Exam qs Flashcards
What needs to be added to single stranded dna dissolved in water to allow synthesis of complimentary strand
Buffer (to get correct pH for enzyme)
Complimentary dna primer
dNTPs
DNA polymerase
Mutations that could stop a trait being observed when cloned by PCR
Insertions/deletions
Introduction of a stop codon
Synonymous substitution of a base for another (don’t alter amino acid sequences- silent mutation)
Non synonymous codon changes (result in a biological change)
How Illumina gets around ambiguity that arises when reading dna with sequence variations
Where as Sanger aggregates the different fragments, leading to ambiguity when reading at the site of a sequence variation, Illumina reads the sequence of a single molecule at each sequencing reaction.
What’s cDNA
Complimentary DNA: DNA synthesised from a single stranded RNA (eg messenger RNA mRNA)
mRNA that has been reverse transcribed into DNA
What enzyme is essential to making cDNAs
Reverse transcriptase
What is a stop codon
A sequence of DNA that is needed to stop translation or making of proteins by stringing amino acids together.
Are catabolism and anabolism linked?
Yes, by high energy intermediates such as ATP
Equations for the complete catabolism of glucose by yeast in the absence of oxygen
Yeast in absence of oxygen means 100% alcoholic fermentation —> glycolysis (glucose to pyruvate) and pyruvate to ethanol
Overall equation:
Glucose + 2 ADP + 2 P -> 2 Ethanol + 2CO2 + 2 ATP
What is ADP
When you remove a phosphate from ATP you get ADP
What are the variable regions on an antibody
The ends of both the heavy and light chains that determine the antigen specificity of the antibody
What binds the different parts of an antibody together
Disulfide Bridges
Ways the structure of an antibody aids its function.
Two recognition sites aids the formation of antibody- antigen complexes
Disulfide Bridges keep the four peptide chains in a robust structure
Light and heavy combination allows more diversity
Variable regions allow for high antigen specificity
What’s humanising an antibody and how can it be achieved
Needed to prevent the human immune system of an antibody having a negative reaction
By: substituting the rodent complimentary determining/variable regions into a human antibody
Engineering recombinant mice that have human antibody genes
How to avoid designing primers that are likely to suffer from “self-priming”
Avoid designing a pair of primers that match each other well, especially ones that are G/C strong. Can be done by shortening the primers to remove the self priming bases
What is dynamic programming
An algorithmic technique used for sequence alignment
What’s the flux control coefficient
The degree of control that each enzyme exerts on a metabolic pathway flux
The percentage change in the pathway flux when a 1% change in the activity of a pathway enzyme is achieved
What is regulation of metabolic pathways and why is it needed
Regulation of an enzyme in a pathway is how it’s activity is increased and decreased in response to signals
Needed to react to changing external conditions to maintain a constant set of internal conditions, homeostasis
What is allosteric regulation
The regulation of an enzyme by binding an effect or molecule at a site other than the enzyme’s active site
Two types of metabolic regulation
Mass action and Allosteric
Mass action - regulated by the chain speed as a whole
Allosteric - signal is sent back
Factors that can reduce the yield in a metabolic pathway
Side products being formed
Allosteric regulation that inhibits previous steps
What is the elasticity coefficient
A local property indicating how responsive the rate of a reaction is to a fractional change in one of its substrates/products or effectors.
Methods to increase the yield in a metabolic pathway
Optimisation and modulation of pathways
Enzyme engineering
Synthetic protein scaffold
Ways to calculate the yield of a new pathway/effect of a modification without running experiments
Flux balance analysis (FBA)/ Metabolic Flux analysis (MFA) using knowledge of the stoichiometry of the system.
This will give optimal solution so may not be exact to real life.
Examples of proteins that recognise specific DNA sequences
Restriction enzymes
RNA polymerase
DNA recombinase
What does NADH do in metabolism
Carries electrons from one reaction to another
What group does ATP carry in its high energy linkage
Phosphate
What does yeast do in aerobic conditions
Enters TCA cycle
What does yeast produce in anaerobic conditions
Ethanol and/or lactate
What is an operon
A functioning unit of DNA containing a cluster of genes under the control of a single promoter
What enzyme transcribes the information in an operon
RNA polymerase
2 steps in protein synthesis
Transcription and translation
Transcription is where DNA is unwound and RNA polymerase creates a complimentary a single strand of mRNA (remember RNA has U instead of T nucleotides)
Translation is where the mRNA is sent to cytoplasm where it binds with ribosomes, the site of protein synthesis.
Where does RNA polymerase on the operon
The promoter - sequence of DNA where RNA polymerase can bind
What does RNA polymerase make
mRNA which can be used to make a protein
What can bind at the operator position on the operon to stop RNA polymerase transcribing the gene
A Repressor - stops mRNA being made therefore no proteins
What is the purpose of the lac operon
Lac Z,Y,A are the genes in the lac operon and encode for enzymes that break down lactose into sugars so that it can be metabolised by bacteria
What does the lac I gene do in the context of the lac operon
The Lac I gene has its own promoter and encodes for the repressor for the lac operon, in the situation where lactose isn’t present so the enzymes needed to break it down aren’t necessary
What is the RBS on an operon
The ribosome binding site is a sequence of nucleotides upstream of the start codon of an mRNA transcript that recruits the ribosome during the initiation of protein translation.
Factors that can contribute to the failure of a gene to express
Weak promoter: inefficient transcription.
Weak RBS: inefficient translation.
Inefficiency/incompatibility of genetic elements from different organisms
Simple way to increase gene expression of a particular gene of an operon
If you want to produce twice as much of something, have the gene twice on the operon!
