Biogenius: Gene Cloning and Expression [Theory & Practice]

Explore how the lac operon coordinates bacterial gene expression through promoter and operator control, repressor binding, and lactose-activated beta-galactosidase production to process lactose.

Modify the lac operon by replacing lacZYA with a gene of interest, using the promoter and operator to control transcription, with IPTG as an inducer to avoid lactose metabolism.

Explore expression plasmids by configuring a promoter, operator, ribosome binding site, start and stop codons, terminator, a multiple cloning site, and an origin of replication with a selectable marker.

Explain the bacterial ribosome binding site and Shine-Dalgarno sequence AGGAGG, the 5 to 9 nucleotide spacing before the start codon, and start codons ATG and GTG, plus stop codons.

Compare host promoters such as p lac, p tac, p bad, and rhamnose, with phage promoters like t7 and t3, highlighting strength and regulation.

Examine pbad arabinose promoter and the rhamnose-based pbad hybrid, detailing how arabinose binding and low glucose activate transcription via the cap cap complex, with tight versus basal regulation shaping expression.

Explore phage promoters like T7, T5, and PL, including T7's very high expression requiring T7 RNA polymerase, T5's use of host RNA polymerase, and PL's temperature-controlled expression via CI repressor.

Demonstrate a two-step system by combining T7 promoter with lac or pbad promoter on a plasmid, inducing T7 polymerase via IPTG or arabinose to drive high-level expression.

Learn how replication origin in plasmids determines copy number, with pUC from the University of California yielding the highest copies, about 500–700 per bacterial cell, unlike pac1 plasmids.

Explore co-expression from two plasmids in the same bacterial cell, producing two different proteins, and its use in yeast two-hybrid systems to study protein-protein interactions with bait and prey.

Analyze transcriptional and translational expression vectors, with promoters, operators, terminators, ribosome binding sites, start codons, restriction sites, ligation, PCR, and directional cloning.

Explore the pros of bacterial expression systems—fast growth, cheap media, and simple host biology. Understand cons like codon bias, limited post-translational modifications, and endotoxin risks requiring careful purification.

Explore the goals of expression: maximize soluble, properly folded protein while preserving cell viability. Adjust inducer level, timing, and temperature to reduce aggregation and aid purification.

Explore expression and purification of fusion proteins using his-tag and GST-tag strategies, including affinity chromatography with Ni-NTA, protease cleavage, IPTG induction, and MCS placement.

Learn how pull-down assays use GST and GSH to capture protein A and its partner B, then elute, run SDS-PAGE, and perform sequencing, while reviewing signal peptides for secretion.

Topoisomerase drives topo cloning by nicking and relaxing DNA to enable ligation without a separate ligase, using A overhangs and strand invasion for directional cloning.

Explore a range of Escherichia coli expression strains, including BL21 variants with T7 polymerase, arabinose induction, and basal expression control to optimize protein expression.

Explore the uses of GFP, or green fluorescent protein, to label proteins, visualize localization in vivo, and design biosensors like the arsenite biosensor in bacteria, plus examples of transgenic animals.

Extract jellyfish RNA, synthesize cDNA, amplify GFP by PCR with restriction primers, assemble recombinant plasmids by digestion and ligation, transform bacteria, induce expression, and observe fluorescence.

Extract total RNA from jellyfish with Trizol, containing guanidinium thiocyanate and phenol, perform di phasic separation with chloroform, precipitate with isopropanol, wash with ethanol, and elute in a T buffer.

Quantify RNA quality and concentration using a Nanodrop spectrophotometer, evaluating 260/280 and 260/230 ratios to ensure purity and guide next steps.

Learn to synthesize cDNA from RNA using a poly T primer binding to poly A tail and reverse transcriptase, perform first- and second-strand synthesis, nick translation, and end-blunting with ligase.

Navigate to the NCBI nucleotide database, locate the GFP coding sequence, and export the CDS in FASTA format to prepare primers for PCR and gene insertion.

Design good primers for PCR using the Pet 22 B+ vector to express GFP, understanding the T7 promoter, lac repressor, IPTG induction, and directional cloning with selected restriction enzymes.

Use Neb cutter to select restriction enzymes that avoid cutting your gene, then design forward and reverse primers with BamH1 and EcoR1 sites, preserving the open reading frame.