Week 2 HW: DNA read / write /edit π§¬
Part I: Benchling & In-silico Gel Art I created my benchling account and imported the sequence GEN BANK CODE:J02459.1 from the NCBI website. I added the sequence to my benchling proyect folder. Then I simulated Restriction Enzyme Digestion with adding the following enzymes and then cutted the DNA in the restricted places
I created my benchling account and imported the sequence GEN BANK CODE:J02459.1 from the NCBI website. I added the sequence to my benchling proyect folder.
Then I simulated Restriction Enzyme Digestion with adding the following enzymes and then cutted the DNA in the restricted places
PROTEIN: Phospholipase A2 Apis mellifera - PA2 APIME
I’ve chosen phospholipase A2 originating from Apis mellifera due to being a key enzymatic factor of honeybee venom and for their study as potential neuroprotective and immunomodulatory agents. Bee venom phospholipase A2 has been reported as having properties that reduce neuroinflammation and protect dopaminergic neurons in animal models of Parkinson’s Disease.
I obtained the protein sequence from Uniprot
sp|P00630|PA2_APIME Phospholipase A2 OS=Apis mellifera OX=7460 PE=1 SV=3 MQVVLGSLFLLLLSTSHGWQIRDRIGDNELEERIIYPGTLWCGHGNKSSGPNELGRFKHT DACCRTHDMCPDVMSAGESKHGLTNTASHTRLSCDCDDKFYDCLKNSADTISSYFVGKMY FNLIDTKCYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKY
Using a reverse translation tool, I converted my amino acid sequence into a nucleotide sequence based on the standard genetic code. Because multiple codons can encode the same amino acid, the resulting sequence represents one of several possible valid sequences that could produce this protein. I obtained the nucleotides using the following tool available on internet: https://www.bioinformatics.org/sms2/rev_trans.html
reverse translation of sample sequence to a 501 base sequence of most likely codons. atgcaggtggtgctgggcagcctgtttctgctgctgctgagcaccagccatggctggcag attcgcgatcgcattggcgataacgaactggaagaacgcattatttatccgggcaccctg tggtgcggccatggcaacaaaagcagcggcccgaacgaactgggccgctttaaacatacc gatgcgtgctgccgcacccatgatatgtgcccggatgtgatgagcgcgggcgaaagcaaa catggcctgaccaacaccgcgagccatacccgcctgagctgcgattgcgatgataaattt tatgattgcctgaaaaacagcgcggataccattagcagctattttgtgggcaaaatgtat tttaacctgattgataccaaatgctataaactggaacatccggtgaccggctgcggcgaa cgcaccgaaggccgctgcctgcattataccgtggataaaagcaaaccgaaagtgtatcag tggtttgatctgcgcaaatat
I need to optimize my codons so that the chosen organism can express them accurately.
Chosen organism: E. coli
I optimized my sequence for expression in E. coli K12 usign the following tool: JCat I realized that the Codon Adaptation Index improve from a value of 0.58 to 1.0, indicating optimal codon usage for the host organism. The GC content of the optimized sequence (51.3%) is consistent with the genomic GC content of E. coli.
Translation of the optimized sequence confirmed that the amino acid sequence remained unchanged because it matches with the previous protein equence I obtained in part 3.1 from Uniprot.
Using an expression vector, the recombinant DNA sequence (which has an optimized codon) can be cloned into a host organism. The host cell transcribes this new DNA sequence into mRNA, which is then translated into protein. Protein production can be done using either cell-based or cell-free expression systems; however, toxic proteins should generally be produced using a cell-free expression system to avoid harming the host organism.
(i) What DNA would you want to sequence (e.g., read) and why? (ii) What technology or technologies would you use to perform sequencing on your DNA and why?
