Part 1: Benchling & In-silico Gel Art

Lambda DNA visualization with restriction enzymes using benchling

Part 3: DNA Design Challenge

3.1 Choose your protein.

For my protein of interest, I have chosen Apolipoprotein E (ApoE). ApoE plays a critical role in transporting cholesterol and other lipids throughout the body, especially in delivering essential nutrients to the brain. Notably, certain genetic variants of ApoE are associated with an increased risk of neurodegenerative diseases, such as Alzheimer’s disease. I selected ApoE because understanding its structure and function could provide valuable insights into how lipid transport impacts brain health and the mechanisms underlying these disorders.

sp|P02649|APOE_HUMAN Apolipoprotein E OS=Homo sapiens OX=9606 PE=1 SV=1 MVPGGSHGKGARIRAEWAGGPGGTLTRKWAQVQGRKHLDRSKVYAEVKQIFAPVTHSQTFE GTKPSKILVGGTLAVTLPDYSWRKQAQHSRRPSSRIDRDSMLHNDVSTGRAEFLNDASENPQHVRFAEVEDFCFDK

3.2 Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

apolipoprotein (ApoE) protein DNA sequence atggtgccgggcggcagccatggcaaaggcgcgcgcattcgcgcggaatgggcgggcggc ccgggcggcaccctgacccgcaaatgggcgcaggtgcagggccgcaaacatctggatcgc agcaaagtgtatgcggaagtgaaacagatttttgcgccggtgacccatagccagaccttt gaaggcaccaaaccgagcaaaattctggtgggcggcaccctggcggtgaccctgccggat tatagctggcgcaaacaggcgcagcatagccgccgcccgagcagccgcattgatcgcgat agcatgctgcataacgatgtgagcaccggccgcgcggaatttctgaacgatgcgagcgaa aacccgcagcatgtgcgctttgcggaagtggaagatttttgctttgataaa

3.3 Codon optimization.

Codon optimization is used to avoid slow translation and other complex unwanted structures. I optimized the protein for human. The reason is that ApoE is commonly produced in cells or in cell-free system. Since it will not be made in bacteria I chose human for the optimization.

ApoE DNA sequence with Codon-Optimization ATGGTGCCTGGCGGCTCTCACGGCAAGGGAGCCAGAATTCGCGCCGAGTGGGCCGGCGGCCCCGGCGGAACTCTGACCAGAAAGTGGGCCCAGGTGCAGGGCAGAAAGCATCTGGATAGATCTAAAGTGTACGCAGAGGTGAAGCAGATTTTCGCCCCTGTGACACACTCCCAGACCTTCGAGGGCACAAAGCCCTCCAAAATCCTGGTGGGCGGAACACTGGCTGTGACCCTGCCTGATTACAGCTGGAGGAAGCAGGCTCAGCACAGCAGGAGGCCATCCTCAAGGATCGACAGAGATTCCATGCTGCACAATGACGTGTCCACCGGGAGAGCCGAGTTCCTGAATGACGCTAGCGAGAACCCCCAGCACGTGCGGTTCGCCGAGGTTGAGGACTTCTGCTTCGACAAG

3.4 You have a sequence! Now what?

For producing ApoE protein, both cell-based and cell-free methods can be employed:

Cell-Dependent Methods: I would use cells that naturally produce ApoE, such as astrocytes or microglia, potentially derived from induced pluripotent stem cells (iPSCs). These cells would be transfected with a plasmid encoding a codon-optimized version of the ApoE gene. Once the DNA is inside the cell, the host's transcription machinery converts it into mRNA, which is then translated by ribosomes into the ApoE protein. This expression can be transient if the plasmid remains episomal, or stable if the DNA integrates into the genome.

Cell-Free Methods: Alternatively, the DNA template can be used in an in vitro transcription/translation system. In this setup, all the necessary components (RNA polymerase, ribosomes, tRNAs, and amino acids) are provided in a reaction mixture, allowing the DNA to be directly transcribed and translated into ApoE protein without the need for living cells.

Part 4: DNA Read/Write/Edit

4.1 DNA read

(i) What DNA would you want to sequence (e.g., read) and why?