Difference between revisions of "Team:Kent/Results"

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<p>With the aim to investigate and track mRNA localization in vivo, we engineered a gene construct
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containing a GFP attached via a linker sequence, allowing conformational flexibility, to a dCas13a coding
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1Rose, J. (2011). GFP with Standard 25 Prefix/Suffix. [WWW Document]. URL http://parts.igem.org/Part:BBa_K648013
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gene. Additionally, two Nuclear localization sequences are on either side of the construct, which
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navigate the fusion protein back to the cell nucleus after protein synthesis in the ER is completed. In the
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2Addgene (2016). p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A. [WWW Document]. URL http://www.addgene.org/83485/
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nucleus, the protein construct recognizes the CRISPR incorporated sequence and attaches to it. When
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the targeted mRNA is consequently exported, the GFP+dCas13a tags along, allowing the ‘tracking’ of
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3New England Biolabs (2017). Gibson Assembly® Master Mix. [WWW Document]. URL https://www.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
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mRNA up until its translation in the ER. The unbound fusion protein constructs are retained in the
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nucleus and do not localize within the cell cytoplasm, reducing background noise.
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4Agilent genomics (2017). Quikchange. [WWW Document]. URL http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160
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5Thomas P., Smart, T.G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51: 187-200.
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6Avraam Buskila, A.., Kannaiah, S. & Amster-Choder, O. (2014). RNA localization in bacteria. RNA Biology, 11(8): 1051-1060.
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7Pyke, C., et al. (1992). Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization. Cancer Research, 52: 1336-1341.
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8Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D. (1998). RNA localization in development. Annu Rev Biochem, 67: 335-94.  
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Revision as of 04:04, 16 December 2017


Results

Our plan of creating our biobrick was pursued in two separate ways. First we attempted to clone the GFP with Standard 25 Prefix/Suffix from the biobrick library (BBa_K6480131) as well as the dCas13a we obtained from Addgene (L. buccalis C2C2, addgene #834852). We also ordered the whole gene through Integrated DNA Technologies (IDT)

dCAS13a

Cas13a is a RNA guided endonuclease which degrades RNAs based on alignment of its CRISPR derived crRNA. We used dead Cas13a with four mutations, two in HEPN1 and two in the HEPN2 domains, which abort its nuclease activity, but maintain its helicase properties, resulting instead in attachment of dCAS13a to the targeted RNA sequence.

gRNAs

Simultaneously, guide RNAs constructs (for crRNA expression) destined for incorporation into the bacterial genome within CRISP repeats was designed. These constructs contain a U6 promoter followed by coding sequences for human β-Actin, pkp4, inpp-1, and Rab13 (individually) and terminated by an oligo (T) repeat. The protein sequences are chosen based on susceptibility to mutations and expression in disease states.

References
  • 1Rose, J. (2011). GFP with Standard 25 Prefix/Suffix. [WWW Document]. URL http://parts.igem.org/Part:BBa_K648013
  • 2Addgene (2016). p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A. [WWW Document]. URL http://www.addgene.org/83485/
  • 3New England Biolabs (2017). Gibson Assembly® Master Mix. [WWW Document]. URL https://www.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information
  • 4Agilent genomics (2017). Quikchange. [WWW Document]. URL http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange/?cid=AG-PT-175&tabId=AG-PR-1160
  • 5Thomas P., Smart, T.G. (2005). HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51: 187-200.
  • 6Avraam Buskila, A.., Kannaiah, S. & Amster-Choder, O. (2014). RNA localization in bacteria. RNA Biology, 11(8): 1051-1060.
  • 7Pyke, C., et al. (1992). Localization of Messenger RNA for MT 72,000 and 92,000 Type IV Collagenases in Human Skin Cancers by in Situ Hybridization. Cancer Research, 52: 1336-1341.
  • 8Bashirullah, A., Cooperstock, R.L., Lipshitz, H.D. (1998). RNA localization in development. Annu Rev Biochem, 67: 335-94.