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Application of biotechnology Expression in E. coli

Application of biotechnology Expression in E. coli. Dr Muhammad Imran. We may not be aware but……. Expression in E. coli needs signals. Three important signals needed for expression Promoter RBS Transcription terminator. Three important signal elements. Promoter. Strong promoters.

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Application of biotechnology Expression in E. coli

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  1. Application of biotechnologyExpression in E. coli Dr Muhammad Imran

  2. We may not be aware but……

  3. Expression in E. coli needs signals Three important signals needed for expression • Promoter • RBS • Transcription terminator

  4. Three important signal elements

  5. Promoter

  6. Strong promoters Weak promoters Constitutive promoter Regulated promoters Induction and repression.

  7. Strong and week promoters

  8. Induction and repression

  9. Misfolding • Forces that help protein fold.. H bonds, hydrophobic AA, ionic interactions, etc • Rate of transcription, translation and folding • Fusion partner role • Chaperon GroEl and GroES, DNAJK • pH • Ligand for folding

  10. Di-sulphide bonds 1- Origami strain 2- Shuffle strain 3- Periplasmiclocalization signal Highlights 1- Constitutively expresses a chromosomal copy of the disufide bond isomeraseDsbC 2- DsbCpromotes the correction of mis-oxidized proteins into their correct form (1,3) 3- The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds (4) 4- DsbA in periplasm express in cytoplasm 5- thioredoxins and glutaredoxinsreductaces maintain a reducing environment in cytoplasm

  11. Signal/localization sequences • Periplasm localization signal

  12. mRNA stability • It is normal cellular process • mRNA formation and mRNA degradation determines the over all level at a given time. • mRNA Length does matter • Secondary structures Rnase E mutated Toxic proteins C41

  13. Rare codons • pRare 2 plasmid • Synthetic genes Secondary structure optimized Rare codon optimized Rnase cleavage site removed

  14. Toxicity • Tight control on expression • Expression in stationary phase • Pre-protein • Weak promoter • Inclusion body and refolding • Rifampicin blocking

  15. Leaky expression • pLysis S and L plasmids • Arabad promoter strong repression

  16. Solubility tags

  17. Purification and detection tags

  18. Expression in inclusion bodies

  19. Optimization of expression strain media effect

  20. Inducer concentration and temperature

  21. Organisms • Gram negative sources genes express better in E coli compared to gram positive organism sources

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