Difference between revisions of "Team:Georgia State/Composite Part"
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<p>Dino III GFP Plasmid<p> | <p>Dino III GFP Plasmid<p> | ||
<p><li><a href="http://parts.igem.org/Part:BBa_K3042005">Part:BBa_K3042005</a></li><p> | <p><li><a href="http://parts.igem.org/Part:BBa_K3042005">Part:BBa_K3042005</a></li><p> | ||
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<p>Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693<p> | <p>Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693<p> | ||
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Revision as of 01:05, 22 October 2019
Dino III RFP Plasmid
pCB302-gfp-MBD plasmid
*This part is not in the registry*
pCB302-gfp-MBD Plasmid
Our predicted sequence created in SnapGene
The actual plasmid map
pCB302-GFP-MBD Binary Vector
pCB302 is a plant targeted plasmid containing gfp encoding fusions with a microtubule-binding domain (MBD) . pCB302-GFP-MBD Binary Vector is harbored in Agrobacterium tumefaciens to evaluate the heterologous expression of the photosynthetic dinoflagellate, Symbiodinium mircoadriaticum. The plasmid was derived from Dr. Boris Voigt from The Institute for Cellular and Molecular Botany, University of Bonn, Germany who donated it to the Instituto de Biotecnología, UNAM (Cuernavaca, Morelos, México) who then donated it to iGEM GSU. After pCB302-GFP-MBD in Agrobacterium tumefaciens was introduced into the Symbiodinium mircoadriaticum, the cells lost their photosynthetic pigments and were left unable to reproduce. A successful transformation of the Symbiodinium mircoadriaticum, done by UNAM, was measured by GFP expression frequency measured as green fluorescence emission yielding 640 per every 1,000,000 cells. Despite losing photosynthetic ability, pCB302-GFP-MBD Binary Vector is the first step to a stable Symbiodinium mircoadriaticum transformation (Ortiz-Matamoros, 2015).
- LB- LB T-DNA repeat
- Left border repeat from nopaline C58 T-DNA
- TGGCAGGATATATTGTGGTGTAAAC
- OriV- incP
- Origin of replication
- AGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGCCCGGTGAGCGTC
- nptIII-
- Kanamycin resistance gene
- CTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCAT
- TrfA-
- Gene for trans-acting replication protein that binds to and activates OriV
- CTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGGCGTGTTCCACCAGGCCGCTGCCTCGCAACTCTTCGCAGGCTTCGCCGACCTGCTCGCGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTCCAGCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATAGTCGAACATCCGTCGGGCCGTCGGCGACAGCTTGCGGTACTTCTCCCATATGAATTTCGTGTAGTGGTCGCCAGCAAACAGCACGACGATTTCCTCGTCGATCAGGACCTGGCAACGGGACGTTTTCTTGCCACGGTCCAGGACGCGGAAGCGGTGCAGCAGCGACACCGATTCCAGGTGCCCAACGCGGTCGGACGTGAAGCCCATCGCCGTCGCCTGTAGGCGCGACAGGCATTCCTCGGCCTTCGTGTAATACCGGCCATTGATCGACCAGCCCAGGTCCTGGCAAAGCTCGTAGAACGTGAAGGTGATCGGCTCGCCGATAGGGGTGCGCTTCGCGTACTCCAACACCTGCTGCCACACCAGTTCGTCATCGTCGGCCCGCAGCTCGACGCCGGTGTAGGTGATCTTCACGTCCTTGTTGACGTGGAAAATGACCTTGTTTTGCAGCGCCTCGCGCGGGATTTTCTTGTTGCGCGTGGTGAACAGGGCAGAGCGGGCCGTGTCGTTTGGCATCGCTCGCATCGTGTCCGGCCACGGCGCAATATCGAACAAGGAAAGCTGCATTTCCTTGATCTGCTGCTTCGTGTGTTTCAGCAACGCGGCCTGCTTGGCCTCGCTGACCTGTTTTGCCAGGTCCTCGCCGGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGTGTCGATGGTCATCGACTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAACGCTCCACGGCGGCCGATGGCGCGGGCAGGGCAGGGGGAGCCAGTTGCACGCTGTCGCGCTCGATCTTGGCCGTAGCTTGCTGGACCATCGAGCCGACGGACTGGAAGGTTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCGGAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATTCAT
- MBD- Microtubule binding domain
- of the mammalian microtubule-associated protein 4 (MAP4) gene
- GFP- Green fluorescent protein;Plant optimized
- Reporter gene
- Binds to microtubules in vitro when fused to MBD
- ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAG TAA
- Primers for PCR
- 5’-GATGAGTAAAGGAGAAGAAC-3’
- 5’-TAT TTGTATAGTTCATCCATGCC-3’
- Tnos-
- Nopaline Synthase Terminator and poly(A) signal
- GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC
- Bar-
- Phosphinothricin acetyl transferase
- Confers resistance to bialophos or phosphinothricin
- ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATCTGA
- Pnos-
- Nopaline Synthase Promoter
- GAACCGCAACGATTGAAGGAGCCACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCA
- RB- RB T-DNA repeat
- Right border repeat from nopaline C58 T-DNA
- GTTTACCCGCCAATATATCCTGTCA
- Forward Primer:
- WDF1: 5’-GCGGATCCATGGCCGAGGGACTC-3’
- Reverse primers:
- 5’-GCACTAGTGTAACGACCAATACCCCA-3’
Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693