Week 2 HW: Principles and Practices

Week 2 Homework: Read, Write and Edit

Part 1: Benchling practice and DNA gel art design

Anchor pattern in 5x5 grid with ladder in 3rd column

Part 2: Restriction digests and gel electrophoresis lab

Lab 1: Gel electrophoresis demonstration Lab 2: LAMBDA EcoRI

Part 3: DNA Design

3.1 Human Pancreatic Lipase (PNLIP)

PNLIP is an essential enzyme for human fat digestion and metabolism. It also has properties which give it significant industrial applications. It’s used in detergents to target fat-based stains on clothing, in the food industry to change fat content of foods and enhance the flavour of cheese, and in the production of pharmacutical products, making it a useful candidate for synthetic biology.

Amino acid sequence (from UniProt)

sp|P16233|LIPP_HUMAN Pancreatic triacylglycerol lipase OS=Homo sapiens OX=9606 GN=PNLIP PE=1 SV=2 MLWVFALLLGAVALSLGKEGCRIPAVPFPNGNVNQMDKSLAAYWREVVGQLSLFSTYGYG SGRGKSTQGAYIFINSFEGNYPSVKALVLLHGYGDDSYNFWSNDASTIHLVPMWNNGMRV WDLFASGNAVPVDPKRQSVFVSLHPQYPKPSRFVKLLMWHDLDPTASFRKARAFAGFSRK ENIIFGLDRLANLGFYTEGNYSLVGSKDSYPLNRYLQVNPGTAYILYPDEIASKLGLASY NITLMDLYFGTRFLRAMASVAKASGKVTIIRHTHGYELHFIFEDGIRYSADLAKFMTEGP KNYVMLDVSLISLMSDKGGLGVAARAVHLVQLNMWVPTWLLFKNGKLYYNSLADLANWAV EEVVNSSLLPNILLGFTGNDVFMVCHKVGRVNCEIENVLGECPFPVNAKACLLSGVEILA DGVHLYLPNPFTVMDITYFRT

3.2 Reverse translate

To produce this protein in a lab setting we reverse translate the amino acids into nucleotides, which leaves us with: ATGCTGTGGGTTTTTGCACTGCTCCTGGGAGCAGTGGCTCTCTCCTTAGGCAAAGAGGGATGCCGCATCCCTGCGGTACCCTTCCCCAATGGCAACGTGAACCAGATGGACAAGTCCCTGGCTGCCTACTGGAGAGAAGTGGTAGGCCAGCTGAGCCTCTTCAGCACCTATGGATATGGATCTGGAAGAGGCAAAAGCACCCAAGGAGCCTACATCTTCATCAACTCCTTTGAAGGAAACTATCCCTCTGTGAAGGCACTCGTCCTGCTGCATGGCTATGGAGATGACTCCTACAACTTCTGGAGCAACGATGCCTCCACCATCCACCTGGTGCCCATGTGGAACAACGGGATGAGGGTCTGGGACCTCTTTGCCAGTGGCAATGCAGTCCCTGTGGACCCCAAGAGACAGAGCGTGTTTGTGTCCCTGCACCCACAGTACCCCAAACCCAGCCGCTTTGTCAAACTCCTCATGTGGCATGATCTTGACCCCACGGCCTCATTCCGCAAAGCCCGGGCCTTTGCTGGTTTCAGTCGCAAAGAAAACATCATCTTTGGCCTGGACAGGCTTGCAAACCTGGGATTCTACACAGAGGGTAACTACAGCTTGGTGGGGTCGAAGGACAGCTATCCCCTCAACAGATACCTGCAGGTGAACCCTGGCACGGCCTACATCCTCTATCCCGATGAGATCGCCTCCAAGCTGGGCCTGGCCTCCTACAACATCACCCTCATGGACCTCTACTTTGGCACCCGCTTCCTCAGAGCCATGGCCTCTGTGGCCAAGGCCTCGGGCAAGGTCACCATCATCCGCCACACCCATGGCTATGAACTCCACTTCATCTTCGAAGATGGCATCAGGTACAGTGCTGACTTGGCCAAGTTTATGACAGAGGGCCCCAAGAACTATGTGATGCTGGATGTCTCCCTCATCTCCCTCATGTCCGACAAGGGAGGGCTGGGGGTGGCCGCAAGGGCTGTCCACCTGGTGCAGCTGAACATGTGGGTGCCCACCTGGCTGCTCTTCAAGAATGGGAAGTTGTACTACAACTCCCTGGCTGACCTGGCCAACTGGGCTGTGGAGGAAGTGGTGAACAGCTCCCTGCTGCCCAACATCCTGCTGGGCTTCACAGGCAACGATGTCTTCATGGTGTGCCACAAGGTGGGTCGGGTGAACTGTGAGATTGAGAATGTGCTCGGGGAGTGTCCCTTCCCTGTCAACGCCAAGGCCTGCCTGCTCAGTGGGGTGGAGATCCTGGCTGATGGCGTCCACCTGTACCTGCCCAACCCCTTCACGGTGATGGACATCACCTACTTCCGCACATGA

3.3 Codon optimisation

Codon optimisation increases the efficiency of gene expression in a heterologous host organism. Different organisms have different preferred codons for the same amino acid which makes translation less efficient and increases the chance that a non-functional protein is produced. Codon optimisation involves translating codons into the format preferred by the host organism.

Improved DNA (using VectorBuilder):

ATGCTGTGGGTGTTCGCGCTGCTGCTGGGCGCGGTGGCACTGAGCCTGGGCAAAGAAGGCTGCCGCATCCCGGCGGTGCCGTTTCCTAATGGCAATGTGAACCAGATGGATAAGAGCCTGGCGGCGTATTGGCGCGAAGTGGTGGGTCAGCTGAGTCTGTTTAGCACCTATGGCTATGGCAGCGGCCGCGGCAAAAGCACCCAGGGCGCGTATATTTTTATTAATAGCTTCGAAGGCAATTACCCGTCTGTGAAAGCGCTGGTGCTGCTGCACGGCTATGGCGATGATAGCTATAACTTCTGGAGCAATGATGCGAGCACCATTCATCTGGTGCCGATGTGGAACAATGGCATGCGCGTGTGGGATCTGTTTGCCAGCGGCAATGCCGTGCCGGTGGATCCGAAACGCCAAAGCGTGTTCGTGAGCCTGCATCCGCAGTATCCGAAACCGAGCCGTTTTGTTAAACTGCTGATGTGGCATGATCTGGATCCGACCGCGAGCTTTCGTAAAGCCCGTGCCTTTGCGGGCTTTTCTCGCAAAGAAAACATTATTTTTGGCCTGGATCGCCTGGCGAACCTGGGTTTTTATACCGAAGGCAACTATAGCCTGGTGGGCAGCAAAGATAGTTATCCGCTGAATCGTTACCTGCAGGTCAACCCGGGCACCGCGTATATTCTGTATCCGGATGAAATTGCGAGCAAACTGGGCCTGGCGAGCTATAACATCACCCTGATGGATCTGTATTTCGGCACCCGCTTTCTGCGCGCAATGGCCAGCGTTGCCAAAGCAAGCGGCAAAGTGACCATTATTCGCCATACCCATGGCTATGAACTGCATTTTATTTTCGAAGATGGCATTCGCTATAGCGCAGATCTGGCCAAATTTATGACCGAAGGCCCGAAAAATTACGTGATGCTGGATGTCAGCCTGATTAGCCTGATGTCAGATAAAGGTGGCTTAGGCGTGGCGGCGCGTGCCGTGCATCTGGTGCAGCTGAATATGTGGGTCCCGACCTGGCTGCTGTTTAAAAACGGCAAACTGTACTATAACAGCCTGGCGGACCTGGCGAATTGGGCGGTGGAAGAAGTGGTTAACAGCAGCCTGCTGCCGAACATTCTGCTGGGCTTTACCGGCAACGACGTTTTTATGGTGTGCCACAAAGTGGGTCGTGTTAACTGTGAAATTGAAAACGTGCTGGGTGAATGCCCGTTTCCGGTGAATGCGAAAGCGTGCCTGCTGAGCGGCGTGGAAATTCTGGCGGATGGCGTGCACCTGTACCTGCCGAATCCGTTTACCGTGATGGATATTACCTACTTCCGCACATAA

3.4

What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.