Team:Georgia State/Parts

Team:Georgia State - 2016.igem.org

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Team: Georgia State IGEM

PARTS

Basic parts

LTNF10- BBa_K1915000

LTNF10 (Lethal Toxin Neutralizing Factor) is a 10 residue peptide that has been shown to neutralize toxins from snakes, scorpions, and bacteria. LTNF peptides were first isolated from opossum (Didephis virginiana) serum. This part contains the LTNF10 peptide as well as C-term Myc and 6xHis tags that can be used for purification and then later removed using the HRV (Human Rhinovirus) protease.

LTNF 15- BBa_K1915002

LTNF15 (Lethal Toxin Neutralizing Factor) is a 10 residue peptide that has been shown to neutralize toxins from snakes, scorpions, and bacteria. LTNF peptides were first isolated from opossum (Didephis virginiana) serum. This part contains the LTNF15 peptide as well as C-term Myc and 6xHis tags that can be used for purification and then later removed using the HRV (Human Rhinovirus) protease.

Mambalgin-1- BBa_K1800000

This codes for the peptide Mambalgin-1, an analgesic. Mambalgin is a protein component of the venom of Dendroaspis polylepis, better known as the Black Mamba. Mambalgin-1 peptide that is a powerful analgesic that directly blocks pain transmission in the peripheral nervous system (Diochot et al, 2012) by targeting acid-sensing ion channels within nociceptors beneath the epidermis. Because Mambalgin acts on pain receptors within the skin rather than on opioid receptors in the brain, this peptide has great potential as a medication for pain treatment that is non-addicting and non-habit forming. Furthermore, recombinant purification of Mambalgin could assist in developing anti-venom without the attendant risk of harvesting venom directly from snakes. This part is designed for expression in E. coli.

CBDA Synthase- BBa_K1800003

Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic-acid(CBGA) into cannabidiolic acid or CBDA(Futoshi et.al, 2007). Cannabidiol (CBD) is a non-psychotropic constituent of the fiber-type cannabis plant which can be found in CBD oil, and it is obtained from non-enzymatic decarboxylation of CBDA (Takeda et.al, 2012). It is important to produce the CBGA to CBDA pathway synthetically independent of cannabis plants in order to insure the creation of CBD and not psychoactive tetrahydrocannabinol (THC). This is important because CBD oil can be used as an effective form of treatment for various disorders such as: seizures, cancer, anxiety, post-traumatic stress disorder, and Crohn’s disease.Since the CBDA synthase is 83.9% similar to THCA synthase in its 544-amino acid overlap, it should be possible to synthesize CBDA synthase in a similar fashion THCA synthase which has been synthesized multiple times(Futoshi et.al, 2007).

Composite parts

Inducible Lac promoter + LTNF15- BBa_K1915003

This is a generator part for BBa_K1915002. This part includes an IPTG inducible promoter, RBS J61101, and the LTNF15 peptide coding sequence.

Inducible Lac promoter + LTNF10- BBa_K1915001

This is a generator part for BBa_K1915000. This part includes an IPTG inducible promoter, RBS J61101, and the LTNF10 peptide coding sequence.

CBDAs + promoter- BBa_K1915004
TBD

Parts collection

Validated part
The IDT part CBDA synthase was inserted into psb1c3 plasmid, and then chemically transformed into E. Coli. A colony PCR and restriction digest confirmed the transformations were successful. The part was validated by DNA sequencing.