccgccgccgccaccgcc
gccgccatgggcgccgacgaggagggcgccaagcccaaggtgagccccagcctgctgccc
ttcagcgtggaggccctgatggccgaccacagaaagcccggcgccaaggagagcgccctg
gcccccagcgagggcgtgcaggccgccggcggcagcgcccagcccctgggcgtgcccccc
ggcagcctgggcgcccccgacgcccccagcagccccagacccctgggccacttcagcgtg
ggcggcctgctgaagctgcccgaggacgccctggtgaaggccgagagccccgagaagccc
gagagaaccccctggatgcagagccccagattcagccccccccccgccagaagactgagc
ccccccgcctgcaccctgagaaagcacaagaccaacagaaagcccagaacccccttcacc
accgcccagctgctggccctggagagaaagttcagacagaagcagtacctgagcatcgcc
gagagagccgagttcagcagcagcctgagcctgaccgagacccaggtgaagatctggttc
cagaacagaagagccaaggccaagagactgcaggaggccgagctggagaagctgaagatg
gccgccaagcccatgctgccccccgccgccttcggcctgagcttccccctgggcggcccc
gccgccgtggccgccgccgccggcgccagcctgtacggcgccagcggccccttccagaga
gccgccctgcccgtggcccccgtgggcctgtacaccgcccacgtgggctacagcatgtac
cacctgacc3.3 Codon Optimization Full codon-optimized sequence:
ATG GCT CCT GCC GCT GAC ATG ACA TCC CTC CCT CTG GGC GTG AAA GTC GAA GAC TCT GCC TTC GGA AAA CCA GCT GGA GGA GGT GCA GGC CAA GCG CCC TCA GCC GCC GCC GCA ACT GCC GCG GCA ATG GGC GCG GAT GAA GAA GGA GCA AAG CCT AAA GTC TCA CCC TCT TTG CTC CCC TTC TCT GTT GAG GCA CTC ATG GCC GAC CAC AGG AAA CCT GGC GCC AAA GAG TCA GCA CTT GCT CCA TCT GAG GGC GTG CAG GCT GCC GGT GGG TCT GCC CAG CCA CTC GGC GTT CCT CCT GGG TCT CTC GGT GCC CCC GAC GCC CCT AGC TCT CCA CGC CCT CTT GGG CAC TTT AGC GTG GGC GGG CTG CTG AAA CTT CCA GAA GAC GCA CTC GTT AAG GCC GAA AGT CCT GAG AAA CCC GAG CGA ACC CCT TGG ATG CAG TCA CCC AGG TTC TCA CCC CCT CCC GCT AGG AGG CTC TCC CCC CCA GCA TGT ACT CTC CGG AAA CAT AAG ACA AAT AGA AAA CCC CGC ACC CCG TTT ACC ACC GCC CAG CTG CTG GCC CTT GAG AGA AAG TTC CGG CAG AAG CAG TAC CTC TCC ATC GCC GAA CGG GCT GAG TTC TCC TCC TCC TTG TCC CTC ACC GAG ACA CAG GTT AAG ATT TGG TTC CAG AAC CGC CGG GCA AAG GCA AAA CGG CTG CAA GAA GCC GAG CTG GAG AAG CTT AAG ATG GCA GCT AAA CCC ATG CTC CCT CCA GCA GCG TTT GGC CTC AGT TTT CCA CTG GGC GGC CCA GCT GCA GTG GCA GCT GCG GCC GGC GCC TCC CTC TAT GGA GCC TCC GGG CCG TTC CAA CGG GCC GCA CTT CCC GTA GCA CCA GTC GGG TTG TAC ACT GCA CAT GTC GGC TAC AGC ATG TAC CAC CTG ACC4. Expression Cassette Engineering Full Expression Cassette Sequence TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCCATTAAAGAGGAGAAAGGTACCatggcccccgccgccgacatgaccagcctgcccctgggcgtgaaggtggaggacagcgccttcggcaagcccgccggcggcggcgccggccaggcccccagcgccgccgccgccaccgccgccgccatgggcgccgacgaggagggcgccaagcccaaggtgagccccagcctgctgcccttcagcgtggaggccctgatggccgaccacagaaagcccggcgccaaggagagcgccctggcccccagcgagggcgtgcaggccgccggcggcagcgcccagcccctgggcgtgccccccggcagcctgggcgcccccgacgcccccagcagccccagacccctgggccacttcagcgtgggcggcctgctgaagctgcccgaggacgccctggtgaaggccgagagccccgagaagcccgagagaaccccctggatgcagagccccagattcagccccccccccgccagaagactgagcccccccgcctgcaccctgagaaagcacaagaccaacagaaagcccagaacccccttcaccaccgcccagctgctggccctggagagaaagttcagacagaagcagtacctgagcatcgccgagagagccgagttcagcagcagcctgagcctgaccgagacccaggtgaagatctggttccagaacagaagagccaaggccaagagactgcaggaggccgagctggagaagctgaagatggccgccaagcccatgctgccccccgccgccttcggcctgagcttccccctgggcggccccgccgccgtggccgccgccgccggcgccagcctgtacggcgccagcggccccttccagagagccgccctgcccgtggcccccgtgggcctgtacaccgcccacgtgggctacagcatgtaccacctgaccCATCACCATCACCATCATCACTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGAnnotated Elements Promoter
TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCCATTAAAGRBS
Spacer
6×His Tag
Terminator
AGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGBenchling Construct Map Figure 2. Benchling construct showing CDS, His-tag, and terminator.
5. DNA Read · Write · Edit 5.1 DNA Read Target genome: Secretariat (racehorse)
Workflow:
DNA extraction Purification Library preparation Sequencing Base calling Assembly 5.2 DNA Write Technology: Phosphoramidite synthesis and gene synthesis providers
Steps:
Oligo synthesis Gibson Assembly Validation sequencing Limitations:
Fragment size limits Time and cost Assembly complexity 5.3 DNA Edit Technology: CRISPR knock-in
Mechanism:
Guide RNA design Cas9 cleavage Homology-directed repair Screening Limitations:
Off-target effects Efficiency variability 