3.1 My protein: DNA topoisomerase 1 [Homo Sapiens]
[add intro]
Source: https://www.ncbi.nlm.nih.gov/protein/NP_003277.1
Why this: because I was doing PCR in school and just thought of this (?
[mini question: I used the E.coli codon table while reverse translating, but my sequence alignment results still showed differences between the optimized sequence and the RT sequence, why so?]
3.4 Production
Cell dependent: insert the gene on a plasmid into E.coli via transformation, electroporation, or conjugation; let the cell replicate in culture medium; transfer to larger fermentation tanks and add IPTG to induce protein production; protein extraction and confirmation with SDS-PAGE.
[mini question: would using a prokaryotic cell to express mammalian proteins create too much metabolic stress for them? Also if they don’t have the splicing mechanism then is PTM feasible at all; or is it feasible because the sequence we are using is RV from the protein sequence, which has already gone through splicing]
Cell-free protein synthesis: a solution containing ribosomes, tRNAs, enzymes, cofactors, amino acids, etc. is used to transcribe and translate the target sequence
[mini question: technically we can use systems extracted from mammalian cells? Would this make CFPS advantageous compared to using a conventional chassis (E.coli) or is there other limitations?]
(bacteria grow faster
3.5
question a: post-transcriptional modification/splicing strategies (like, exon skipping and intron retention?; different reading frames?; protein isoforms
[mini question: protein isoforms would be impacted by the cellular environment (pH and other factors) and the presence of isomerases right?]
reverse translated and codon-optimized sequence alignment