Oligo 3 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 4 and 5.
Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.
pRS313 yeast shuttle vector with Biobrick MCS
A shuttle vector for yeast. The vector is compatible with RFC10 standard and contains an ADH terminator behind the RFC10 cloning site. Ampicilin resistance for bacteria and URA3 gene for yeast selection.
contains an ADH Terminator and restrictions sites for the RFC10 standard only in the multiple cloning site, no restrictions sites for RCF10 enzymes in the backbone.
This part is the cDNA of a mutated calcium ion channel protein.
This part encodes the gene of Calcium release-activated calcium channel protein 1 (Orai1), which is a part of calcium selective ion channel in humans. Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE).Store-operated calcium (Ca2+) entry (SOCE) is activated in response to cellular stimuli that deplete the sarco/endoplasmic reticulum (ER) lumen of Ca2+, a prerequisite event that opens exquisitely Ca2+ selective plasma membrane (PM) channels, augmenting cytosolic Ca2+ levels and refilling ER stores. The principal molecular components of SOCE in many cell types include the ER-inserted stromal interaction molecule-1 (STIM1)3,4 and the Orai1 PM channel subunits5,6,7,8, which physically interact at ER???PM junctions in a functional Ca2+ release-activated Ca2+ (CRAC) channel complex. SOCE through CRAC channels is integral to myriad signalling pathways in electrically excitable and non-excitable cells, the most prominent being immune cells in which inheritable mutations in STIM1 and Orai1 have been linked to devastating immunodeficiency diseases. Therefore, we use this part to help with eliminating Ca2+ in hard water.
Usually, the protein Orai1 works only when the STIM1 channel is activated. However, we select the mutated protein that did not require the presistence of STIM1, which changed the 102th amino acid valine to alanine.
This part comes from the cDNA of mutated Orai1 protein (V102A).
Oligo 2 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 3, 4 and 5.
pRS315 yeast shuttle vector with Biobrick MCS
A Shuttle vector for yeast. The vector is compatible with RFC10 standard and contains an ADH terminator behind the RFC10 cloning site. Ampicilin resistance for bakteria and URA3 gene for yeast selection.
contains an RFC10 compatible multiple cloning site, no restrictions sites for RFC10 in the Backbone. ADH Terminator after MCS.
Coding Sequence of IscA2 pigeon homologue (codon-optimized for humans)
The gene Iron-Sulfur cluster assembly 2 (IscA2) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to add the start codon since this second isoform did not have methionine built in.
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005502393.1), and codon-optimized for humans with IDT's tools.
All Oligos for Tetrahedral Nanostructure
This will be used to build and express our desired functional nanostructure in E. coli that target micro RNA biomarkers. This plasmid encodes all the ssDNA that will make the tetrahedral nanostructure, i.e. Oligos 1, 2, 3, 4 and 5.
This will be use to build our desired nanostructure. This ssDNA contains also split G-quadruplex sequences that allow the oligo itself to function as an DNAzyme under specific conditions.
Plasmid Construction: Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7. Oligo Construction: Nakatsuka, K., Shigeto, H., Kuroda, A., & Funabashi, H. (2015). A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids. Biosensors and Bioelectronics, 74, 222-226.
Oligo 5 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 3 and 4.
Oligo 4 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 3 and 5.
This part contains the WT loxP site, which is recognised by Cre recombinase (Part BBa_K1680007), in forward orientation.
software generated kanamycin aptamer (candidate 2)
Aptamer binding kanamycin created by the MAWS Software from iGEM Team Heidelberg
MAWS software from iGEM Team Heidelberg (created with the pre-alpha version)
For further details on the in silico aptamer generation see:
software generated kanamycin aptamer (candidate 1)
Coding Sequence of IscA1 pigeon homologue (codon-optimized for humans)
Michael Becich, Thomas Lau, Sreyas Misra
The gene Iron-Sulfur cluster assembly 1 (IscA1) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005508102.1), and codon-optimized for humans with IDT's tools.
Ray Paul Laureano-Mercado & Mark Kirollos
This part is the coding sequence for the enzyme melamine deaminase, optimized for usage in E. coli. This enzyme eliminates an amine group from melamine to produce ammonia and ammeline and is able to remove another amine group at a rate 10 times slower than the first reaction to bring the compound to ammelide. The enzyme does not react to a measurable extent with this last compound, so it metabolizes melamine by consecutive and predictable deaminations.
The TriA gene???s codons were modified from the original and optimized for E. coli to a Codon Adaptation Index (CAI) of .76. After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744).
pRS316 yeast shuttle vector with Biobrick MCS
A Shuttle vector for yeast. The vector is compatible with RFC10 standard and contains an ADH terminator behind the RFC10 cloning site. Ampicilin resistance for bacteria and URA3 gene for yeast selection.
Has an RFC10 compatible MCS, with ADH Terminator. No further RFC10 restrictions sites in the backbone.
All Oligos for simplified Tetrahedral Nanostructure
This will be used to build and express our desired functional nanostructure in E. coli that target micro RNA biomarkers, but a simplified 2-strand nanostructure. This plasmid encodes the two ssDNA that will make the nanostructure, i.e. Oligos 1 and 5, from parts BBa_K2054001 and BBa_K2054005 respectively.