Genomics 7-8

Question 1

What are the 4 types of recombinant technology ?

Answer 1

Type 2 restriction enzyme cloning
Polymerase Cycling assembly
Transformation associated recombination
Laboratory Automation

Question 2

What was the first full synthetic genome assembled from and how was it assembled ?

Answer 2

Oligonucleotides

PCA>PCR>Ligation>Transformation in e.coli

Question 3

What is good about the gibson master mix ?

Answer 3

All enzymes work at 55 degrees

Question 4

What is beneficial about the reuse of stop codons ?

Answer 4

Gives a free codon which allows unnatural amino acid incorporation at 100% efficiency.
Can be used against bacteriophage infection
Protection against HGT

Question 5

What does genome refactoring allow ? And how does it do it ?

Answer 5

Makes organisms easier to engineer
Removes: overlapping genes, specific RE sites, Destabilising elements

Adds: DNA barcodes, Specific recombination sites and safe harbours

Question 6

What method was used to designed Sc2.0 ? And what was necessary to allow this method ?

Answer 6

Swap-in method

Recode restriction endonucleases

Question 7

What was deleted in the design of the Sc2.0 ?

Answer 7

Repeats
tRNAs placed on neochromosome
Remove all introns apart from one

Question 8

How are megachunks 30-60kb assembled ?

Answer 8

By transformation associated assembly

Question 9

Why are tRNAs placed on the neochromosome ?

Answer 9

They are transposition hotspots and major areas of DNA rearrangement

Question 10

How does SCRAMBLE provide an alternative route to minimise the genome ?

Answer 10

Puts LoxPsym (Recombinase sites) sites into the synthetic chromosome allowing the genome to be shuffled at will.

Question 11

What protein is put into the plasmid or genome on a promoter that is only active in mitosis ?

Answer 11

Cre protein stuck to estradiol binding domain.

Question 12

What does the pangeonome contain ?

Answer 12

The full complement of genes in a species including all strains

Question 13

What does the core genome contain ?

Answer 13

All the genes found in all strains (shared genes)

Question 14

What does the accessory genome contain ?

Answer 14

Genes present in one or more strain

Question 15

What does the extended genome contain ?

Answer 15

Includes genes present in atleast 99% of the sampled genome.

Question 16

What are whole genomes compared for ?

Answer 16

To study:
Biological similarities and differences
Evolutionary relationships

Question 17

How are novel species characterised ?

Answer 17

Through polyphasic taxonomy

Question 18

List 5 parameters polyphasic taxonomy takes into account ?

Answer 18

Phenotypic features 
Morphology 
physiology 
biochemistry
Chemotaxonomic markers

Question 19

What are the limitations of the pilot project ?

Answer 19

Number of sequenced genomes was biased towards genomes with medical and biotechnological purpose

Many of the genomes did not include 16s rRNA sequence

Question 20

What is the aim of GEBA

Answer 20

To sequence 1 genome of atleast 1 representative strain of every bacterial and archeae species that has a valid published name

Question 21

What digital tool does GEBA use instead of wet labs DDH ?

Answer 21

ANI: average of nucleotide sequence identity

95% ani = 70% DDH

Question 22

What are the benefits of GEBA ?

Answer 22

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

Question 23

Describe the primers used in PCA and what type of primer produces the final construct ?

Answer 23

ssDNA 50bp primers with a 20 bp overhang

Terminal primer

Question 24

What enzyme fills in the gaps in PCA

Answer 24

DNA polymerase

Question 25

What are e.cole and s.cerevisiae able to do if DNA is inserted via electroporation ?

Answer 25

Homology dependent repair of DNA (transformed associated recombination)

Question 26

How did nichols aid our understanding of the E.coli phenome ?

Answer 26

Used the keio strain of e.coli and put them under 113 different stresses.

Question 27

How did nichols measure the fitness of the keio strain and what type of plate was used ?

Answer 27

High density agar plate

Used a camera to measure growth rate and turned that into a fitness score.