Team:Georgia State/Composite Part

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Composite Parts

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Composite Parts
Dino III GFP Plasmid	Part:BBa_K3042005
Dino III RFP Plasmid	Part:BBa_K3042006
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

OriV- incP

Origin of replication

nptIII-

Kanamycin resistance gene

TrfA-

Gene for trans-acting replication protein that binds to and activates OriV

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

Primers for PCR

5’-GATGAGTAAAGGAGAAGAAC-3’

5’-TAT TTGTATAGTTCATCCATGCC-3’

Tnos-

Nopaline Synthase Terminator and poly(A) signal

Bar-

Phosphinothricin acetyl transferase

Confers resistance to bialophos or phosphinothricin

Pnos-

Nopaline Synthase Promoter

RB- RB T-DNA repeat

Right border repeat from nopaline C58 T-DNA

Forward Primer:

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