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| == The big picture == | | == The big picture == |
| * We can now synthesize relatively long stretches of DNA
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| * There are techniques for large-scale re-arrangement of existing chromosomes
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| * This makes it possible to consider synthesizing chromosomes from scratch, or building new chromosomes by rearranging existing ones. For example, chromosomes I, III and VI in S. cerevisiae are < 350kb long, and are candidates for being synthesized from scratch.
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| This leads to the $64,000 question: ''If you were to rebuild a yeast chromosome, what changes would you make to it ?'' or ''If you were to build a new yeast chromosome, what would you put on it ?''
| | == Initial ideas == |
| | | * Build a self assembling and degradable scaffold |
| If you're interested in understanding large-scale chromosome structure and its effects, there are (at least) a couple of different overarching goals that can drive the answer to this question:
| | ** Self assembly would get rid of the use of extraneous material to form proper bonds. |
| | | ** If the material is degrabale, it can degrade leaving the scaffold behind. |
| * The ''Science'' goal: Investigate how chromosomes are currently organized, and the importance of various elements of their organization. | | * Find coating to cover degradable scaffold that can select for viruses or cells that excrete scaffold material (collagen, calcuim bicarbonate etc.) |
| * The ''Engineering'' goal: Investigate how to build a chromosome with a particular set of capabilities independent of the actual genes on the chromosome, like a low overall recombination rate. | | * Find coating to bind proteins needed to assemble outer scaffold layer. |
| | | ** Coating must not alter assembly or binding of scaffold |
| At the moment, I'm leaning towards having an engineering goal.
| | ** Coating must be very selective for protein or cells needed to form outer non-degradable layer of scaffolding. |
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| == Initial ideas ==
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| === "Science" ideas === | | === "Science" ideas === |
| * Build rearranged/jumbled chromosomes: preserve functional elements (eg gene + associated promoter), but change gene order, orientation & strand. Could be done via some combination of the fragmentation, rearrangement and fusion techniques described in the papers below. Profile gene expression, histone location, replication origin activity etc, and use the data as input to data mining algorithm that tries to find correlations between various aspects of chromosome structure and whatever was profiled. Idea is that re-arranged chromosomes give you a larger data set to mine and allows you to make stronger conclusions than data from just the WT chromosomes.
| | === "Engineering" ideas === |
| ** Generating specific rearrangements: there is an algorithm for calculating minimal sequence of inversions, translocations etc needed to transform one permutation (ie ordering) of genes into another, by [http://www.cse.ucsd.edu/groups/bioinformatics/GRIMM/ Pavel Pevzner's group at UCSD]. Extending this algorithm to take into account practical issues that would arise when trying to rearrange a chromosome, like having to make sure that you don't remove any essential genes during a rearrangement step, might be an interesting engineering problem.
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| * Disrupt all occurrences of TF binding sequences that occur in coding sequence (by disrupting the motif but keeping the same amino acid sequence) and then profile gene expression patterns. Would help to determine whether in-gene binding sites are biologically relevant, eg by acting as “titrating” sites (along TK’s theory).
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| ** Specifically: pick a transcription factor that has a well-known, unique overexpression phenotype and disrupt all of its intragenic binding sites. If these binding sites acted to titrate the TF away from the “real” binding sites, then you should see the same phenotype as when the TF is overexpressed
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| * Remove all “inert” DNA ie non-coding, not promoters etc; see whether yeast is still alive.
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| * Remove all ORFs of unknown/duplicated function, see whether yeast is alive or not.
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| * Remove all introns
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| ** Not sure what this would really tell us. Would it make yeast easier to manipulate ?
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| * Change all codon usage to be “optimal” (if there are some non-optimal codons) & see whether fitness (by some measure of fitness) improves
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| ** Problem is that you’d (probably) have to do this across all chromosomes, not just a single chromosome, in order to see an effect on fitness
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| * Put all genes in pheromone response pathway on their own chromosome & remove the endogenous copies, to test current model of pathway
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| ** Problem is that there are >50 genes involved in the pathway and removing all the endogenous copies would be a lot of work, and it's not even clear whether the yeast would still be alive.
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| ** But, might be able to use an orthogonal transcription factor (leaving native copies under Ste12 control, in a Ste12 del strain)
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| * Rebuild chromosome by moving promoters + ORFs associated with recombination hotspots around, re-profile recombination hotspots and see whether they’ve moved with the promoters/ORFs.
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| * Engineer photosynthesis into yeasts.
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| ** Not clear what the point of doing so would be, other than “because we can”.
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| * Explore yeast mating-type switching, since all genes involved are on chromosome III
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| ** Not appealing because lots of experiments with chromosome architecture and location of the loci have already been done; also, behavior doesn’t seem to be sequence-specific, with exception of the RE element. See also [http://www.pnas.org/cgi/reprint/101/52/18069 Galgoczy et al], which seems like a pretty thorough, low-level dissection of mating-type switching.
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| === "Engineering" ideas ===
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| Design chromosome that:
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| * Is resistant to disruption by Ty1: try to design “Super Ty1” transposon (similar to what [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15152256&itool=iconabstr&query_hl=11 Han and Boeke] did with human LINE1 transposons) that disrupts WT chromosomes a lot and then design chromosomes that are resistant to being invaded by this Super Ty1.
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| * Undergoes meiotic recombination only rarely
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| * Gets replicated very quickly/slowly; not sure why you'd want that, though, other than as a way of being able to shorten/lengthen S phase.
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| * Has custom chromatin structure eg
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| ** doesn’t have any closed regions of chromatin
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| ** has chromatin structure that varies with, say, cell cycle
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| ** has uniform chromatin structure, so that differences in gene expression are determined only by promoter sequence & levels of TF and thus (in theory, at least) easier to model.
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| ** has nucleosomes made up only of custom histones that don’t respond in a standard way to the usual acetylation/methylation events, or have a custom histone code to allow extended programming of chromatin structure.
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| ** ''Caveat: unclear to what extent you can really control chromatin structure via DNA sequence.''
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| === Papers === | | === Papers === |
| * Genome organization
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| **[http://compbiol.plosjournals.org/archive/1553-7358/2/1/pdf/10.1371_journal.pcbi.0020002-L.pdf "Long-range periodic patterns in Microbial Genomes Indicate Significant Multi-Scale Chromosomal Organization"]
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| *Paradigms for redesign
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| **"Refactoring bacteriophage T7" Nature/EMBO Molecular Systems Biology (2005).[[Media:Macintosh HD-Users-nkuldell-Desktop-T7redesign MolSysBiol05.pdf | download pdf]]
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| **"Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome" Proc Natl Acad Sci U S A. (2005) 102(44):15971-6.[[Image:Macintosh HD-Users-nkuldell-Desktop-yeast rebuild-twogenomes PNAS05.pdf | download pdf]] [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16236728&query_hl=9&itool=pubmed_docsum]]
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| *Deletion
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| **"Engineering a reduced Escherichia coli genome" Genome Res. (2002) 12(4):640-647 [[Media:Macintosh HD-Users-nkuldell-Desktop-yeast rebuild-ReducedGenome Ec GenomRes02.pdf | download pdf]] [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11932248&query_hl=6&itool=pubmed_docsum]]
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| **"Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome" Mol Microbiol. (2005) 55(1):137-149. [[Image:Macintosh HD-Users-nkuldell-Desktop-yeast rebuild-ReducedGenome Ec MolMicro05.pdf | download pdf]] [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]]
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| ** [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16434698&itool=iconpmc&query_hl=1&itool=pubmed_docsum "A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast", Hirashima et al, Nucleic Acids Res. 2006 Jan 24;34(2):e11.]
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| *Fragmentation
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| **"PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae"Biotechniques (2005) 38(6):909-914. [[Media:Macintosh HD-Users-nkuldell-Desktop-yeast rebuild-ChromSplitting yeast Biot05.pdf |download pdf]] [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]]
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| *Rearrangement
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| **"Effects of reciprocal chromosomal translocations on the fitness of Saccharomyces cerevisiae" EMBO Rep. (2004) 5(4):392-398. [[Media:Macintosh HD-Users-nkuldell-Desktop-yeast rebuild-rearrange yeast EMBORep04.pdf | download pdf]] [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]]
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| **[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12621434&itool=iconabstr&query_hl=34 Engineering evolution to study speciation in yeasts]: The authors rearranged the S.cerevisiae genome to be collinear with that of S.mikatae in order to allow them to study the constraints on mating between these two yeast species.
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| *Fusion
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| **"A method for fusing chromosomes in S.cerevisiae", Journal of Fermentation and Bioengineering, Vol. 83, no. 2, pp. 125-131. 1997.
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| <table border="1">
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| <tr>
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| <th> Transformation Factor
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| </th><th> FY2068 colonies/plate
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| </th></tr>
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| <tr>
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| <td> PRS416
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| </td><td> 330
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| </td></tr>
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| <tr>
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| <td> no Temp PCR
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| </td><td> 0
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| <tr>
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| <td> + Temp PCR
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| </td><td> 12
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| </td></tr></table>
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| <br>
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| <br>
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| | === Module 4 tables === |
| {| border="1" | | {| border="1" |
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