Week 7 Lab: Cell-free systems

Protein synthesis requires transcription and translation

Transcription

eukaryotes or in cytoplasm in prokaryotes RNA polymerase DNA nucleotides to make RNA polymerase will bind promoter and in the space will

Translation

tRNA, amino acids and ribosomes, mRNA inside nucleas, splicing take introns and leave extrons

RBS (ribosome binding site) - attach to small subunit - mRNA to merge with small subunit tRNA will bind start codon (AUG/ ATG) (complementary to start codon) codons are 3 nucleotides EPA E P A are three sites of tRNA working on codons [exit, peptide, amino acid] bind to start, peptides growing protein on P site, then amino acid on site and leave ribosome

can happen outside of cells

TX transcription TL translation CFPS (Cell free protein synthesis)

Cell lysate

ribosomes for translations tRNA Initiation, transcription, and trasnslation factors Microsomes (membranes phospholipid bilayer)

Template

plasmid DNA (more stable) linear PCR

Supplements

Nucleotides amino acids ATP (transcription) GTP (translation)

buffer stabilize the pH

Prokaryotes transcription and translation can happen at the same time Eukaryotes separate (extrons and introns) you need specific machinery enzymes that will take out the introns

Cell free systems will give code sequence no need introns and extrons plasmid -coding sequence - use messenger RNA to produce insulin

Endosymbiotic theory (using mitochondria will be bactera - circular DNA too complex, use more energy keep mitochondria working)

-no time-consuming cloning steps required -reaction conditions can be fully controlled and modified -proteins that are toxic to cells can still be produced

Tx-TL system can be classified by the source of the cell extract

bacterial cell-free system E Coli eukaryotic yeast, mammalian, insect, plant

Insulin are made by disulfide bond make two polypeptide chains

Cell lysis and a lot of purification

Chromoproteins just have colors

GFP Green florescent protein requires blacklight

RFP

purification filters the protein you want

his-tag + protein of interest –> the tag will bind to another metal ion

Fusion protein | His-tag (histadine) | Ligand | Bead

Magnetic block

Today’s protocol:

GFP, RFP, mix Affinity Purification to isolate our samples Mixture –> will get separation

1 GFP 2 RFP 3 mix 4 mix