Protein Expression + Engineering Flashcards
What is the promoter
What is an operator
What is the RBS
What is the ATG site
What is the antibiotic resistance gene for in plasmids
What are the different genetic elements involved in expression of a plasmid encoded protein
How does the lac operon work
What are the 4 phases of bacterial growth
Lag phase
Exponential phase
Stationary phase
Decline/ death phase
What is the lag phase
What is the exponential phase
What is the stationary phase of bacterial growth
When is the optimal time for expressing a protein of interest
What are the limitations to recombinant protein expression + solution
What is auto induction
Why can expression of recombinant proteins be toxic to transformed bacteria
How can the problem of expression of recombinant proteins being toxic to the transformed bacteria be solved
Give some examples of E. coli strains and their key features
What are the steps of cloning
How does traditional cloning work
What does ligand independent cloning do
What is the process of ligand independent cloning
How does gateway technology cloning work
What is a fusion protein
What are commonly used tags in cloning
Explain this E. coli expression vector
How are forward primers designed
How are reverse primers designed
How can you isolate the tagged protein
When using a tagged protein, how can you remove the tag
Why are bacteria expression systems not suitable for many proteins
What are the different types of expression systems
What factors should you consider when choosing an expression system
Why are the characteristics of yeast expression systems
What is the process of using a yeast expression system
What is the disadvantages of using a yeast expression system
What are the characteristics of using a saccharomyces cerevisiae expression system
What are the issues with working with yeast expression systems + yeast cell wall structure
What is a yeast cell disruptor
What is an insect cell expression systems characteristics
What is the process of using an insect cell expression system
What is a titer assay
What is a shuttle vector
What at the different types of mammalian cell strains
What at the different ways to transfect mammalian cells
What is stable transfection
What is cell free expression
What are the advantages and disadvantages of bacteria expression systems
PTMS= post translational modifications
What are the advantages and disadvantages of yeast expression systems
What are the advantages and disadvantages of insect cell expression systems
What are the advantages and disadvantages of mammalian expression systems
What are the advantages and disadvantages of cell free expression systems
What are the purposes of protein engineering
What is the most common approach for mutagenesis
What is alanine scanning
Test function of protein to see the impact of the mutation using a functional assay
What is random mutagenesis
How is plasmid based mutagenesis done
How are overlap extension methods done
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.
What can alanine scanning be used for
What is chemical mutagenesis
How can PCR be used for random mutagenesis
How can plasmids in E. coli be used for random mutagenesis
How can DNA shuffling be used for random mutagenesis
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.
Why is random mutagenesis an iterative process
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
How can you screen for variants when doing mutagenesis
What is iterative saturation mutagenesis
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
