Part:BBa_K1598008:Experience

User Reviews

There is an incredible story behind this biobrick, and I am not sure of where to share it, so here we go:

We like to consider this biobrick our little miracle, and have numerous people to thank for it. The idea to express Pregnenolone using E Coli. came to us in late August, a few weeks before wiki freeze. It was a command that came straight from the most important people in the world, the people suffering from conditions like anxiety, schizophrenia, and depression, who were the reason we were doing this project in the first place. In all the interviews we did for the Human Practices aspect of the project, there was one recurring theme- debilitating side-effects.

Whenever we asked any mentally distressed if they'd consume our psychobiotics instead of the conventional drugs they've been on, they always had one answer. People were willing to take something as novel and as controversial as genetically modified bacteria that make neuroactive compounds only, and only if it was a significant improvement to their current medication. The antidepressants and anxiolytics of today are riddled with problems. The consumer develops a tolerance to them over time, so the dosage has to be constantly increased, resulting in extravagant costs. Withdrawal anxiety worse than any hangover is experienced when the treatment is discontinued, and finally they have crippling side effects which include weight gain, memory loss, sedation, and hyperactivity.

Relief of Side-effects from Depression/Anxiety drugs:

Pregnenolone and its derivates prevent the development of tolerance, and augment recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice. Benzodiazepines, such as Valium, are one of the most popular antidepressant/anti-anxiety drugs in the world! Basically, this removes the need to constantly increase the dosage, and reduces a plethora of side-effects, such as sedation, memory loss, etc., of various antidepressants through its interactions with receptors in the brain such as GABA (A), and the production of other neuro-active compounds.[1]

Schizophrenia Treatment

Emerging preclinical and clinical evidence suggests that pregnenolone may be a promising novel therapeutic candidate in schizophrenia. Pregnenolone is a neurosteroid with pleiotropic actions in rodents that include the enhancement of learning and memory, neuritic outgrowth, and myelination. Further, pregnenolone administration results in elevations in downstream neurosteroids such as allopregnanolone, a molecule with neuroprotective effects that also increases neurogenesis, decreases apoptosis and inflammation, modulates the hypothalamic-pituitary-adrenal axis, and markedly increases GABA(A) receptor responses. In addition, pregnenolone administration elevates pregnenolone sulfate, a neurosteroid that positively modulates NMDA receptors. There are thus multiple mechanistic possibilities for pregnenolone as a potential therapeutic agent in schizophrenia, including the amelioration of NMDA receptor hypofunction (via metabolism to pregnenolone sulfate) and the mitigation of GABA dysregulation (via metabolism to allopregnanolone). [2]

Memory, Cognition, and Neurotransmission

Neurosteroids derived from Pregnenolone modulate several neurotransmitter systems such as gamma-aminobutyric acid type A (GABA(A)), N-methyl-D-aspartate (NMDA) and acetylcholine receptors. As physiologic consequences, they are involved in neuronal plasticity, learning and memory processes, aggression and epilepsy, and they modulate the responses to stress, anxiety and depression. There is evidence for a common mechanism of action between neurosteroids and sigma1-receptor ligands and focus on the potential therapeutic interests of such interaction in the physiopathology of learning and memory impairments, stress, depression and neuroprotection. [3]

Steroid Biosynthesis

Pregnenolone is the precursor to all steroids, and the conversion of cholesterol into Pregnenolone is the rate limiting step of the biosynthesis of all steroids (http://www.sciencedirect.com/science/article/pii/S0143416006000856). Hence, this biobrick unlocks steroidogenesis, and paves the way for a innumerable applications in the fields of hormone replacement, fitness, birth control, immunorepression, etc.

(Source:https://en.wikiversity.org/wiki/Wikiversity_Journal_of_Medicine/Diagram_of_the_pathways_of_human_steroidogenesis)

Characterization

High Performance Liquid Chromatography was used to charachterize the biobrick. This was done by using the HPLC system Series 1200 (Agilent, USA) at 50˚C and a flow rate of 1 ml/min. The stationary phase used was a Symmetry column (Waters, USA) 250 mm × 4.6 mm (with a guard column 20 mm × 3.9 mm) packed with reverse phase ODS (5 µm). The liquid phase used was a solution of 52% acetonitrile, 48% H2O and 0.01% acetic acid. A buffer of 0.01% acetic acid was used for elution.

Peak detection was carried out by UV absorbance at 200 nm. Identification of the peaks and the quantification of pregnenolone was carried out using standard technique.

Results As shown by the image below, pregenolone is seen as a clear peak at around 11mins.
A standard curve of area under the peak vs. concentration of pregnenolone in standard solution was plotted based on the peaks, as shown below.

HPLC reference results

References were taken for different concentrations of pregnenelone

Results

Therefore, a maximum pregnenolone production of 0.007 mg/ml within 4 hours of substrate addition was observed! This was approximately 17.5 times greater than the amount quantified in past research [4]. Furthermore, this is possibly the first time Human FDX1 gene, Human FDXR gene, and Human CYP11A1 were expressed together in E Coli., as no literature on this particular combination of genes being expressed by E. Coli. was found.

Cell Growth

As illustrated by the graphs above the cell growth rate of the E. Coli with the part in 3 different backbones was studied using OD600 measurements taken every 10 minutes, overnight. This was used to get a better estimate of the best backbone for the part, and as seen in the graphs above, there is no significant difference in cell growth caused by a different backbone.

UNIQ01b821bba85480c5-partinfo-00000006-QINU UNIQ01b821bba85480c5-partinfo-00000007-QINU

References

• [1] Reddy DS and Kulkami SK Neurosteroid coadministration prevents development of tolerance and augments recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice. in Methods Find Exp Clin Pharmacol. 1997 Jul-Aug;19(6):395-405
• [2] Marx CE et al. Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and clinical evidence. in Neuroscience. 2011 Sep 15;191:78-90. doi: 10.1016/j.neuroscience.2011.06.076. Epub 2011 Jul 1.
• [3] Maurice T et al. Neuroactive neurosteroids as endogenous effectors for the sigma1 (sigma1) receptor: pharmacological evidence and therapeutic opportunities. in Jpn J Pharmacol. 1999 Oct;81(2):125-55.
• [4] Desislava S. Makeeva Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli in American Journal of Molecular Biology, 2013, 3, 173-182