Team:Georgia State/Experiments
EXPERIMENTS
Awesome Opossum
Our furry friend the opossum, while being the only North American marsupial, is also one of only a few animals on earth that are naturally able to neutralize multiple types of snake venom. This remarkable ability has been attributed to a series of peptides known as Lethal Toxin Neutralizing Factors (LTNF) (Lipps, 1999). These factors when tested in mice were found to complete neutralize multiple venoms as well as other toxins, such as ricin and botulism). Currently the only way to obtain LTNF for experimentation is through a complex and time consuming protein purification using High pressure chromatography on Opossum Serum (Lipps, 1999). Our plan is to take the genetic sequence for producing these peptides and clone it into E.coli cells for expression. Which is an easier and more cost effective route for obtaining LTNF for the purpose of producing a universal anti-venom and toxin neutralizer.
Construct Design Bba_k1915001, Bba_k1915003
For the design, we first codon optimized the LTNF10 (Bba_k1915001) and LTNF15 (Bba_k1915003) for Escherichia coli using the IDT codon optimization tool. Restriction sites were then removed from the coding sequence that were not iGEM compatible. We added RFC 10 prefix and suffix and also included a start codon in the beginning of the sequence. Also included in the construct was a prokaryotic RBS (BBa_J61101), IPTG inducible promoter (BBa_R0011), an HRV3C Protease Site ( and Myc and 6xHis epitopes. This protein was designed specifically for easy expression in E. coli.
Experimental Design
In our experiment we decided to attempt to isolate the two most commonly worked with versions of the LTNF peptide, LTNF-10 and LTNF-15. To achieve this we inserted the sequence for each of these into a separate PSB1C3 vectors. Using heat shock these vectors were then transformed into the BL21 expression strain E. coli, which was IPTG induced in liquid cell culture.
Lipps, B. V. (1999). Anti-Lethal Factor From Opossum Serum Is A Potent Antidote For Animal, Plant And Bacterial Toxins. Journal of Venomous Animals and Toxins, 5(1). doi:10.1590/s0104-79301999000100005
Mambalgin
Mambalgin is a potent analgesic protein found in the venom of the Black Mamba snake (Dendroaspis polylepis). Mambalgin-1, the version found here, is a 3-finger toxin consisting of 57 amino acid residues forming 3 loops around a core in the shape of a hand. This protein has been found to inhibit acid-sensing ion channels (ASICs) in the central and peripheral nervous systems of mice through intraplanter and intrathecal injections. The inhibition of ASICs – important contributors to the pain pathway in both mice and humans – decreases sensitivity of nociceptive neurons to the perception of pain. The potency of mambalgin has been compared to the drug morphine. Unlike morphine, however, mambalgin has not shown an increase in tolerance over time (Diochot et al, 2012). Because of its potency and non-addictive properties, the potential of mambalgin as a pain reducer in humans is enticing. Synthetically producing this protein in large quantities would invalidate the need to harvest and extract directly from snakes – a dangerous and costly process.
Experimental process
The 2015 iGem team explored the possibility of synthetically producing the analgesic mambalgin-1 protein using E. coli. Currently, the most effective form of extraction involves milking the venom from Dendroaspis polylepis (black mamba) – a dangerous and costly process. For E. coli, they inserted the mambalgin E. coli construct into the pSB1C3 vector to create a recombinant plasmid. This was then transformed into BL21 expression strain E. coli, which was IPTG induced in liquid cell culture. The team was able to successfully express and characterize the mambalgin protein in E. coli. After inducing with IPTG overnight, the cell cultures were centrifuged and disrupted with a French press. Mambalgin was isolated using affinity chromatography, utilizing the 6x His tag in the construct. A SDS-PAGE was done, followed by a Ponceau S. and coomassie stain to visualize the proteins. A band indicating a protein of ~9 kda – the size of the mambalgin for E. coli construct – was seen in the eluate fraction. This year the iGem team repeated this process and successfully utilized the Myc tag as well to further characterize the mambalgin protein. Protein expression was also optimized this year by varying induction time as well as altering concentrations of IPTG.
CBDA
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).
Experimental Design
This past year we worked on inserting the cDNA sequence for CBDA synthase into the pORE binary vector system. This would allow us to transfect tobacco plant tissue with Agrobacteria that code for CBDA synthase creating transgenic tobacco plants that will produce CBDA synthase in their roots. The roots of these plants will then be introduced to CBDA synthase substrate, Cannabigerolic acid, which will be catalyzed to form Cannabidiolic acid. The Cannabidiolic acid will then be separated and heated at 120°C for 20min to form Cannabidiol. Because of the difficulties with producing CBDA synthase, we decided to use the same procedure to produce Horseradish Peroxidase (HRP) in the roots of transgenic tobacco as a proof of concept. One of the benefits of this is the bioluminescence that is produced when it catalyzes its substrate and allows us to prove that the same process could work for production of CBDA synthase.
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