Synthetic Genome/ Metabolism Flashcards
What is the history of genome sequencing?
Phi X174 was the first virus genome to be sequenced in 1977
H. Influenzae was the first bacterial genome to be sequenced in 1995
Yeast was the first eukaryote genome sequenced in 1996
Arabidopsis was the first plant genome sequenced in 2000
The Human Genome Project took 15 years and cost $2.7 billion in 2001
MinIon from Nanopore can sequence a human genome in less than a day for less then $1000
Sequencing has become faster, cheaper and more accessible
Describe Phi X174 and what were the challenges in sequencing?
A bacteriophage that invades E. coli
Technical challenge -> being 5-6 kb in size was overcome using gel purification to collect the correct size DNA
Technical challenge -> difficult to assemble large constructs accurately and quickly was overcome using ligation under stringent annealing conditions
Technical challenge -> difficulty avoiding incorrect length oligonucleotides and proofreading was overcome by using assembly PCR
Describe Syn 1.0
1 kbp from M. mycoides synthetic genome was implanted into an M. capricolum cell, which then took on the implanted genome’s phenotype.
This created almost an exact copy of the M. mycoides genome.
This was technically reverse engineering.
Comparative genomics was used to get a hypothetical minimum number of genes necessary for life.
Oligonucleotides were created and annealed into 1.4 kbp dsDNA fragments which were inserted into E. coli plasmid vector (7 kbp in each vector) with 15 vectors then being assembled in yeast to make 1 large plasmid.
The plasmid was then extracted from yeast using rolling circle amplification in vitro. This created 1/8 of the needed genome thus had to be repeated 7 more times.
The genome was then either methylated or placed into a host that didn’t have a restriction system.
The whole process took 3 weeks and ended up not working which showed that we didn’t know enough to build a genome.
Syn 1.0 was planktonic
What were the problems in the methods with creating Syn 1.0?
Methods for transferring genome treatments into M. capricolum
Methods for extracting genome fragments from yeast
Methods for stitching large fragments together (Gibson assembly)
How was it decided what improvements to make to Syn 1.0?
Tn5 puromycin resistance transposon mutagenesis screen was performed on Syn 1.0 and 80,000 colonies were pooled together
PCR and sequencing was used to identify where in the genome the mutations were and were categorised as follows;
Genes with no mutations -> essential
Genes with frequent hits -> non-essential
Genes with hits but growth impairments -> quasi-essential
However paralog genes also exist with the same function.
Describe Syn 2.0
Analysis identified 26 genes to add back in to the genome after Tn5 puromycin resistance transposon mutagenesis screen to create 2.0 which is considered to be the first bacteria with a functional reduced genome
Describe Syn 3.0
Further analysis identified 42 genes for removal using Tn5 puromycin resistance transposon mutagenesis screening.
3.0 has 438 protein coding genes and 35 RNA coding genes, 79 of which cannot be assigned a function
Retained all genes required for the synthesis and processing of macromolecules but loss of genes involved in biosynthesis pathways of small molecules. All transport proteins were also retained.
It was found that rearranging the genes by type had no impact on the phenotype or functionality.
Slow growing filamentous mats were created thus is polymorphic to 1.0
Describe Sc 2.0
Yeast S. cerevisae synthetic genome project
~ 6000 genes across 16 chromosomes
Unlike Syn, this approach utilised the natural capacity of yeast for homologous recombination
30-60 kb sections were replaced at a time
Describe HGP 2.0
First HGP was about reading and the second is about writing ie. Whole genome engineering of human cell lines
So much remains unknown after HGP 1.0
Also allows the testing of controversial ideas such as recoding human codon usage to make us virus resistant
There are also many ethical concerns
Describe malaria and artemisinin
Malaria caused half a million deaths a year and A. annua (sweet wormwood) contains both artemisinin and dihydroartemisinin which can be biosynthesised (Tu Youyou Nobel Prize, 2015)
What are the problems with getting artemisinin from sweet wormwood?
Sweet wormwood takes between 6-8 months to harvest
Yields are variable due to weather as drought can affect seedlings and damp conditions can cause lodging
Price varies wildly which is unfair in places where malaria is prevalent
Describe the first iteration of the artemisinin biosynthesis pathway
The synthetic pathway is put into yeast and amplified using fermentation.
The mevalonate pathway begins with acetyl coA and ends with farnesyl pp. The farnesyl pp is converted to amorphadiene using amorphadiene synthase amorphadiene is then converted to artemisinic acid using cytp450 and cytp450 reductase. The artemisinic acid is then transported out of the yeast cell.
The artemisinic acid is then chemically converted to artemisinin.
Describe how the second iteration improved the artemisinin pathway
Increased the carbon flux to artemisinic acid by increasing the cellular activity of the key enzymes and restricting/removing unwanted enzymes (HMGR was x2 in the mevalonate pathway and farnesyl pyrophosphate synthetase was upregulated leading to a decrease in squalane (carbon competition) production.
Pathway was completed in a UPC2-1 semi dominant mutant background
How much products were produced after the 2nd iteration artemisinin biosynthesis?
150 mg/l amorphadiene in shake flasks
100 mg/l artemisinic acid in yeast
Describe the 3rd iteration improvements artemisinin pathway
3x HMGR
Use of strong Gal promoters
CENPK.2 industrial yeast strain which increased the amorphadiene production but not the artemisinic acid production so fermentation conditions were changed
Copper repression of squalane which was cheaper than methionine repression
CPR1 copy number decreased as it positively interacted with p450s
