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Karas BJ, Molparia B, Jablanovic J, et al. Assembly of eukaryotic algal chromosomes in yeast. J Biol Eng. 2013;7(1):30doi: 10.1186/1754-1611-7-30.
Karas, B. J., Molparia, B., Jablanovic, J., Hermann, W. J., Lin, Y. C., Dupont, C. L., Tagwerker, C., Yonemoto, I. T., Noskov, V. N., Chuang, R. Y., Allen, A. E., Glass, J. I., Hutchison, C. A., Smith, H. O., Venter, J. C., & Weyman, P. D. (2013). Assembly of eukaryotic algal chromosomes in yeast. Journal of biological engineering, 7(1), 30. https://doi.org/10.1186/1754-1611-7-30
Karas, Bogumil J, et al. "Assembly of eukaryotic algal chromosomes in yeast." Journal of biological engineering vol. 7,1 (2013): 30. doi: https://doi.org/10.1186/1754-1611-7-30
Karas BJ, Molparia B, Jablanovic J, Hermann WJ, Lin YC, Dupont CL, Tagwerker C, Yonemoto IT, Noskov VN, Chuang RY, Allen AE, Glass JI, Hutchison CA, Smith HO, Venter JC, Weyman PD. Assembly of eukaryotic algal chromosomes in yeast. J Biol Eng. 2013 Dec 10;7(1):30. doi: 10.1186/1754-1611-7-30. PMID: 24325901; PMCID: PMC4029449.
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J Biol Eng. 2013 Dec 10;7(1):30. doi: 10.1186/1754-1611-7-30.

Assembly of eukaryotic algal chromosomes in yeast.

Journal of biological engineering

Bogumil J Karas, Bhuvan Molparia, Jelena Jablanovic, Wolfgang J Hermann, Ying-Chi Lin, Christopher L Dupont, Christian Tagwerker, Isaac T Yonemoto, Vladimir N Noskov, Ray-Yuan Chuang, Andrew E Allen, John I Glass, Clyde A Hutchison, Hamilton O Smith, J Craig Venter, Philip D Weyman

Affiliations

  1. Department of Synthetic Biology and Bioenergy, J, Craig Venter Institute, 10355 Science Center Dr,, San Diego, CA 92121, USA. [email protected].

PMID: 24325901 PMCID: PMC4029449 DOI: 10.1186/1754-1611-7-30

Abstract

BACKGROUND: Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (> ~ 150 kb), high G + C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G + C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G + C content. The model diatom Phaeodactylum tricornutum has an average G + C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast.

RESULTS: We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency.

CONCLUSIONS: Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G + C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.

References

  1. Nucleic Acids Res. 2012 Nov 1;40(20):10375-83 - PubMed
  2. Science. 2010 Jul 2;329(5987):52-6 - PubMed
  3. Nucleic Acids Res. 2010 May;38(8):2570-6 - PubMed
  4. Appl Microbiol Biotechnol. 2009 Feb;82(2):195-201 - PubMed
  5. ACS Synth Biol. 2012 Jul 20;1(7):267-73 - PubMed
  6. Methods Mol Biol. 2006;313:107-20 - PubMed
  7. PLoS One. 2011 Jan 21;6(1):e16214 - PubMed
  8. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):491-6 - PubMed
  9. Mar Biotechnol (NY). 1999 May;1(3):239-251 - PubMed
  10. Nucleic Acids Res. 2010 May;38(8):2558-69 - PubMed
  11. Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15971-6 - PubMed
  12. Methods Mol Biol. 2012;852:133-50 - PubMed
  13. Nucleic Acids Res. 2012 Mar;40(6):2782-92 - PubMed
  14. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4123-7 - PubMed
  15. Proc Natl Acad Sci U S A. 2007 May 1;104(18):7705-10 - PubMed
  16. Biotechniques. 2011 Nov;51(5):335-6, 338 - PubMed
  17. Mol Gen Genet. 1984;197(2):345-6 - PubMed
  18. Methods Mol Biol. 2004;255:69-89 - PubMed
  19. Gene Ther. 1999 Sep;6(9):1634-7 - PubMed
  20. Genomics. 1998 Aug 15;52(1):1-8 - PubMed
  21. Mol Biol Evol. 2005 May;22(5):1260-72 - PubMed
  22. Curr Protoc Mol Biol. 2011 Apr;Chapter 3:Unit3.22 - PubMed
  23. Nat Methods. 2010 Nov;7(11):901-3 - PubMed
  24. ACS Synth Biol. 2012 Jan 20;1(1):22-8 - PubMed
  25. Nature. 2011 Sep 14;477(7365):471-6 - PubMed
  26. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4256-60 - PubMed
  27. Nat Methods. 2013 May;10(5):410-2 - PubMed
  28. Plant Signal Behav. 2008 Feb;3(2):140-1 - PubMed
  29. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4559-63 - PubMed
  30. Nature. 2008 Nov 13;456(7219):239-44 - PubMed
  31. J Bacteriol. 2012 Dec;194(24):7007 - PubMed
  32. Plant Physiol. 2005 Feb;137(2):500-13 - PubMed
  33. Biotechnol Biofuels. 2012 Jun 06;5(1):40 - PubMed
  34. Nat Rev Genet. 2006 Oct;7(10):805-12 - PubMed
  35. Bioeng Bugs. 2012 May-Jun;3(3):168-71 - PubMed
  36. Mol Gen Genet. 1996 Oct 16;252(5):572-9 - PubMed
  37. Nucleic Acids Res. 1997 Oct 1;25(19):3959-61 - PubMed

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