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Chlamydomonas BAC vectörlerinin RED/ET yöntemiyle tek basamakta modifikasyonu

Year 2019, Volume: 32 Issue: 1, 49 - 55, 01.04.2019
https://doi.org/10.29136/mediterranean.508032

Abstract

Tüm genom
sekansının tamamlanması üzerine,
Chlamydomonas
BAC’ları fonksiyonel genomik analizlerinde yaygın bir şekilde
kullanılmaktadır. Fakat bu vektörlerin hazırlanmalarında kullanılan aşağıdaki
sebeplerden dolayı bu tip analizlerde kullanımları optimalden daha düşük
olmaktadır: (1) Bu BAC’larda komplementasyon analizlerinde elde edilen
transformantların direk seçimini sağlayan bir gen yoktur. (2) BAC ve bağımsız
bir vektörün ko-transformasyon (birlikte transformasyon) olarak kullanımında
birlikte transformasyon oranı düşüktür. (3) BAC’ların çoğu birden fazla gen
içermektedir, bu da her genin farklı bir vektöre sub-klon (alt klonlama)
yapılmasını gerektirmektedir (bu da restriksiyon enzimlerine bağımlılık
demektir). Bu çalışmada, BAC transformasyonu işlemini kolaylaştırmak için,
RED/ET tekniğini kullanarak
Chlamydomonas
BAC’larına 2 selectable marker cassette (seçilebilir ekspresyon kaseti)
eklenmiştir. Bir ökaryotik ve bir prokaryotic ekpresyon kaseti ligasyon edilmiş
ve BAC’ların iki basamak modifikasyonu yerine, tek basamakta modifiye edilmesi
sağlanmıştır ve bu kasetlerin ekspresyonu kanıtlanmıştır. Böylelikle BAC’ların
Chlamydomonas’da fonksiyonel genomik analizlerinde kullanımları için harcanacak
zaman süresi azalacaktır.

References

  • Aksoy M, Pootakham W, Grossman AR (2014) Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation. The Plant Cell 26(10): 4214–29.
  • Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim JS, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports, 6(30620; doi: 10.1038/srep30620).
  • Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153: 401-412.
  • Blaby IK, Blaby-Haas CE, Tourasse N, Hom EF, Lopez D, Aksoy M, Prochnik S (2014) The Chlamydomonas genome project: a decade on. Trends in Plant Science 19(10): 672–680.
  • Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiology 137: 545-556.
  • Ferenczi A, Pyott DE, Xipnitou A, Molnar A (2017) Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. Proceedings of the National Academy of Sciences 114(51): 13567–72.
  • Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, Greenbaum E, Melis A (2000) Microalgae: a green source of renewable H2. Trends in Biotechnology 18: 506-511.
  • Gonzalez-Ballester D, de Montaigu A, Galvan A, Fernandez E (2005) Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA. Analytical Biochemistry 340: 330-335.
  • Harris E (1989) The Chlamydomonas Sourcebook. 1st edition, Academic Press, San Diego, California.
  • Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP (2014) Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryotic Cell 13(11): 1465–69.
  • Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America, 87: 1228-1232.
  • Li X, Zhang R, Patena W, Gang SS, Blum SR, Ivanova N, Jonikas MC (2016) An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii. The Plant Cell 28: 367–87.
  • Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal 14(7): 441-447.
  • Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard, L. et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245-250.
  • Muyrers JP, Zhang Y, Stewart AF (2001) Techniques: Recombinogenic engineering-new options for cloning and manipulating DNA. Trends in Biochemical Sciences 26(5): 325-331.
  • Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20: 123-128.

One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system

Year 2019, Volume: 32 Issue: 1, 49 - 55, 01.04.2019
https://doi.org/10.29136/mediterranean.508032

Abstract

With the availability of the complete genome, Chlamydomonas BACs are being used extensively in functional genomics analysis. The following aspects of their construction, however, make them less than optimal for some types of analysis. (1) These BACs do not contain a gene to allow direct selection of transformants in complementation analysis. (2) Co-transformation using the BAC and an independent vector with a selectable marker has a low efficiency. (3) Most BACs have more than one gene, necessitating sub-cloning of each gene into a different vector (relying on the use of restriction enzymes). To simplify this process, we modified Chlamydomonas BACs by inserting 2 selectable marker cassettes, using the RED-ET system. We ligated a eukaryotic and a prokaryotic selectable marker cassette and used it in a one-step modification instead of a two-step counter selection protocol and showed the expression of both cassettes. This method will decrease the time needed for use of BACs in functional genomics analysis in Chlamydomonas.




References

  • Aksoy M, Pootakham W, Grossman AR (2014) Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation. The Plant Cell 26(10): 4214–29.
  • Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim JS, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports, 6(30620; doi: 10.1038/srep30620).
  • Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153: 401-412.
  • Blaby IK, Blaby-Haas CE, Tourasse N, Hom EF, Lopez D, Aksoy M, Prochnik S (2014) The Chlamydomonas genome project: a decade on. Trends in Plant Science 19(10): 672–680.
  • Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiology 137: 545-556.
  • Ferenczi A, Pyott DE, Xipnitou A, Molnar A (2017) Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. Proceedings of the National Academy of Sciences 114(51): 13567–72.
  • Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, Greenbaum E, Melis A (2000) Microalgae: a green source of renewable H2. Trends in Biotechnology 18: 506-511.
  • Gonzalez-Ballester D, de Montaigu A, Galvan A, Fernandez E (2005) Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA. Analytical Biochemistry 340: 330-335.
  • Harris E (1989) The Chlamydomonas Sourcebook. 1st edition, Academic Press, San Diego, California.
  • Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP (2014) Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryotic Cell 13(11): 1465–69.
  • Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America, 87: 1228-1232.
  • Li X, Zhang R, Patena W, Gang SS, Blum SR, Ivanova N, Jonikas MC (2016) An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii. The Plant Cell 28: 367–87.
  • Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal 14(7): 441-447.
  • Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard, L. et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245-250.
  • Muyrers JP, Zhang Y, Stewart AF (2001) Techniques: Recombinogenic engineering-new options for cloning and manipulating DNA. Trends in Biochemical Sciences 26(5): 325-331.
  • Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20: 123-128.
There are 16 citations in total.

Details

Primary Language English
Subjects Agricultural Engineering
Journal Section Makaleler
Authors

Münevver Aksoy 0000-0002-0798-5805

Charlene Forest This is me

Publication Date April 1, 2019
Submission Date January 4, 2019
Published in Issue Year 2019 Volume: 32 Issue: 1

Cite

APA Aksoy, M., & Forest, C. (2019). One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences, 32(1), 49-55. https://doi.org/10.29136/mediterranean.508032
AMA Aksoy M, Forest C. One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences. April 2019;32(1):49-55. doi:10.29136/mediterranean.508032
Chicago Aksoy, Münevver, and Charlene Forest. “One Step Modification of Chlamydomonas Reinhardtii BACs Using the RED/ET System”. Mediterranean Agricultural Sciences 32, no. 1 (April 2019): 49-55. https://doi.org/10.29136/mediterranean.508032.
EndNote Aksoy M, Forest C (April 1, 2019) One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences 32 1 49–55.
IEEE M. Aksoy and C. Forest, “One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system”, Mediterranean Agricultural Sciences, vol. 32, no. 1, pp. 49–55, 2019, doi: 10.29136/mediterranean.508032.
ISNAD Aksoy, Münevver - Forest, Charlene. “One Step Modification of Chlamydomonas Reinhardtii BACs Using the RED/ET System”. Mediterranean Agricultural Sciences 32/1 (April 2019), 49-55. https://doi.org/10.29136/mediterranean.508032.
JAMA Aksoy M, Forest C. One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences. 2019;32:49–55.
MLA Aksoy, Münevver and Charlene Forest. “One Step Modification of Chlamydomonas Reinhardtii BACs Using the RED/ET System”. Mediterranean Agricultural Sciences, vol. 32, no. 1, 2019, pp. 49-55, doi:10.29136/mediterranean.508032.
Vancouver Aksoy M, Forest C. One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences. 2019;32(1):49-55.

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