Protein Expression + Engineering Flashcards

Question

Explain this E. coli expression vector

Answer 1

An expression system is a biological setup or combination of elements used to produce a specific protein or gene product within a host organism Components of an expression system: Gene of interest Promoter Host organism eg bacteria Vector Selection marker

Answer 2

A mammalian cell expression system is a type of biological system used to produce proteins within mammalian cells, typically from species like humans, mice, or hamsters.

Answer 3

Transfection is a process used to introduce foreign nucleic acids (DNA or RNA) into eukaryotic cells In transient transfection, the foreign DNA or RNA remains in the cell temporarily

Answer 4

PTMS= post translational modifications

Answer 5

Test function of protein to see the impact of the mutation using a functional assay

Answer 6

Overlap extension is a technique used in molecular biology, particularly in PCR (Polymerase Chain Reaction), to create specific mutations, fuse two DNA sequences, or assemble multiple DNA fragments. This method involves using complementary "overlapping" regions in the DNA fragments to join them through a series of PCR steps Steps: Design Primers with Overlapping Sequences: Two or more DNA fragments are required, each with overlapping regions that are complementary to one another at their ends. Primers are designed to include the overlapping sequences. These primers are used in separate PCR reactions to amplify the individual DNA fragments. The overlapping sequences at the ends of the fragments are designed to match, so they can anneal (hybridize) to each other in the next step. Initial PCR Amplification: The separate DNA fragments are first amplified using conventional PCR. Each fragment is amplified with the specific primers designed to include the overlapping sequences. The result is two or more DNA fragments with complementary ends. Overlap Extension (Fusion) PCR: In the second round of PCR, the two DNA fragments are mixed together without external primers initially. During the denaturation, annealing, and extension cycles, the overlapping complementary regions at the ends of the fragments hybridize to each other. The DNA polymerase extends these overlaps, generating a full-length DNA product that contains the fused DNA fragments. Amplification of the Full-Length Product: After the overlap extension step, external primers (that bind to the non-overlapping ends of the two fragments) are added to amplify the entire fused DNA sequence in a final round of PCR. This results in a single, full-length DNA fragment containing the desired mutations or fused sequences.

Answer 7

Chemical mutagenesis PCR approaches Plasmid in E. coli cells DNA shuffling

Answer 8

The basic idea is to create a library of new, potentially improved variants of a gene by breaking apart the original gene (or a set of related genes), randomly recombining the fragments, and then amplifying the shuffled sequences. This process introduces both random mutations and recombinations that mimic natural evolutionary processes.

Answer 9

Random mutagenesis is an iterative process because it typically requires multiple rounds of mutation, selection, and screening to achieve significant improvements or desired traits in genes or proteins. Each round builds on the previous one, accumulating beneficial mutations while eliminating less favorable ones. Here's why random mutagenesis is done iteratively

Answer 10

ISM focuses on specific amino acid positions (often identified as critical for function) and explores all possible mutations at those positions in an iterative, stepwise manner The first step in ISM is to identify key positions in the protein sequence that are likely to influence its function Saturation mutagenesis is performed at one or more of the identified key positions. In saturation mutagenesis, all 20 possible amino acids are tested at a specific position, allowing researchers to explore how each amino acid substitution affects the protein's function After introducing the mutations, the variants are expressed in a suitable system (e.g., bacterial or mammalian cells), and they are screened or selected for the desired property The best-performing variants are chosen for further rounds of mutagenesis. the process is iterative, meaning that after identifying beneficial mutations at one site, the best variant is used as the starting point for the next round of saturation mutagenesis at another position. The process is repeated, moving through several key positions in a stepwise manner. Each iteration builds on the improvements made in the previous rounds, allowing for the accumulation of beneficial mutations over time