What is the question for the minimal genome project
What is the minimum bumber of protein-coding genes required for life ( at least for bacteria growinf under labratroy conditions)
Which bioinformatic approach was used to search for the minimum number of protein-coding genes by Mushegian and Koonin in 1996?
What was the result?
looked fo all ortholog genes conserved among th 3 complete bacterial genomes available at the time
H. influenzae
M. genitalium
E. coli
256 genes in minimal set
What where the limitation of the estimation from Mushegian & Koonin?
with distantly-related species it is hard to identify orthologs
some functions can be performed by non-orthologous genes
also known as NOD (non orthologous gene displacment)
results will change as new genome sequences becoma available
no ecidence that minimal set alone is sufficient for life
What was the result of COG (Cluster of Orthologous groups) for minimal genome
63 genes are common to all of life
91 genes are common to Archaea and Bacteria
217 genes are common ro Bacteria
Das COG-Prinzip (Der Dreiecks-Test): Ein Gen gehört erst dann zu einem COG, wenn es ein konsistentes Dreieck bildet:
Gen A aus Organismus 1 findet Gen B aus Organismus 2 als besten Treffer.
Gen B findet Gen C aus Organismus 3 als besten Treffer.
Gen C findet wiederum Gen A aus Organismus 1 als besten Treffer.
What was the initial experiment approach to find minmal genome performed by Craig Venter & colleagues in 1999?
Start with closely-related species
Mycoplasma genitalium
smallest genome known (580 kb, 480 protein encoding genes)
M. pneumoniae (816 Kb, 480 M. genitalium orthologs + 197 unique genes = 677 total genes)
Used TE insetioaln mutagenesis to randomly knock out genes in both species -> sequenced DNA, flanking the TE to determine where it inserted & which gene was knocked out
if TE was inserted within first 80% of protein coding region & beyond nucelotide 9 of the coding sequence it was considered a gene disruption (hit)
only cells that survived could be detected -> hit genes must be non-essential
How many hits were found for M.genitalium & M.pneumoniae
M.genitalium : 93
M.pneumoniae: 150 (57 had M.genitalium orthologs, 93 unique)
Is the hit result then the min genome
no
480 - 93 =387 would be an overestimation
the screen was not saturated, some genes were not hit ( just by chance)
How did Craig Venture and collegues estimate then minimal genome?
M. gentialium : 480 genes -> hits 93
M. pneumoniae: 480 + 197 = 677 -> hits 150 (57 +93)
The 197 genes are of course non essentail for M.genitalium but for the hits only 93 were found so we know only 47 % were hit.
In the next step we calculate N with 0.47N =4,793 kp 57 (ortholog hits) and get 121 for N, what is the numver of non-essentail genes
-> 480 - 121 =359 359 essentail genes
the authors estimate this even lower to 265 - 350 genes
for 111 the function is unknown
What is the problem with the minimal genome estimate from Craig
not all 197 M. pneumonia-Specific genes may be non essentail
genes were knocked out individually. What about synthetic lethals? ( two genes can be knocked out individually with no effect but the cell dies if both genes are knocked out together)
Which bacterium did the use for the experminetal approach from Kobayashi in 2003:
Bcillus subtilis - 4100 genes
Explain the experminetal approach from Kobayashi in 2003:
using bacillius subtilis
knocked out genes using targeted, homologous recombiantion approach
combined result with the previous studies
result: 217 (6%total) genes were essential for growth under optimal labroratory conditions, reminaing 3830 (94%) not
What happend to the results of the kobayashi study?
essential genes were classified into functional categories
50 % involved in DNA/RNA metabolism
essential genes were well conserved in other bacterial species (& also Archaea & eucaryotes) -> but not all over all known genomes or even all known bacterial genomes
-> these genes would not show up for bioinformatics approaches
How many essential genes found by Kobayashi were conserved
30 % conserved across 54 bacterial
20% conserved across 18 Archaea & Eukaryotic
Limitation of Kobayashis work
genes were knocked out individually
snythethic lethals
can organsim with only 271 genes be engineered?
Is the number of essential genes correlated with total number of genes in the genome?
-> negative correlation between percentage of essentail genes & the total number of genes in genome
few genes -> high proportion of essential
less genes -> los proportion
Is it possible to transplant entire bacterial genomes and engineer synthetic life ? (example?)
yes
Approach has been used to make synthetic strain of Mycoplasma that has only 472 genes
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