Yeast synbio Flashcards
Saccharomyces cerevisiae
- 5-10um diameter
- 12 Mio Bp, 6275 genes, 16 chromosomes
- only 5800 functional genes
- 31% homolog humans
- Chromosomes A-P
Naming of genes in yeast (Saccharomyces cerevisiae) chromosomes
Genes have systematic names: e.g YGL118W
Y = Species (yeast gene)
G= The Chromosome on which gene is located ,
L = Left (L) or right (R)
118 = Sequence number of the gene Gene/ORF on this arm starting at centromere,
W = Whether the coding sequence is on Watson/Crick strand
e.g: YBR134C –> right arm of chromosome 2 and is the 134. ORF on that arm starting from the CEN. The coding sequence is on the Crick strand of the DNA.
The Watson strand of DNA is the strand that has the 5´end at the short-arm telomere
The Crick strand of DNA is the strand that has its 5´end at the long-arm telomere
Why is yeast (Saccharomyces cerevisiae) a good model organism? Key features?
- Single-cell organism
- Short generation time (doubling time 1.25-2 h at 30°C)
- Easy cultivation - multiple speciemens can be cultured in parallel at low cost
- Easy manipulation add or delete genes via homolgous recombination
- can grow as a haploid which simplifies the creation of gene knockout strains. (Two form of yeast cells exist: haploid and diploid)
- Mean replicative lifespan is about 26 cell divisions
- All strains can grows aerobically on glucose, maltose and trehalose and fail to grow on lactose and cellbiose
- can grow under anaerobic conditions on galactose and fructose –> produces ethanol (wine making)
- Can use Ammonia and Urea as the sole nitrogen source –> can not use nitrate due to alck of ability to reduce nitrate to ammonium ions
- can use most amino acids and small peptide as N source
- No protease excretion
What is the the Synthetic Yeast Genome Project = Sc2.0
Yeast can be easily genetic modified
Simple process - cell as chassis:
* Original cell –> delete all gene you do not need to grow under certain conditions
* Chassis cell (Cell that has modifed properties)
* Add new genes (syntheic or from other organism) to chassis cell
* New optimized cell for medical use or biotechnology
Dream: yeast strain with all 16 chromosomes re-synthesized (not there yet)
The Synthetic Yeast Genome Project = SC2.0
Aim:
to establish a designer, customizable, synthetic genome from scratch that is more stable than the WT
Remove:
All Transposons, repetitive elements and many introns
Change
All UAG Stop-Codons are changed to UAA
(removement of 1 stop-codon, out of 3)
tRNA genes are moved to a novel neochromosome
- Integration of PCRTags into ORF, by recoding 20 nucleotides of the coding sequence of an ORF, without altering the amino acid sequence
- PCR primers specific to the yeast having the new sequence can be designed. Proteins are not new but sequence is new.
How to build a synthetic yeast genome?
In yeast we have the entire sequence of all chromosomes.
Every single nucleotide is known and in data bases
- Single strand DNA
–> Oligonucleotides (80nt long) - Assemble Double strand DNA
–> assemble oligonucleotides to longer builing blocks (they have overlapping sequences = sequence complementary) (750 nt long) - Ligation
–> into bigger chunks (10kb long) - Chunks assembled into super chunks ( 30kb long)
- Super chunk –> make them bigger to a chromosme arm
- 2 Chromome arm + centromere
super chunks have a part of sequence that is identical to wild type, a marker gene LEU2 and a modified sequence.
–> By introducing super chunk into yeast cell, the yeast perfome homologues recmobination between identicall sequences. At the end the super chunk will be inserted into chromose.
Modified chromosome has modiefied sequence, LEU 2 and wt sequence.
Take new super chunk and repeat steps until you resynthesized complete chromosome.
What are Neochromosomes?
A neochromomse is a chromomse normally not present in nature.
Yeast has 16 chromosomes - if you add a 17th chromosome it is the neochromosome.
Also called “supernummary chromosome”
Very little is currently knwon about:
* the stability of neochromomes
* requirements of their designe
* the physiology of the cell carrying a neochromosome
