MICR 302 Lecture Notes - Lecture 18: Selectable Marker, Telomere, Epitope
1 May 2018
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Lecture 18
SYNTHETIC BIOLOGY
THE NEXT FRONTIER: making yeast genomes
How to Build a Genome:
IDEA: by inserting a knockout in place of normal coding sequence and epitope tag to follow it
then undergo homology recombination to incorporate it we can systematically walk down the
chromosome and replace the native sequence with a new sequence
-practical on a genome-wide scale in yeast in chunks because synthesizing DNA chemically
has become very cheap
1. Selecto for a single cross using strategic markers in the yeast strain (has KanMX before,
add in Leu2)
- if transformation works correctly the strain will be Leu+ and Kanomycin sensitive
2. add in the next chuck at regions of homology upstream of leu2 and use a different counter
selectable marker (Ura3), the strain is now Ura+ and Leu-
3. Use Leu again —>recycling same markers to add in new sequence
4. results in entirely synthetic DNA
the chunks:
-oligo 75 bp piece
-reconstitute and mixed together the fragments (coplementary and overlapping)
next:
-building block —> 10-15 olgios = 750 bp, denature and anneal
-can purchase enzyme that fills in the blanks in vitro
next:
-make many overlapping building blocks and fill in blanks in test tube to generate a
mINICHUNK
next:
-mini chunk is synthesized into chunks in the e coli.
Chromosome 3 (Sc 2.0)
-let statistics come up with combinations of genes with DNA sequences that have features you
wouldn't have known of in the first place
-“evolution on steroids” —> cell undergoes million of years of evolution in HOURS to creat
massive diversity (genetic diversity is the best recourse to look for new function, election
overtime dictates functions)
WHAT THEY DID TO CHROMOSOME 3
1. Telomeres (genome stability
background:
-telomeres are repetitive sequences that can often be the site of recombination and contribute
to the stability of the genome.
find more resources at oneclass.com
find more resources at oneclass.com
WHY:
-they wondered if there was something special about the lengthy sequences of telomeres as
they take up space in the genome.
WHAT THEY DID:
-replaces every telomere and sub-telomere with functional mini-telomere
HOW:
-made the sequence, cut out the telomeres and added in the mini telomeres to see if they
thrived or not
2. tRNA genes
Background:
-tRNA genes are unstable regions of the genome as they are highly transcribed DNA that
remains unpackaged and more open
-tRNAs are hot spots for recombination as they have similar sequences that are scattered all
across the genome
-more translocations near tRNAs near anywhere else
WHAT THEY DID:
-took tRNAs and put them all on the same place
HOW:
- made one circular plasmid with all the tRNAs
3. Retrotransposons
Background:
-viruses and transposons can jump around in a form of mutagenesis which contributes heavily
to darwinian evolution
-trade-off = opportunity to get new functions but a risk of lethal mutations
-yeast have 21 retrotransposons that hop around
-good evidence is that stress prompts this jumping —> chasing functions in stress conditions
WANTED TO KNOW:
-do yeasts need transposons to be fit?
-may be more genetically stable without or vice versa
4. TAG codon
Background:
-3 different stop codons are present in the yeast genome
WHAT THEY DID
-decided to free them up and make a new codon
HOW:
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Practical on a genome-wide scale in yeast in chunks because synthesizing dna chemically has become very cheap: selecto for a single cross using strategic markers in the yeast strain (has kanmx before, add in leu2) 4. results in entirely synthetic dna the chunks: Reconstitute and mixed together the fragments (coplementary and overlapping) next: Building block > 10-15 olgios = 750 bp, denature and anneal. Can purchase enzyme that lls in the blanks in vitro next: Make many overlapping building blocks and ll in blanks in test tube to generate a minichunk next: Mini chunk is synthesized into chunks in the e coli. Let statistics come up with combinations of genes with dna sequences that have features you wouldn"t have known of in the rst place. Evolution on steroids > cell undergoes million of years of evolution in hours to creat massive diversity (genetic diversity is the best recourse to look for new function, election overtime dictates functions)
