Genomics 7-8 Flashcards
What are the 4 types of recombinant technology ?
Type 2 restriction enzyme cloning
Polymerase Cycling assembly
Transformation associated recombination
Laboratory Automation
What was the first full synthetic genome assembled from and how was it assembled ?
Oligonucleotides
PCA>PCR>Ligation>Transformation in e.coli
What is good about the gibson master mix ?
All enzymes work at 55 degrees
What is beneficial about the reuse of stop codons ?
Gives a free codon which allows unnatural amino acid incorporation at 100% efficiency.
Can be used against bacteriophage infection
Protection against HGT
What does genome refactoring allow ? And how does it do it ?
Makes organisms easier to engineer
Removes: overlapping genes, specific RE sites, Destabilising elements
Adds: DNA barcodes, Specific recombination sites and safe harbours
What method was used to designed Sc2.0 ? And what was necessary to allow this method ?
Swap-in method
Recode restriction endonucleases
What was deleted in the design of the Sc2.0 ?
Repeats
tRNAs placed on neochromosome
Remove all introns apart from one
How are megachunks 30-60kb assembled ?
By transformation associated assembly
Why are tRNAs placed on the neochromosome ?
They are transposition hotspots and major areas of DNA rearrangement
How does SCRAMBLE provide an alternative route to minimise the genome ?
Puts LoxPsym (Recombinase sites) sites into the synthetic chromosome allowing the genome to be shuffled at will.
What protein is put into the plasmid or genome on a promoter that is only active in mitosis ?
Cre protein stuck to estradiol binding domain.
What does the pangeonome contain ?
The full complement of genes in a species including all strains
What does the core genome contain ?
All the genes found in all strains (shared genes)
What does the accessory genome contain ?
Genes present in one or more strain
What does the extended genome contain ?
Includes genes present in atleast 99% of the sampled genome.
What are whole genomes compared for ?
To study:
Biological similarities and differences
Evolutionary relationships
How are novel species characterised ?
Through polyphasic taxonomy
List 5 parameters polyphasic taxonomy takes into account ?
Phenotypic features Morphology physiology biochemistry Chemotaxonomic markers
What are the limitations of the pilot project ?
Number of sequenced genomes was biased towards genomes with medical and biotechnological purpose
Many of the genomes did not include 16s rRNA sequence
What is the aim of GEBA
To sequence 1 genome of atleast 1 representative strain of every bacterial and archeae species that has a valid published name
What digital tool does GEBA use instead of wet labs DDH ?
ANI: average of nucleotide sequence identity
95% ani = 70% DDH
What are the benefits of GEBA ?
Improved identification of protein families and ortholog groups across species allowing for better annotation of other genomes.
Gene discovery
Improve correlations between phenotype and genotype
Describe the primers used in PCA and what type of primer produces the final construct ?
ssDNA 50bp primers with a 20 bp overhang
Terminal primer
What enzyme fills in the gaps in PCA
DNA polymerase
What are e.cole and s.cerevisiae able to do if DNA is inserted via electroporation ?
Homology dependent repair of DNA (transformed associated recombination)
How did nichols aid our understanding of the E.coli phenome ?
Used the keio strain of e.coli and put them under 113 different stresses.
How did nichols measure the fitness of the keio strain and what type of plate was used ?
High density agar plate
Used a camera to measure growth rate and turned that into a fitness score.
